Biological material extraction magnetic bead

Abstract

To provide a biological material extraction magnetic bead that has favorable biological material extraction efficiency and is less likely to lower examination accuracy of an extracted biological material.SOLUTION: A biological material extraction magnetic bead comprises: a magnetic metal particle; an inorganic oxide layer that covers a surface of the magnetic metal particle and contains an inorganic oxide; a base layer that is provided on a side opposite to the magnetic metal particle of the inorganic oxide layer and contains a metal element forming a gold ion reducing agent or a catalyst for a gold ion reduction reaction; a gold layer that covers a surface of the base layer and contains gold; and an immobilization layer that is bound to the gold layer via an Au-S bond and contains a ligand or a ligand-reactive group, wherein a mass ratio M / Au of a content of the metal element to a content of the gold is 0.03 or more and 19.0 or less.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to magnetic beads for extracting biological substances. [Background technology]

[0002] In recent years, there has been an increasing demand for testing biological materials in the fields of medical diagnosis and life science. Among biological material testing methods, the polymerase chain reaction (PCR) method is a method in which nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are extracted and specifically amplified for detection. In the process of testing such biological materials, it is first necessary to extract the material to be tested from a specimen. To extract this biological material, for example, an extraction carrier capable of binding to the biological material is used.

[0003] Patent Document 1 cites ligands as an example of binding species capable of binding to cell surface receptors or viruses. It then discloses the detection of biomolecules using colloids bearing the ligands. It also discloses that a self-assembled monolayer composed of thiols is formed on the surface of gold-coated particles, and that the self-assembled monolayer presents a binding partner for the ligand. It also discloses the use of magnetic beads as particles. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2017-122728 Summary of the Invention [Problem to be solved by the invention]

[0005] In the extraction of biological materials, various physical and chemical loads are applied to the extraction support. When such loads are applied to the colloid carrying the ligand described in Patent Document 1, there is a concern that the extraction efficiency of the biological material may decrease due to detachment of the biological material bound to the extraction support or aggregation of the extraction support.

[0006] Furthermore, there is room for improvement in the dispersibility of the colloid described in Patent Document 1 in the dispersion medium. If the dispersibility is low, the efficiency of capturing the biological material decreases, and as a result, the efficiency of extracting the biological material decreases.

[0007] Furthermore, when magnetic beads are used as the particles described in Patent Document 1, metal ions contained in the magnetic beads may be eluted into the dispersion medium, which may hinder the testing of biological materials.

[0008] Therefore, the realization of magnetic beads for extracting biological substances that have good extraction efficiency for biological substances and that do not reduce the accuracy of testing the extracted biological substances has become an issue. [Means for solving the problem]

[0009] The magnetic beads for extracting biological substances according to the application example of the present invention are: Magnetic metal particles; an inorganic oxide layer that coats the surfaces of the magnetic metal particles and contains an inorganic oxide; a base layer provided on the opposite side of the inorganic oxide layer from the magnetic metal particles, the base layer containing a metal element that constitutes a reducing agent for gold ions or a catalyst for a reduction reaction of gold ions; a gold layer that covers a surface of the underlayer and contains gold; an immobilization layer comprising a ligand or a ligand-reactive group bonded to the gold layer via an Au-S bond; and The mass ratio M / Au of the content of the metal element to the content of gold is 0.03 or more and 19.0 or less. [Brief explanation of the drawings]

[0010] [Figure 1]FIG. 1 is a process diagram illustrating an example of a biological material extraction method. [Figure 2] FIG. 2 is a schematic diagram for explaining the biological substance extraction method shown in FIG. [Figure 3] FIG. 2 is a schematic diagram for explaining the biological substance extraction method shown in FIG. [Figure 4] FIG. 2 is a schematic diagram for explaining the biological substance extraction method shown in FIG. [Figure 5] FIG. 1 is a cross-sectional view showing a magnetic bead according to an embodiment. [Figure 6] FIG. 6 is a partial enlarged view of the coating film shown in FIG. [Figure 7] FIG. 6 is a cross-sectional view showing a modified example of the magnetic beads of FIG. [Figure 8] FIG. 1 is a schematic diagram showing a state in which a compound having a functional site is bound to a gold layer via an Au—S bond. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, preferred embodiments of the magnetic beads for extracting biological substances of the present invention will be described in detail with reference to the accompanying drawings.

[0012] The magnetic beads for biological material extraction according to the embodiment are a group of particles that adsorb biological materials and are used for magnetic separation. Magnetic separation is a technique for separating the solid and liquid phases by applying an external magnetic field to a container containing a solid phase containing magnetic beads for biological material extraction and a liquid phase containing a dispersion medium, thereby magnetically attracting the solid phase. In the following description, the magnetic beads for biological material extraction may be simply referred to as "magnetic beads."

[0013] Biological materials refer to substances such as nucleic acids such as DNA and RNA, proteins, sugars, various cells such as cancer cells, peptides, bacteria, and viruses. Nucleic acids may be present in biological samples such as cells and biological tissues, or in a state contained in viruses and bacteria. The biological material extraction method extracts such biological materials through the steps of dissolution / adsorption, washing, and elution, and the biological materials can be purified and extracted by utilizing the magnetic separation described above during this process.

[0014] 1. Biological material extraction method An example of a method for extracting a biological material using magnetic separation will be described below, taking the case where the biological material is nucleic acid as an example.

[0015] Fig. 1 is a process chart for explaining an example of a biological material extraction method, and Figs. 2 to 4 are schematic diagrams for explaining the biological material extraction method shown in Fig. 1.

[0016] The biological material extraction method shown in Fig. 1 includes a dissolving / adsorbing step S102, a washing step S108, and an eluting step S110. Each step will be explained below in order.

[0017] 1.1.Dissolution / adsorption process In the dissolution and adsorption step S102, a specimen sample containing nucleic acid, a liquid 3 containing a dissolution and adsorption solution, and magnetic beads 2 are placed in a container 1 shown in FIG. 2. The contents of the container 1 are then mixed. As a result, the magnetic beads 2 are dispersed in the liquid 3 in the container 1, as shown in FIG. 2. Nucleic acids are typically encapsulated in cell membranes or nuclei. Therefore, when the so-called outer shells of cell membranes and nuclei are dissolved and removed by the dissolution action of the dissolution and adsorption solution, the nucleic acids are extracted from the specimen. The nucleic acids are then adsorbed to the magnetic beads 2 by the adsorption action of the dissolution and adsorption solution. The magnetic beads 2 with the adsorbed nucleic acids are separated from the liquid phase through magnetic separation and liquid discharge operations, which will be described later.

[0018] The dissolving / adsorbing liquid may be, for example, a liquid containing a chaotropic substance. The chaotropic substance generates chaotropic ions in an aqueous solution, which reduces the interactions of water molecules and destabilizes the structure, contributing to the adsorption of nucleic acids to the magnetic beads 2.

[0019] When extracting nucleic acids, particularly RNA, it is preferable to acidify the liquid 3 in the container 1 by adding an acid, for example. RNA monomers contain ribose, and therefore are more soluble in polar solvents than DNA. Therefore, after adding an acid to the liquid 3 containing the specimen sample from which the outer shell has been dissolved and removed, a nonpolar solvent such as phenol or chloroform is added. This causes the DNA to migrate to the nonpolar solvent, while the RNA remains in the polar solvent. As a result, the RNA and DNA can be separated, and the RNA can be extracted.

[0020] When extracting RNA, the pH of the liquid 3 in the container 1 is preferably 5.0 or less, and more preferably 2.0 to 4.0. This shifts the ionization equilibrium of the phosphate groups contained in the RNA toward the hydroxyl groups, making the RNA particularly soluble in polar solvents. This further increases the efficiency of RNA extraction.

[0021] 1.1.1.Magnetic separation operation In the dissolution / adsorption step S102, an external magnetic field is applied to the magnetic beads 2 to which nucleic acids are adsorbed, causing them to be magnetically attracted. This causes the magnetic beads 2 to move to the inner wall of the container 1 and be immobilized, as shown in FIG. 3. As a result, the magnetic beads 2, which are the solid phase, can be separated from the liquid 3, which is the liquid phase. In this specification, the operation of immobilizing the magnetic beads 2 by applying such an external magnetic field is referred to as the "magnetic separation operation."

[0022] Before magnetic separation, the contents of the container 1 are stirred as needed, thereby increasing the probability that nucleic acids will be adsorbed onto the magnetic beads 2. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. may be used.

[0023] To apply the external magnetic field, for example, a magnet 5 placed on the side of the container is used. The magnet 5 may be an electromagnet or a permanent magnet. When the external magnetic field acts on the magnetic beads 2, the magnetic beads 2 move toward the magnet 5.

[0024] After the magnetic separation operation, acceleration may be applied to the container as needed. This allows the liquid 3 adhering to the magnetic beads 2 to be shaken off, thereby reducing the amount of liquid 3 that has not been separated. The acceleration may be centrifugal acceleration. A centrifuge may be used to apply centrifugal acceleration.

[0025] 1.1.2.Liquid drain operation In the dissolution / adsorption step S102, with the magnetic beads 2 fixed to the inner wall of the container 1, the liquid 3 accumulated at the bottom of the container 1 is aspirated and discharged using, for example, a pipette 6, as shown in FIG. 4. In this specification, this operation of discharging the liquid 3 is referred to as the "liquid discharge operation." After the liquid discharge operation, the magnetic beads 2 with the nucleic acid adsorbed thereon remain in the container 1.

[0026] 1.2.Cleaning process In the washing step S108, the magnetic beads 2 to which nucleic acids are adsorbed are washed. Washing refers to a procedure in which the magnetic beads 2 to which nucleic acids are adsorbed are brought into contact with a washing solution and then separated again to remove impurities adsorbed to the magnetic beads 2.

[0027] Specifically, after pouring a washing solution into the container 1 containing the magnetic beads 2 to which nucleic acids are adsorbed, the above-mentioned magnetic separation operation and liquid discharge operation are carried out again.

[0028] In the magnetic separation operation, the magnetic beads 2 and washing solution in the container 1 are first stirred. This brings the washing solution into contact with the magnetic beads 2, washing the magnetic beads 2 to which nucleic acids have been adsorbed. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. may be used. At this time, the application of the external magnetic field may be temporarily turned off. This allows the magnetic beads 2 to be redispersed in the washing solution, further improving washing efficiency.

[0029] Next, as a liquid discharge operation, the washing solution accumulated at the bottom of the container 1 is discharged while the magnetic beads 2 are fixed to the inner wall of the container 1. By supplying and discharging the washing solution as described above at least once, the magnetic beads 2 are washed. This allows impurities to be removed with high precision.

[0030] The washing liquid is not particularly limited as long as it does not promote the elution of nucleic acids and does not promote the binding of impurities to the magnetic beads 2, but examples include organic solvents such as ethanol, isopropyl alcohol, acetone, or aqueous solutions thereof, and low-salt aqueous solutions.

[0031] The washing solution may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), SDS, etc. The washing solution may also contain a chaotropic substance such as guanidine hydrochloride.

[0032] Furthermore, the cleaning step S108 may be performed as needed, and may be omitted if cleaning is not necessary.

[0033] 1.3.Elution process In the elution step S110, the nucleic acid adsorbed to the magnetic beads 2 is eluted into an elution solution. Elution is an operation in which the magnetic beads 2 to which the nucleic acid is adsorbed are brought into contact with the elution solution, thereby transferring the nucleic acid to the elution solution.

[0034] Specifically, after the eluate is poured into the container 1 containing the magnetic beads 2 to which nucleic acids are adsorbed, the above-mentioned magnetic separation operation and liquid discharge operation are carried out again.

[0035] In the magnetic separation operation, the magnetic beads 2 and eluate in the container 1 are first stirred. This brings the eluate into contact with the magnetic beads 2, and the nucleic acids are eluted into the eluate. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. may be used. At this time, the application of the external magnetic field may be temporarily turned off. This allows the magnetic beads 2 to be redispersed in the eluate, further increasing the elution efficiency.

[0036] Next, as a liquid discharging operation, the eluate that has accumulated at the bottom of the container 1 is discharged while the magnetic beads 2 are fixed to the inner wall of the container 1. This makes it possible to collect the eluate that contains nucleic acids.

[0037] The elution liquid is not particularly limited as long as it is a liquid that promotes the elution of nucleic acids from the magnetic beads 2 to which nucleic acids are adsorbed. For example, in addition to water such as sterilized water or pure water, a TE buffer solution, i.e., an aqueous solution containing 10 mM Tris-HCl buffer and 1 mM EDTA and having a pH of about 8, is preferably used.

[0038] The eluent may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), SDS, etc. It may also contain sodium azide as a preservative.

[0039] In the elution step S110, the eluate may be heated, thereby facilitating the elution of nucleic acids. The heating temperature of the eluate is not particularly limited, but is preferably 70°C or higher and 200°C or lower, more preferably 80°C or higher and 150°C or lower, and even more preferably 95°C or higher and 125°C or lower.

[0040] 2. Magnetic beads for extracting biological materials Next, the magnetic beads 2 (magnetic beads for extracting biological substances according to the embodiment) will be described. The magnetic beads 2 are particles that are magnetic and have the ability to bind to biological substances on their surfaces.

[0041] Figure 5 is a cross-sectional view showing a magnetic bead 2 according to an embodiment. The magnetic bead 2 shown in Figure 5 has magnetic metal particles 22 and a coating film 24. The magnetic metal particles 22 are made of magnetic metal powder. At least the surface of the coating film 24 is composed of a substance or chemical structure that has the ability to bind to biological substances. In this specification, magnetic beads 2 refer to a particle group or a single particle that constitutes the particle group.

[0042] Such magnetic beads 2 enable magnetic separation when the adsorbed biological material is separated from the liquid phase. In magnetic separation, the momentum applied to the adsorbate to which the biological material is adsorbed is kept small compared to other separation methods, such as centrifugation. This reduces the load applied to the adsorbate, making it possible to prevent the biological material and ligand from detaching.

[0043] The saturation magnetization of the magnetic metal particles 22 is preferably 50 emu / g or more, more preferably 80 emu / g or more, and even more preferably 100 emu / g or more. Saturation magnetization is the value of magnetization when a magnetic material exhibits a constant magnetization regardless of the applied magnetic field when a sufficiently strong external magnetic field is applied. If the saturation magnetization is within the above range, the magnetic material can fully function. Specifically, the movement speed of the magnetic beads 2 in a magnetic field can be improved, thereby shortening the time required for magnetic separation. Furthermore, the saturation magnetization of the magnetic metal particles 22 affects the adsorptive strength when immobilized by an external magnetic field. If the saturation magnetization is within the above range, a sufficiently high adsorptive strength can be obtained, preventing the magnetic beads 2 from being discharged with the liquid 3 when the liquid 3 is discharged with the magnetic beads 2 immobilized. This prevents a decrease in nucleic acid yield due to a decrease in the magnetic beads 2.

[0044] The upper limit of the saturation magnetization of the magnetic metal particles 22 is not particularly limited, but is preferably 220 emu / g or less from the viewpoint of easiness in selecting a material that is suitable for balancing performance and cost.

[0045] The saturation magnetization of the magnetic metal particles 22 can be measured using a vibrating sample magnetometer (VSM) or the like. An example of a vibrating sample magnetometer is the TM-VSM1230-MHHL manufactured by Tamagawa Seisakusho Co., Ltd. The maximum magnetic field applied when measuring the saturation magnetization is, for example, 0.5 T or more.

[0046] The average particle size D50 of the magnetic beads 2 is preferably 0.5 μm to 30 μm, more preferably 1 μm to 20 μm, even more preferably 2 μm to 15 μm, and particularly preferably 3 μm to 10 μm. If the average particle size D50 of the magnetic beads 2 is within the above range, the specific surface area of ​​the magnetic beads 2 can be sufficiently increased, and the magnetic beads 2 can generate attractive and adsorbing forces suitable for magnetic separation. Furthermore, aggregation of the magnetic beads 2 can be suppressed, improving dispersibility. If the average particle size D50 of the magnetic beads 2 is below the lower limit, the magnetization value of the magnetic beads 2 decreases and they become more susceptible to aggregation, potentially resulting in a decrease in the nucleic acid extraction efficiency. Furthermore, the movement speed of the magnetic beads 2 may decrease, potentially lengthening the time required for magnetic separation. On the other hand, if the average particle size D50 of the magnetic beads 2 exceeds the upper limit, the specific surface area of ​​the magnetic beads 2 becomes smaller, making it impossible to adsorb a sufficient amount of nucleic acid, potentially reducing the amount of nucleic acid extracted. Furthermore, the magnetic beads 2 tend to settle, which reduces the amount of magnetic beads 2 that can contribute to the extraction of nucleic acids, possibly resulting in a decrease in the efficiency of nucleic acid extraction.

[0047] The average particle size D50 of the magnetic beads 2 can be determined by measuring the volumetric particle size distribution using a laser diffraction and dispersion method and then using the cumulative distribution curve obtained from this particle size distribution. Specifically, the particle size (median diameter) at which the cumulative value from the smallest diameter side in the cumulative distribution curve is 50% is the average particle size D50 of the magnetic beads 2. Examples of devices for measuring particle size distribution using the laser diffraction and dispersion method include the MT3300 series manufactured by Microtrac-Bell. Note that the method is not limited to the laser diffraction and dispersion method, and image analysis and other techniques may also be used.

[0048] The 90% particle diameter of the magnetic beads 2 is defined as D90. The ratio D90 / D50 of the 90% particle diameter D90 to the average particle diameter D50 of the magnetic beads 2 is preferably 3.00 or less, more preferably 2.00 or less, and even more preferably 1.75 or less. This reduces the content of coarse particles, thereby preventing the coarse particles from attracting and agglomerating surrounding smaller particles. If aggregates are formed, they may settle due to their own weight, resulting in reduced extraction efficiency and prolonged testing times for biological materials. Therefore, if the ratio D90 / D50 is within the above range, these problems can be prevented. If the ratio D90 / D50 exceeds the upper limit, the content of coarse particles increases, which may reduce the dispersibility of the magnetic beads 2 and increase the likelihood of aggregation, even when the external magnetic field is turned off.

[0049] The 90% particle diameter D90 of the magnetic beads 2 can be determined from the cumulative distribution curve obtained from the particle size distribution measured on a volume basis by laser diffraction and dispersion. Specifically, the particle diameter at which the cumulative value from the smallest diameter side is 90% on the cumulative distribution curve is the 90% particle diameter D90 of the magnetic beads 2.

[0050] Furthermore, when the average thickness of the coating film 24 is t and the average particle size of the magnetic beads 2 is D50, the ratio of t to D50, t / D50, is preferably 0.0001 or more and 0.05 or less, more preferably 0.001 or more and 0.01 or less. If t / D50 is below the lower limit, the ratio of the thickness of the coating film 24 to the size of the magnetic metal particles 22 becomes too small, which may cause the coating film 24 to be damaged or peeled off when the magnetic beads 2 collide with each other or with the inner wall of the container 1. This may reduce the amount of nucleic acid adsorbed to the surface of the coating film 24 and extracted, potentially reducing extraction efficiency. Furthermore, if fragments of the peeled coating film 24 or magnetic metal particles 22 are present in the extract, they may be mixed in as contaminants when the nucleic acid is extracted. Furthermore, destruction and peeling of the coating film 24 exposes the magnetic metal particles 22, which may lead to the elution of iron ions and other contaminants when the magnetic metal particles 22 come into contact with an acidic solution, potentially resulting in a decrease in the efficiency of nucleic acid extraction. On the other hand, if t / D50 exceeds the upper limit, the volume ratio of the coating film 24 to the total volume of the magnetic beads 2 increases, potentially reducing the magnetization per volume of the magnetic beads 2. This may reduce the movement speed of the magnetic beads 2 when an external magnetic field is applied, potentially lengthening the time required for magnetic separation.

[0051] The thickness of the coating film 24 can be measured from a cross-sectional image of the magnetic beads 2 observed using, for example, a transmission electron microscope or a scanning electron microscope. The average thickness t of the coating film 24 can be calculated by acquiring multiple observation images and averaging the measured values ​​obtained through image processing or the like. For example, the average thickness t is determined by measuring the thickness of the coating film 24 at five or more locations on one magnetic bead 2, calculating the average value, and then averaging the average values ​​for ten or more magnetic beads 2. Alternatively, the intensities of, for example, Si-K characteristic X-rays and Fe-L characteristic X-rays may be compared using an analyzer such as EDX (Energy Dispersive X-ray spectroscopy), and the thickness of the coating film 24 may be calculated from the comparison results. That is, as described below, when the coating film 24 contains silicon and the magnetic metal particles 22 are made of an Fe-based alloy, the intensity ratio of the Si-K characteristic X-rays originating from the coating film 24 to the sum of the Fe-L characteristic X-rays originating from the magnetic metal particles 22 and the Si-K characteristic X-rays originating from the coating film 24 can be converted into the thickness of the coating film 24.

[0052] Furthermore, the coercive force Hc of the magnetic metal particles 22 is preferably 100 A / m or less, more preferably 80 A / m or less, even more preferably 60 A / m or less, and particularly preferably 50 A / m or less. The coercive force Hc refers to the value of an external magnetic field in the opposite direction required to return a magnetized magnetic material to an unmagnetized state. In other words, the coercive force Hc refers to the resistance to an external magnetic field. The smaller the coercive force Hc of the magnetic metal particles 22, the less likely the magnetic beads 2 are to aggregate even when switching from an applied magnetic field to an unapplied magnetic field, allowing the magnetic beads 2 to be more uniformly dispersed in the dispersion liquid. Furthermore, even when switching between applied magnetic fields repeatedly, the smaller the coercive force Hc, the better the redispersibility of the magnetic beads 2, thereby further suppressing aggregation of the magnetic beads 2. Note that the lower limit of the coercive force Hc of the magnetic metal particles 22 is not particularly limited, but is preferably 5 A / m or more from the viewpoint of ease of material selection that balances performance and cost.

[0053] The coercive force Hc of the magnetic metal particles 22 can be measured by a vibrating sample magnetometer, etc., in the same manner as the saturation magnetization described above. The maximum applied magnetic field when measuring the coercive force Hc is, for example, 15 kOe.

[0054] Furthermore, the relative magnetic permeability of the magnetic metal particles 22 is preferably 5 or more. If the relative magnetic permeability of the magnetic metal particles 22 is below the lower limit, the movement speed of the magnetic beads 2 may decrease, and the time required for magnetic separation may become longer. Note that there is no particular upper limit to the relative magnetic permeability of the magnetic metal particles 22, but because the magnetic beads 2 are in powder form, the relative magnetic permeability often takes a value of substantially 100 or less due to the influence of a demagnetizing field.

[0055] 2.1.Magnetic metal particles The magnetic metal particles 22 are magnetic particles and preferably contain at least one of Fe, Co, and Ni as a constituent element. In particular, from the viewpoint of obtaining high saturation magnetization, the composition of the magnetic metal particles 22 is preferably an alloy (Fe-based alloy) whose main component is Fe. Specifically, the Fe content is more preferably 50% or more, and even more preferably 70% or more, in terms of atomic ratio. Examples of Fe-based alloys include Fe-Co based alloys, Fe-Ni based alloys, Fe-Co-Ni based alloys, and compounds containing Fe, Co, and Ni. Furthermore, from the viewpoint of obtaining high magnetization, carbonyl iron powder, Fe-Si based alloy powder, Fe-Si-Cr based alloy powder, and the like, which are composed of substantially 100 atomic % Fe, can be preferably used as the magnetic metal particles 22. Such Fe-based alloys can realize magnetic metal particles 22 that exhibit high saturation magnetization and high magnetic permeability, even with a small particle size. This allows for the realization of magnetic beads 2 that have a high migration speed under the action of an external magnetic field and a strong attractive force when captured by an external magnetic field. As a result, the time required for magnetic separation can be shortened, and the magnetic beads 2 themselves can be prevented from being mixed into the eluate and becoming contaminants.

[0056] In addition to the aforementioned Fe, an element that exhibits ferromagnetism by itself, Fe-based alloys can contain one or more elements selected from the group consisting of Cr, Nb, Cu, Al, Mn, Mo, Si, Sn, B, C, P, Ti, and Zr, depending on the desired properties. Si is a major constituent element of alloy powders, but it also promotes amorphization.

[0057] The Fe-based alloy may contain impurities to the extent that they do not impair the effects of the magnetic metal particles 22. In this embodiment, the impurities are elements that are unintentionally mixed into the raw materials of the magnetic metal particles 22 or during the production of the magnetic beads 2. The impurities are not particularly limited, but examples thereof include O, N, S, Na, Mg, K, etc.

[0058] An example of an Fe-based alloy is an alloy with a Si content of preferably 1.0 atomic % or more and 30.0 atomic % or less, more preferably 1.5 atomic % or more and 13.0 atomic % or less, and even more preferably 2.0 atomic % or more and 7.0 atomic % or less. Such an alloy has a high magnetic permeability and therefore tends to have a high saturation magnetization.

[0059] The Fe-based alloy may also contain at least one of B (boron) with a content of 5.0 atomic % to 16.0 atomic % and C (carbon) with a content of 0.5 atomic % to 5.0 atomic %. These elements promote amorphization and contribute to the formation of a stable amorphous structure or nanocrystalline structure in the magnetic metal particles 22.

[0060] Furthermore, the Fe-based alloy preferably contains Cr (chromium) at a content of 1.0 atomic % or more and 8.0 atomic % or less, which can improve the corrosion resistance of the magnetic metal particles 22.

[0061] The total content of impurities is preferably 1.0 atomic % or less, so that the effect of the magnetic metal particles 22 is not impaired even if impurities are contained.

[0062] A particularly preferred example of an Fe-based alloy is an alloy containing Fe as the main component, with a Si content of 2.0% by mass to 9.0% by mass, a B content of 1.0% by mass to 5.0% by mass, a Cr content of 1.0% by mass to 3.0% by mass, and a C content of 2.0% by mass or less. Such an Fe-based alloy may contain a stable amorphous structure, resulting in low coercivity. Furthermore, the high Fe content results in high saturation magnetization. Furthermore, the inclusion of Cr enhances corrosion resistance and can suppress the elution of iron ions and the like. Since iron ions can adversely affect the testing of biological materials, it is preferable to suppress their elution.

[0063] The constituent elements and composition of the magnetic metal particles 22 can be identified by ICP atomic emission spectrometry as defined in JIS G 1258:2014, spark atomic emission spectrometry as defined in JIS G 1253:2002, or the like. Furthermore, if the magnetic metal particles 22 are coated with a coating film 24, the coating film 24 can be removed by a chemical or physical method, and then the elements and composition can be measured by the above-mentioned method. Furthermore, if it is difficult to remove the coating film 24, the magnetic beads 2 can be cut, and the core portion of the magnetic metal particle 22 can be analyzed using an analytical device such as an EPMA (Electron Probe Micro Analyzer) or EDX (Energy Dispersive X-ray spectroscopy).

[0064] The Vickers hardness of the magnetic metal particles 22 is preferably 100 or more, more preferably 300 or more, and even more preferably 800 or more. The hardness of the magnetic metal particles 22 can be measured, for example, as follows. A plurality of magnetic metal particles 22 are taken out and embedded in resin to prepare a resin-embedded sample, which is then ground and polished to reveal the cross section of the magnetic metal particles 22 on the surface of the resin-embedded sample. An indentation is made on this using a micro Vickers tester or a nanoindenter, and the hardness is measured from the size of the indentation.

[0065] If the Vickers hardness of the magnetic metal particles 22 is less than the lower limit, the magnetic metal particles 22 may be plastically deformed due to the impact of a collision with the magnetic beads 2. If plastic deformation occurs, there is a risk that the coating film 24 may peel off or fall off. The upper limit of the Vickers hardness is not particularly limited, but is preferably 3000 or less from the viewpoint of facilitating the selection of a material that is suitable for balancing performance and cost.

[0066] The main metal structure constituting the magnetic metal particles 22 can take various forms, such as a crystalline structure, an amorphous structure, or a nanocrystalline structure. An amorphous structure is a non-crystalline structure without crystals, and a nanocrystalline structure is a structure primarily composed of fine crystals with a grain size of 100 nm or less. The amorphous structure and nanocrystalline structure impart high hardness to the magnetic metal particles 22. Furthermore, by using an amorphous structure or a nanocrystalline structure, the coercive force of the magnetic beads 2 becomes particularly low, contributing to improved redispersibility of the magnetic beads 2. The volume fraction of the amorphous structure or nanocrystalline structure in the magnetic metal particles 22 is preferably 40% or more, and more preferably 60% or more. This volume fraction is determined from the results of crystal structure analysis using X-ray diffraction. Furthermore, the crystalline structure, amorphous structure, and nanocrystalline structure may exist individually, or two or more of them may be mixed.

[0067] The metallic structure of the magnetic metal particles 22 can be identified by performing crystal structure analysis on the magnetic metal particles 22 using X-ray diffraction. Furthermore, it can be specified by analyzing the structure observation image or diffraction pattern of a cut sample using a transmission electron microscope (TEM). For example, in the case of an amorphous structure, peak analysis using X-ray diffraction does not reveal diffraction peaks derived from metal crystals such as the α-Fe phase. Furthermore, in the case of an amorphous structure, a so-called halo pattern is formed in the electron beam diffraction pattern using TEM, and no crystalline spots are observed. A nanocrystalline structure is composed of a crystalline structure with a grain size of, for example, 100 nm or less, and can be confirmed from a TEM observation image. More precisely, the average grain size can be calculated from multiple TEM structure observation images containing multiple crystals by image processing, etc. Furthermore, the crystal grain size can be estimated using the Sherer method from the diffraction peaks of the target crystalline phase obtained by X-ray diffraction. Furthermore, for crystalline structures with large grain sizes, the crystal grain size can be measured by methods such as observing the cross section using an optical microscope or a scanning electron microscope (SEM).

[0068] To obtain an amorphous structure and a nanocrystalline structure, it is effective to increase the cooling rate when pulverizing the molten raw material and then cooling it during the production of the magnetic metal particles 22. The ease of forming an amorphous structure and a nanocrystalline structure also depends on the alloy composition. A specific alloy system suitable for forming an amorphous structure and a nanocrystalline structure is preferably a composition in which Fe is added with one or more elements selected from the group consisting of Cr, Si, B, C, P, Nb, and Cu.

[0069] The magnetic metal particles 22 are manufactured by a method similar to a general method for manufacturing metal powder. Examples of manufacturing methods include a melting process in which a metal is melted and solidified to produce a powder, a chemical process in which a powder is produced by a reduction method or a carbonyl method, or a mechanical process in which a larger object such as an ingot is mechanically crushed to obtain a powder. Of these, the melting process is suitable for manufacturing the magnetic metal particles 22.

[0070] Atomization is a typical manufacturing method using the melting process, in which a molten metal of a desired composition formed by melting is atomized to form a powder.

[0071] Atomization is a method of rapidly cooling and solidifying molten metal by colliding it with a fluid (liquid or gas) sprayed at high speed to turn it into powder, and can be divided into water atomization, high-pressure water atomization, rotary water jet atomization, gas atomization, etc. depending on the type of coolant and the configuration of the equipment. Atomization can efficiently produce magnetic metal particles 22. Furthermore, in high-pressure water atomization, rotary water jet atomization, and gas atomization, the particle shape of the metal powder becomes nearly spherical due to the effect of surface tension.

[0072] 2.2.Coating film As shown in Fig. 5, the coating film 24 coats the magnetic metal particles 22. The coating film 24 may be formed on at least a part of the surface of the magnetic metal particles 22, but it is preferable that the coating film 24 coats the entire surface.

[0073] FIG. 6 is a partial enlarged view of the coating film 24 shown in FIG. 6, the coating film 24 has an inorganic oxide layer 242, an underlayer 244, a gold layer 246, and an immobilization layer 248. In other words, the coating film 24 has a multilayer structure having these layers. It is preferable that each layer covers the entire underlayer, but discontinuous portions are also acceptable.

[0074] 2.2.1. Inorganic oxide layer The inorganic oxide layer 242 covers the surface of the magnetic metal particles 22 and contains an inorganic oxide. Examples of the inorganic oxide include silicon oxide, magnesium oxide, calcium oxide, aluminum oxide, titanium oxide, zirconium oxide, boron oxide, yttrium oxide, molybdenum oxide, etc., and one or a mixture of two or more of these is used. Since the inorganic oxide layer 242 containing such an inorganic oxide is porous, a large contact area with the underlying layer 244 can be ensured. Thereby, the adhesion of the underlying layer 244 to the inorganic oxide layer 242 can be enhanced.

[0075] Among these, the inorganic oxide is preferably silicon oxide or titanium oxide. Since these are chemically stable, oxidation and corrosion of the magnetic metal particles 22 can be particularly suppressed, and the corrosion resistance of the magnetic beads 2 can be particularly enhanced.

[0076] Silicon oxide is represented by the composition formula SiOx (0 < x ≤ 2), but is preferably SiO2. Further, silicon oxide may form a composite oxide or a composite with one or two or more selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr.

[0077] Titanium oxide is represented by the composition formula TiOx (0 < x ≤ 2), but is preferably TiO2. Further, titanium oxide may form a composite oxide or a composite with one or two or more selected from the group consisting of Si, Al, V, Nb, Cr, Mn, Sn, and Zr.

[0078] The inorganic oxide may contain a substance (impurity) other than the inorganic oxide, for example, at a ratio of 50% by mass or less of the above-described inorganic oxide, within a range not impairing its effect. For example, when silicon oxide is used as the inorganic oxide, examples of the impurity include C, N, P, etc. The composition of the inorganic oxide can be confirmed by, for example, EDX analysis, Auger electron spectroscopy measurement, etc.

[0079] The inorganic oxide layer 242 may cover the surface of one magnetic metal particle 22, or may cover a plurality of magnetic metal particles 22 together.

[0080] FIG. 7 is a cross-sectional view showing a modified example of the magnetic bead 2 of FIG. The magnetic beads 2A shown in FIG. 7 include a plurality of magnetic metal particles 22. Although not shown in FIG. 7, an inorganic oxide layer 242 is provided so as to cover the plurality of magnetic metal particles 22. Although not shown in FIG. 7, an underlayer 244, a gold layer 246, and an immobilization layer 248 are also laminated on the inorganic oxide layer 242. In this manner, the coating film 24 shown in FIG. 7 is configured. Such magnetic beads 2A can also achieve the same effect as the magnetic beads 2 shown in FIG. 5. Note that the inorganic oxide layer 242 may cover each magnetic metal particle 22, while the underlayer 244, the gold layer 246, and the immobilization layer 248 may be provided across the plurality of magnetic metal particles 22. Note that the underlayer 244 may also cover each magnetic metal particle 22, while the gold layer 246 and the immobilization layer 248 may be provided across the plurality of magnetic metal particles 22.

[0081] The number of magnetic metal particles 22 that the magnetic beads 2A have is not particularly limited, but is set to be 2 or more and 100 or less.

[0082] The average thickness of the inorganic oxide layer 242 is preferably 10 nm or more and 200 nm or less, more preferably 20 nm or more and 150 nm or less, and even more preferably 30 nm or more and 100 nm or less. This prevents the inorganic oxide layer 242 from being destroyed or peeled off even when the magnetic beads 2 collide with each other or with the inner wall of the container. As a result, it is possible to prevent the elution of iron ions and the like that occurs when the magnetic metal particles 22 are exposed. Furthermore, it is possible to prevent a decrease in the magnetization per volume of the magnetic beads 2 and a decrease in the movement speed of the magnetic beads 2.

[0083] The average thickness of the inorganic oxide layer 242 is measured in the same manner as the method for measuring the average thickness of the coating film 24 described above.

[0084] Examples of methods for forming the inorganic oxide layer 242 include wet methods such as the sol-gel method and dry methods such as vapor phase deposition. Among these, the Stöber method, which is a type of sol-gel method, and the ALD (Atomic Layer Deposition) method can be preferably used. The Stöber method is a method for forming monodispersed particles by hydrolysis of metal alkoxide. For example, when the inorganic oxide layer 242 is formed of silicon oxide, silicon oxide can be produced by a hydrolysis reaction of silicon alkoxide. Note that before forming the inorganic oxide layer 242, the underlying surface, for example, the surfaces of the magnetic metal particles 22, may be cleaned using water or an organic solvent.

[0085] 2.2.2. Base layer As shown in FIG. 6 , the base layer 244 is a layer that can serve as a base for the gold layer 246. The base for the gold layer 246 refers to a surface on which gold can be favorably deposited when the gold layer 246 is formed by electroless gold plating. Specifically, the base layer 244 contains a reducing agent that reduces gold ions to gold, or a catalyst for a reduction reaction that reduces gold ions to gold. Such a base layer 244 improves the adhesion of the gold layer 246. As a result, the adhesion of the immobilization layer 248 formed on the gold layer 246 can be improved.

[0086] The reducing agent contained in the base layer 244 dissolves in the electroless gold plating solution, ionizes, and releases electrons, thereby reducing the gold ions to gold. Therefore, the base layer 244 containing the reducing agent enables the formation of the gold layer 246 by displacement plating. Examples of the reducing agent include elemental metals such as nickel, palladium, and copper, which have a higher ionization tendency than gold, and alloys containing these metals.

[0087] The catalyst contained in the underlayer 244 has catalytic activity that catalyzes the reduction reaction of gold ions when a reducing agent contained in the electroless gold plating solution causes the reduction reaction. Therefore, the catalyst-containing underlayer 244 enables the formation of the gold layer 246 by autocatalytic or underlayer catalytic reduction plating. Examples of catalysts include elemental metals such as nickel, palladium, and copper, as well as alloys containing these metals. The content of these metals in the underlayer 244 is preferably greater than 50% by mass, and more preferably 70% by mass or greater. Examples of reducing agents contained in the electroless gold plating solution include sulfites, thiosulfates, hydroxylamine and its salts or derivatives, hydrazine, amine borane compounds such as dimethylamine borane, borohydride compounds such as sodium borohydride, sugars such as glucose, and hypophosphites.

[0088] As described above, the base layer 244 contains the metal element M, which acts as a reducing agent or catalyst used in the reduction of gold ions. This allows a gold plating film to be formed on the base layer 244 efficiently and uniformly.

[0089] Examples of such metal element M include nickel, palladium, copper, etc., and one or more of these are used in combination. These can function stably as reducing agents or catalysts. Of these, the metal element M is preferably nickel. Nickel is a particularly stable reducing agent or catalyst, contributing to the efficient deposition of gold. Therefore, if the base layer 244 contains nickel or a nickel alloy, the gold layer 246 can be successfully formed by an electroless gold plating method such as a displacement plating method or an autocatalytic or base catalyst reduction plating method.

[0090] These reducing agents and catalysts may be present in any form. For example, the underlayer 244 may be a plated film, a film formed by a vapor phase deposition method, or a film formed by a liquid phase deposition method. Examples of plated films include electroless plating films. Before forming the underlayer 244, the surface of the underlayer, for example, the inorganic oxide layer 242, may be subjected to surface treatments such as degreasing and acid immersion, or cleaning using water or an organic solvent.

[0091] The average thickness of the underlayer 244 is not particularly limited, but is preferably 10 nm to 300 nm, more preferably 30 nm to 250 nm, and even more preferably 50 nm to 200 nm, which ensures that the underlayer 244 has a sufficient thickness to function as a base.

[0092] The average thickness of the underlayer 244 is measured in the same manner as the method for measuring the average thickness of the coating film 24 described above.

[0093] The average thickness of the base layer 244 may be thinner than the average thickness of the inorganic oxide layer 242, but is preferably set to be thicker. The average thickness of the base layer 244 is more preferably set to be 1.2 to 5.0 times, and even more preferably 1.5 to 4.0 times, the average thickness of the inorganic oxide layer 242. This enhances the base function of the base layer 244, which is to stably form the gold layer 246, and improves the corrosion resistance of the magnetic beads 2. As a result, the immobilization layer 248 can be stabilized, and oxidation and corrosion of the magnetic metal particles 22 can be suppressed, resulting in magnetic beads 2 with particularly good nucleic acid extraction efficiency.

[0094] The base layer 244 may also have a multi-layer structure. For example, the first layer on the inorganic oxide layer 242 side may be a nickel-containing layer, and the second layer may be a palladium-containing layer. Such a multi-layer structure can more reliably suppress oxidation and corrosion of the base layer 244. As a result, the corrosion resistance of the magnetic beads 2 can be further improved.

[0095] 2.2.3. Gold layer As shown in FIG. 6, the gold layer 246 is a layer that can serve as a base for the immobilization layer 248. The base for the immobilization layer 248 refers to a surface that can effectively bind the compounds contained in the immobilization layer 248. Specifically, the gold layer 246 can be made of simple gold or a gold-containing alloy. Because the gold layer 246 is highly corrosion-resistant, it effectively suppresses oxidation and corrosion of the magnetic metal particles 22. This suppresses the decrease in magnetization of the magnetic metal particles 22 and the decrease in nucleic acid extraction efficiency associated with the elution of iron ions, etc. Furthermore, when the magnetic beads 2 are in solution, the surface of the gold layer 246 is often negatively charged. This causes electrostatic repulsion between the magnetic beads 2, suppressing aggregation of the magnetic beads 2.

[0096] The gold content of the magnetic beads 2 is preferably 1% by mass or more and 30% by mass or less, more preferably 3% by mass or more and 25% by mass or less, and even more preferably 5% by mass or more and 20% by mass or less, which ensures a sufficient coverage rate of the gold layer 246 and allows the immobilization layer 248 to be formed evenly.

[0097] The average thickness of the gold layer 246 is preferably 0.5 nm to 50 nm, more preferably 1 nm to 30 nm, and even more preferably 2 nm to 20 nm, which ensures particularly good corrosion resistance of the magnetic beads 2.

[0098] Furthermore, the total average thickness of the inorganic oxide layer 242, the underlayer 244, and the gold layer 246 is preferably 10 nm or more and 400 nm or less, more preferably 50 nm or more and 350 nm or less, and even more preferably 100 nm or more and 300 nm or less. This ensures sufficient corrosion resistance of the magnetic beads 2. Furthermore, the volume ratio of the coating film 24 to the total volume of the magnetic beads 2 can be optimized, and a decrease in the magnetization per volume of the magnetic beads 2 can be suppressed.

[0099] As described above, methods for forming the gold layer 246 include displacement plating and electroless gold plating methods such as autocatalytic or base catalyst reduction plating. Before forming the gold layer 246, the surface of the base, for example, the base layer 244, may be subjected to surface treatments such as degreasing and acid immersion, or cleaning using water or an organic solvent.

[0100] In addition, the mass ratio of the content of the metal element M to the content of gold in the magnetic beads 2 is M / Au. In the magnetic beads 2, the mass ratio M / Au is 0.03 or more and 19.0 or less, preferably 0.10 or more and 7.00 or less, and more preferably 1.00 or more and 3.00 or less.

[0101] This configuration optimizes the quantitative balance between gold and the metal element M. This optimizes the balance of the thicknesses of the gold layer 246 and the base layer 244 containing the metal element M, thereby improving their mutual adhesion. Furthermore, since the coverage of the gold layer 246 is ensured, the coverage of the immobilization layer 248 supported by the gold layer 246 is also improved. Therefore, interlayer adhesion and coverage can be ensured without increasing the thickness of the coating film 24. This prevents a decrease in the magnetization per volume of the magnetic beads 2 while ensuring the functionality of each layer of the magnetic beads 2. As a result, it is possible to prevent elution of iron ions and other substances derived from the magnetic metal particles 22, peeling or damage to the coating film 24, and aggregation of the magnetic beads 2. This allows for the realization of magnetic beads 2 that exhibit good nucleic acid extraction efficiency and are able to suppress the inclusion of contaminants. Furthermore, when testing the extracted nucleic acid, it is possible to prevent a decrease in test accuracy due to elution of iron ions and other substances.

[0102] Furthermore, when a strong external magnetic field is applied to the magnetic beads 2, it is possible to reduce the time required for magnetic separation while preventing peeling or damage to the coating film 24. It is also possible to increase the attractive force against the external magnetic field. This reduces the time required for magnetic separation and prevents the magnetic beads 2 themselves from becoming contaminants in the eluate.

[0103] If the mass ratio M / Au is below the lower limit, the content of the metal element M relative to the content of gold decreases. This reduces the film thickness and coverage of the base layer 244, which serves as the base for the gold layer 246. As a result, the coverage of the gold layer 246 and the coverage of the immobilization layer 248 decrease. In this case, the amount of nucleic acid that can be extracted decreases. On the other hand, if the mass ratio M / Au exceeds the upper limit, the content of gold relative to the content of the metal element M decreases. As a result, the coverage of the gold layer 246 decreases, reducing the amount of immobilized ligand, thereby reducing the amount of nucleic acid that can be extracted. Furthermore, electrostatic repulsion by the gold layer 246 is suppressed, reducing the dispersibility of the magnetic beads 2. In this case, the accuracy of nucleic acid extraction decreases due to impurities captured in the secondary particles. Furthermore, the corrosion resistance of the gold layer 246 decreases, leading to a decrease in the accuracy of nucleic acid testing due to the elution of gold ions and the like.

[0104] The gold content and the metal element M content in the magnetic beads 2 can be determined by ICP optical emission spectrometry as specified in JIS G 1258:2014, spark optical emission spectrometry as specified in JIS G 1253:2002, or the like, respectively.

[0105] In addition, in the magnetic beads 2, the mass ratio of the content of inorganic oxide Z to the content of metal element M is defined as Z / M. In the magnetic beads 2, the mass ratio Z / M is preferably 0.030 or more and 0.300 or less, and more preferably 0.050 or more and 0.200 or less.

[0106] This configuration makes it possible to optimize the quantitative balance between the metal element M and the inorganic oxide Z. This allows for an optimized balance in thickness between the underlayer 244 containing the metal element M and the inorganic oxide layer 242 containing the inorganic oxide Z, thereby improving adhesion between them. Furthermore, the coverage of the inorganic oxide layer 242 can be ensured, improving adhesion between the magnetic metal particles 22 and the underlayer 244. As a result, it is possible to suppress elution of iron ions and the like derived from the magnetic metal particles 22, peeling or damage to the coating film 24, aggregation of the magnetic beads 2, and the like.

[0107] If the mass ratio Z / M is below the lower limit, the content of the inorganic oxide Z will be low relative to the content of the metal element M. This may result in a decrease in adhesion between the base layer 244 and the inorganic oxide layer 242. Furthermore, the coverage of the inorganic oxide layer 242 may be reduced. On the other hand, if the mass ratio Z / M is above the upper limit, the content of the metal element M will be low relative to the content of the inorganic oxide Z. This may result in a decrease in adhesion between the base layer 244 and the inorganic oxide layer 242. Furthermore, the coverage of the base layer 244 may be reduced.

[0108] The content of inorganic oxide Z in the magnetic beads 2 can be determined by EDX analysis, Auger electron spectroscopy, etc. of the inorganic oxide layer 242. In this case, the content of inorganic oxide Z is calculated as the total content of silicon oxide (SiO2) and titanium oxide (TiO2). Specifically, it is assumed that all of the Si and Ti contained in the magnetic beads 2 are present as dioxide, and the total content of silicon oxide (SiO2) and titanium oxide (TiO2) is calculated from the respective contents of Si and Ti. The calculated result is then used as the content of inorganic oxide Z.

[0109] 2.2.4. Fixed layer The immobilization layer 248 contains a compound having a ligand or a ligand-reactive group. A ligand is a moiety that has the function of specifically binding to a target biological substance. A ligand-reactive group is a moiety that can bind to such a ligand. By having such an immobilization layer 248, the magnetic beads 2 can efficiently adsorb the target biological substance.

[0110] Specific examples of the ligand include multidentate ligands such as nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), phenanthroline, terpyridine, bipyridine, triethylenetetraamine, tris(carboxymethyl)ethylenediamine, diethylenetriaminepentaacetic acid, polypyrazolylboronic acid, 1,4,7-triazocyclononane, dimethylglyoxime, diphenylglyoxime, and derivatives thereof.

[0111] Of these, nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) is preferably used as the ligand, and these ligands are particularly useful.

[0112] Specific examples of the ligand-reactive group include, for example, an aldehyde group, a carboxy group, an amide group, N-hydroxysuccinimide (NHS), an amino group, a hydroxy group, -CH=CH-, -(C=O)-CH=CH-, etc. These functional groups are particularly useful as ligand-reactive groups.

[0113] Of these, the ligand-reactive group is preferably an aldehyde group, a carboxy group, an amide group, or N-hydroxysuccinimide (NHS).

[0114] These ligands or ligand-reactive groups are bonded to the gold layer 246 via an optional spacer and an Au-S bond. The strong Au-S bond contributes to the strong fixation of the ligands and ligand-reactive groups to the gold layer 246. This prevents the ligands from detaching even when various physical and chemical loads are applied to the magnetic beads 2 with nucleic acid adsorbed thereon. As a result, a decrease in nucleic acid capture efficiency can be prevented, resulting in magnetic beads 2 with good nucleic acid extraction efficiency.

[0115] Furthermore, the magnetic beads 2 are provided with an underlayer 244 as a base for the gold layer 246. The underlayer 244 improves the adhesion of the gold layer 246. This prevents the gold layer 246 from peeling off, which also helps to prevent ligand desorption. Furthermore, the magnetic beads 2 are provided with an inorganic oxide layer 242 as a base for the underlayer 244. The inorganic oxide layer 242 improves the adhesion of the underlayer 244. This prevents the underlayer 244 from peeling off, which also helps to prevent ligand desorption. Therefore, the coating film 24 having the above-described multilayer structure prevents ligand desorption, enabling magnetic beads 2 to be realized with good nucleic acid extraction efficiency.

[0116] A compound having a ligand or a ligand-reactive group is a compound having a thiol group or a disulfide bond capable of forming an Au-S bond, and is a reaction product of the reaction between a thiol derivative or its salt, as described below, and the gold layer 246.

[0117] 8 is a schematic diagram showing a state in which a compound 10 having a functional moiety X is bonded to a gold layer 246 via an Au—S bond. In FIG. 8, the ligand or the ligand-reactive group is referred to as the “functional moiety X.”

[0118] Compound 10 shown in FIG. 8 is composed of an Au—S bond, a spacer L, and a functional moiety X.

[0119] The spacer L is a moiety (linker) that connects the Au-S bond moiety and the functional moiety X. Examples of unit structures that constitute the spacer L include PEG (polyethylene glycol), PPG (polypropylene glycol), PBG (polybutylene glycol), a composite structure of a carbonate bond and PEG, a composite structure of an amide bond and PEG, methylene, and the like, and also includes a structure in which one or more of these are mixed.

[0120] Furthermore, as shown in FIG. 8, self-organization occurs between adjacent compounds 10 due to interactions between spacers L. This facilitates orientation of the compounds 10. As a result, an immobilization layer 248 in which functional sites X are densely arranged can be realized. In such an immobilization layer 248, the amount of biomaterial adsorbed per unit amount of magnetic beads 2 can be increased. Note that the compounds 10 may have a branched molecular chain structure.

[0121] The following formula (1) is an example of the structure of a thiol derivative that produces compound 10. R 1 -(CH2) x -(C2H4O) y -R 2 … (1) [In the above formula (1), x is an integer of 2 or more and 18 or less, and y is an integer of 0 or more and 100 or less. In addition, in the above formula (1), R 1 is a thiol group or a disulfide bond, and R 2 is a ligand or a ligand-reactive group.

[0122] The thiol derivative having the structure represented by the above formula (1) or its salt reacts with the gold layer 246 to produce a compound 10 capable of arranging ligands or ligand-reactive groups at a high density, thereby obtaining magnetic beads 2 with particularly good nucleic acid extraction efficiency.

[0123] The structure represented by the formula (1) above is the whole or a part of the molecular structure of the thiol derivative, and therefore the molecular structure of the thiol derivative may contain the structure represented by the formula (1) above and another structure.

[0124] Examples of salts of thiol derivatives include alkali metal salts such as lithium salts, sodium salts, and potassium salts, and alkaline earth metal salts such as magnesium salts and calcium salts.

[0125] A thiol group is a bonding functional group represented by -SH. A disulfide bond is a bond represented by -SS-, and is converted into a thiol group by reduction. Therefore, R in the above formula (1) 1 is a disulfide bond, the thiol derivative can be obtained by the above formula (1) 1 The thiol group reacts with gold to form a strong Au-S bond. The product of this reaction is compound 10.

[0126] Also, -(CH2) x -(C2H4O) y The - corresponds to the spacer L described above. The thiol derivative represented by the above formula (1) may contain methylene or PEG as the spacer L. These contribute to the self-assembly of compound 10 when bound to the gold layer 246. This allows compound 10 to be oriented, enabling the high-density arrangement of functional moieties X.

[0127] In the above formula (1), x, which represents the number of methylene groups, is 2 to 18, preferably 3 to 10, and more preferably 4 to 6. This stabilizes compound 10 and suppresses deterioration of the immobilization layer 248 associated with magnetic separation and liquid discharge. If x is below the lower limit, the length of spacer L becomes shorter, which may reduce the functionality of functional moiety X, i.e., the ligand or ligand-reactive group may become less functional. Furthermore, a shorter length of spacer L may reduce the self-assembly of compound 10 and reduce the amount of biomaterial adsorption. On the other hand, if x exceeds the upper limit, the molecular chain of compound 10 may become too long, which may make it difficult for compound 10 to orient, thereby preventing the density of functional moiety X from being sufficiently increased.

[0128] In the above formula (1), y, which represents the number of PEGs, is 0 to 100, preferably 1 to 30, and more preferably 2 to 10. This stabilizes compound 10. If y is below the lower limit, the length of spacer L becomes shorter, which may reduce the functionality of functional moiety X. Furthermore, a shorter length of spacer L may reduce the self-assembly effect of compound 10. On the other hand, if y is above the upper limit, the molecular chain of compound 10 may become too long, which may make it difficult for compound 10 to orient, and may prevent the density of functional moiety X from being sufficiently increased.

[0129] Specific examples of the thiol derivative having a ligand include compounds represented by the following formulas (A-1) to (A-7).

[0130] [ka]

[0131] [ka]

[0132] [ka]

[0133] [ka]

[0134] [ka]

[0135] [ka]

[0136] [ka]

[0137] Specific examples of the thiol derivative having a ligand-reactive group include compounds represented by the following formulas (B-1) to (B-4).

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] Such thiol derivatives are synthesized, for example, by using a linker compound in which various functional groups are attached to a PEG chain or an alkyl chain, and linking them via the PEG chain or the alkyl chain.

[0143] 3. Effects of the above embodiment As described above, the magnetic beads 2 serving as magnetic beads for extracting biological substances according to the embodiment include magnetic metal particles 22, an inorganic oxide layer 242, a base layer 244, a gold layer 246, and an immobilization layer 248. The inorganic oxide layer 242 covers the surfaces of the magnetic metal particles 22 and contains an inorganic oxide Z. The base layer 244 is provided on the side of the inorganic oxide layer 242 opposite the magnetic metal particles 22 and contains a metal element M that constitutes a reducing agent for gold ions or a catalyst for a reduction reaction of gold ions. The gold layer 246 covers the surface of the base layer 244 and contains gold. The immobilization layer 248 contains a ligand or a ligand-reactive group bonded to the surface of the gold layer 246 via an Au-S bond. The mass ratio M / Au of the content of the metal element M to the content of gold is 0.03 or more and 19.0 or less.

[0144] This configuration provides magnetic beads 2 that can suppress ligand detachment even when a load is applied, and can also suppress oxidation and corrosion of the magnetic metal particles 22. This provides magnetic beads 2 that have good extraction efficiency for biological substances and are less likely to reduce the accuracy of testing the extracted biological substances.

[0145] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the immobilization layer 248 contains a reaction product of the gold layer 246 and a thiol derivative represented by the following formula (1) or a salt thereof.

[0146] R 1 -(CH2) x -(C2H4O) y -R 2 … (1) [In the above formula (1), x is an integer of 2 or more and 18 or less, and y is an integer of 0 or more and 100 or less. In addition, in the above formula (1), R 1 is a thiol group or a disulfide bond, and R 2is a ligand or a ligand-reactive group.

[0147] The thiol derivative having the structure represented by the above formula (1) or its salt can produce compound 10, on which ligands or ligand-reactive groups can be densely arranged, by reacting with gold layer 246. This allows for the production of magnetic beads 2 that have particularly good extraction efficiency for biological materials.

[0148] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, R 2 is a ligand, nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA), or a ligand reactive group, an aldehyde group, a carboxy group, an amide group, or N-hydroxysuccinimide (NHS). These are particularly useful as ligands or ligand-reactive groups.

[0149] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the inorganic oxide Z is silicon oxide or titanium oxide.

[0150] These are chemically stable, and therefore can particularly inhibit oxidation and corrosion of the magnetic metal particles 22, thereby particularly enhancing the corrosion resistance of the magnetic beads 2.

[0151] Furthermore, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the mass ratio Z / M of the content of the inorganic oxide Z to the content of the metal element M is 0.030 or more and 0.300 or less.

[0152] With this configuration, the balance of the thicknesses of the underlayer 244 containing the metal element M and the inorganic oxide layer 242 containing the inorganic oxide Z can be optimized, thereby improving adhesion between them. In addition, the coverage of the inorganic oxide layer 242 can be ensured, improving adhesion between the magnetic metal particles 22 and the underlayer 244.

[0153] In addition, in the magnetic beads 2 serving as the magnetic beads for extracting biological substances according to the embodiment, the metal element M is nickel.

[0154] In this configuration, nickel serves as a particularly stable reducing agent or catalyst, contributing to the efficient deposition of gold. Therefore, since the base layer 244 contains nickel, the gold layer 246 can be successfully formed by an electroless gold plating method such as displacement plating or autocatalytic or base catalytic reduction plating.

[0155] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the total average thickness of the inorganic oxide layer 242, the base layer 244 and the gold layer 246 is 10 nm or more and 400 nm or less.

[0156] This configuration can sufficiently ensure the corrosion resistance of the magnetic beads 2. In addition, the volume ratio of the coating film 24 to the total volume of the magnetic beads 2 can be optimized, and the magnetization per volume of the magnetic beads 2 can be prevented from decreasing.

[0157] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the average thickness of the base layer 244 is 1.2 times or more and 5.0 times or less the average thickness of the inorganic oxide layer 242 .

[0158] This configuration enhances the function of the base layer 244 as a base for stably depositing the gold layer 246, and also improves the corrosion resistance of the magnetic beads 2. As a result, the immobilization layer 248 can be stabilized, and oxidation and corrosion of the magnetic metal particles 22 can be suppressed, resulting in magnetic beads 2 with particularly good extraction efficiency for biological substances.

[0159] Moreover, the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment have an average particle size of 0.5 μm or more and 30 μm or less.

[0160] This configuration allows the specific surface area of ​​the magnetic beads 2 to be sufficiently large, and allows the magnetic beads 2 to generate attractive and adsorbing forces suitable for magnetic separation. In addition, aggregation of the magnetic beads 2 can be suppressed, and dispersibility can be improved.

[0161] In addition, in the magnetic beads 2 as the magnetic beads for extracting biological substances according to the embodiment, the saturation magnetization of the magnetic metal particles 22 is 50 emu / g or more, and the coercive force of the magnetic metal particles 22 is 100 A / m or less.

[0162] This configuration can improve the movement speed of the magnetic beads 2 in the magnetic field, thereby shortening the time required for magnetic separation. Furthermore, because a sufficiently high adsorptive force can be obtained, when the liquid 3 is discharged with the magnetic beads 2 fixed, the magnetic beads 2 can be prevented from being discharged together with the liquid 3. This can prevent a decrease in the yield of the biological material due to a decrease in the magnetic beads 2. Furthermore, even when the application of the magnetic field is repeatedly switched, aggregation of the magnetic beads 2 can be prevented.

[0163] While the magnetic beads for biological substance extraction of the present invention have been described above based on the illustrated embodiments, the present invention is not limited thereto. For example, the magnetic beads for biological substance extraction of the present invention may be those having optional components added to the above-described embodiments. Specifically, optional layers may be added between the layers of the above-described embodiments. [Example]

[0164] Next, specific examples of the present invention will be described. 4. Preparation of Magnetic Beads Example 1 First, a metal powder having an amorphous structure as the main structure, manufactured by water atomization, was prepared as the magnetic metal particles, and the prepared metal powder was washed with ultrapure water and ethanol.

[0165] Next, a silicon oxide (SiO2) film was formed on the surface of the magnetic metal particles using the Stöber method to obtain an inorganic oxide layer. Tetraethoxysilane (TEOS) was used as the silicon alkoxide. The magnetic metal particles with the inorganic oxide layer were then degreased and immersed in acid.

[0166] Next, a nickel film was formed on the surface of the inorganic oxide layer by electroless nickel plating to obtain an underlayer. The magnetic metal particles on which the underlayer was formed were subjected to a degreasing treatment.

[0167] Next, a gold film was formed on the surface of the underlayer by displacement electroless gold plating to obtain a gold layer. The magnetic metal particles on which the gold layer was formed were washed with ultrapure water and ethanol.

[0168] Next, a thiol derivative having a structure represented by the symbol in Table 1 was dissolved in ethanol to prepare an ethanol solution. Subsequently, the magnetic metal particles on which the gold layer was formed were immersed in the ethanol solution and subjected to a stirring treatment. This caused the thiol derivative to react with the gold layer, resulting in an immobilized layer.

[0169] The configuration of the magnetic beads (average particle size of the magnetic beads, saturation magnetization and coercive force of the magnetic metal particles, and conditions for forming the coating film, etc.) is as shown in Table 1. The symbols representing the thiol derivatives shown in Table 1 correspond to the above-mentioned formula (A-1).

[0170] 4.2. Examples 2 to 8 and Comparative Examples 1 and 2 Magnetic beads were obtained in the same manner as in Example 1, except that the composition of the magnetic beads was as shown in Table 1.

[0171] 4.3. Examples 9 to 15 and Comparative Examples 3 and 4 Magnetic beads were obtained in the same manner as in Example 1, except that the composition of the magnetic beads was as shown in Table 2.

[0172] 4.4. Examples 16 to 19 Magnetic beads were obtained in the same manner as in Example 1, except that the composition of the magnetic beads was as shown in Table 3.

[0173] 4.5. Comparative Example 5 Magnetic beads were obtained in the same manner as in Example 1, except that the inorganic oxide layer was omitted and the magnetic beads had a structure as shown in Table 3.

[0174] 4.6. Comparative Example 6 Magnetic beads were obtained in the same manner as in Example 1, except that the underlayer was omitted and the magnetic beads had the structure shown in Table 3.

[0175] 4.7. Comparative Example 7 Except for using gold colloid particles instead of magnetic metal particles, magnetic beads were obtained in the same manner as in Example 1. Note that the gold colloid particles are particles composed only of gold and do not include an inorganic oxide layer or an underlayer.

[0176] 4.8. Comparative Example 8 Magnetic beads were obtained in the same manner as in Example 1, except that ferrite-containing particles were used instead of magnetic metal particles and the composition of the magnetic beads was as shown in Table 3. The ferrite-containing particles are particles in which ferrite particles are dispersed in silica.

[0177] 4.9. Comparative Examples 9 and 10 Magnetic beads were obtained in the same manner as in Example 1, except that a silane coupling agent was used instead of the thiol derivative and the composition of the magnetic beads was as shown in Table 3. The symbols representing the silane coupling agents shown in Table 3 correspond to the silane coupling agents represented by the following formulas (C-1) and (D-1).

[0178] [ka]

[0179] [ka]

[0180] 4.6. Examples 20 to 29 Magnetic beads were obtained in the same manner as in Example 1, except that the composition of the magnetic beads was as shown in Table 4. The symbols representing the thiol derivatives shown in Table 4 correspond to the thiol derivatives represented by the above-mentioned formulas (A-2) to (A-7) and formulas (B-1) to (B-4).

[0181] 5. Evaluation of Magnetic Beads 5.1. Purification yield of biological material First, Protein A, the target biological substance, was dispersed in pure water to prepare a sample solution in a microtube. The concentration of Protein A was 50 μg / mL.

[0182] Next, the magnetic beads of each Example and Comparative Example were dispersed in the sample aqueous solution to a concentration of 30% by mass.

[0183] Next, a magnet was placed close to the side of the microtube, a magnetic field was applied, and the supernatant was removed. The magnetic beads remaining in the microtube were then collected. This allowed the target biological material to be adsorbed onto the magnetic beads. The collected magnetic beads were then dispersed in a PBS buffer solution to prepare a dispersion. The PBS buffer solution was a phosphate buffer solution containing 137 mmol / L NaCl, 8.1 mmol / L Na2HPO4, 2.7 mmol / L KCl, and 1.5 mmol / L KH2PO4.

[0184] The prepared dispersion was then stirred by end-over-end stirring for 30 minutes, after which the supernatant was recovered and the BCA assay test solution was added. The absorbance of the dispersion to which the test solution had been added was then measured using a UV-vis spectrophotometer (U-3900H spectrophotometer). The abs.-562 nm value of the sample aqueous solution alone was used as the reference value, and the ratio of the abs.-562 nm value of the supernatant obtained using the magnetic beads of each Example and Comparative Example was calculated. The closer this ratio was to 1.0, the greater the amount of biological material purified by the magnetic beads. The calculated ratios were then evaluated according to the following evaluation criteria. The evaluation results are shown in Tables 1 to 4.

[0185] A: The ratio of the supernatant abs.-562nm value to the reference value is 0.8 or more. B: The ratio of the supernatant abs.-562nm value to the reference value is 0.7 or more and less than 0.8 C: The ratio of the supernatant abs.-562nm value to the reference value is 0.6 or more and less than 0.7 D: The ratio of the supernatant abs.-562nm value to the reference value is 0.3 or more and less than 0.6 E: The ratio of the supernatant abs.-562nm value to the reference value is less than 0.3

[0186] 5.2. Redispersibility First, the magnetic beads of each Example and Comparative Example were dispersed in a PBS buffer solution to a concentration of 30% by mass to prepare a dispersion. The PBS buffer solution was the same as that used in 5.1.

[0187] Next, the prepared dispersion was placed in a laser diffraction / scattering particle size distribution analyzer, and the volumetric average particle size D50 was measured. The obtained average particle size D50 was taken as the value before the separation of the target biological material, and this was used as the reference value.

[0188] Next, magnetic beads were dispersed in the same manner as in 5.1. in the sample aqueous solution prepared in the same manner as in 5.1., and magnetic separation was performed to recover the magnetic beads that had adsorbed the biological material. The recovered magnetic beads were then dispersed in a PBS buffer solution in the same manner as in 5.1. to prepare a dispersion. The volume-based average particle size D50 was then measured in the same manner as above, and this measurement value was used as the value after the separation of the target biological material.

[0189] Next, the ratio of the value after separation to the reference value was calculated. The closer this ratio is to 1.0, the better the redispersibility of the magnetic beads is. The calculated ratios were then evaluated according to the following evaluation criteria. The evaluation results are shown in Tables 1 to 4.

[0190] A: The ratio of the value after separation to the standard value is less than 1.1. B: The ratio of the value after separation work to the standard value is 1.1 or more and less than 1.3 C: The ratio of the value after separation to the reference value is 1.3 or more and less than 1.5 D: The ratio of the value after separation work to the reference value is 1.5 or more and less than 2.0 E: The ratio of the value after separation to the reference value is 2.0 or more.

[0191] 5.3.Corrosion Resistance First, the magnetic beads of each Example and Comparative Example were dispersed in a PBS buffer solution to a concentration of 30% by mass to prepare a dispersion. The PBS buffer solution was the same as that used in 5.1.

[0192] Next, the prepared dispersion was left to stand for 10 days under conditions of 1013 hPa pressure and 70°C temperature, and then the element concentration of the supernatant was measured using an ICP analyzer, 5100 ICP-OES, manufactured by Agilent Technologies.

[0193] The element concentration of the PBS buffer solution alone was used as the reference value, and the difference between this and the supernatant ion concentration was calculated. The smaller the difference between the supernatant ion concentration and the reference value, the less magnetic component was eluted. The evaluation results are shown in Tables 1 to 4.

[0194] A: The difference in ion concentration is less than 10 ppm B: The difference in ion concentration is 10 ppm or more but less than 20 ppm C: The difference in ion concentration is 20 ppm or more and less than 35 ppm D: The difference in ion concentration is 35 ppm or more but less than 100 ppm E: The difference in ion concentration is 100 ppm or more

[0195] 5.4.Magnetic Separation Speed First, a dispersion was prepared by dispersing the magnetic beads of each Example and Comparative Example in pure water at 25°C to a concentration of 0.1% by mass. Next, the dispersion was placed in a spectroscopic cell and stirred by ultrasonic irradiation or using a vortex mixer. The stirring time was 1 minute. Next, the stirred spectroscopic cell was quickly placed in the cell holder of the spectrophotometer. A magnet had been attached to the cell holder in advance, aligned with the position where the spectroscopic cell would be placed. The shortest distance between the magnet and the outer wall of the spectroscopic cell placed in the cell holder was 2.0 mm, and the magnet used had a surface magnetic flux density of 180 mT.

[0196] Next, the spectroscopic cell was allowed to stand, and at the same time, the absorbance of the spectroscopic cell at a wavelength of 550 nm was measured. The time until the measured absorbance decayed to 10% of the initial absorbance was measured, and this measurement result was used as an evaluation index for evaluating the magnetic separation speed. The obtained evaluation index was then evaluated in accordance with the following evaluation criteria. The evaluation results are shown in Tables 1 to 4.

[0197] A: The evaluation index for magnetic separation speed is less than 20 seconds. B: The evaluation index for magnetic separation speed is 20 seconds or more and less than 30 seconds C: The evaluation index for magnetic separation speed is 30 seconds or more and less than 45 seconds D: The evaluation index for magnetic separation speed is 45 seconds or more and less than 60 seconds E: The evaluation index for magnetic separation speed is 60 seconds or more.

[0198] [Table 1]

[0199] [Table 2]

[0200] [Table 3]

[0201] [Table 4]

[0202] From the evaluation results shown in Tables 1 to 4, the following can be seen. The magnetic beads of each example yielded a large amount of purified biological material and had good redispersibility even after magnetic separation. The magnetic beads of each example had good corrosion resistance even when immersed in an acid-containing PBS buffer solution for 10 days. It was also confirmed that the magnetic beads of each example had a sufficiently high magnetic separation speed.

[0203] From the above results, it can be seen that the present invention can provide magnetic beads for extracting biological substances that have good extraction efficiency for biological substances and that do not reduce the accuracy of testing the extracted biological substances. [Explanation of symbols]

[0204] 1...container, 2...magnetic beads, 2A...magnetic beads, 3...liquid, 5...magnet, 6...pipette, 10...compound, 22...magnetic metal particles, 24...coating film, 242...inorganic oxide layer, 244...underlayer, 246...gold layer, 248...immobilization layer, S102...dissolution / adsorption step, S108...washing step, S110...elution step, L...spacer, X...functional site

Claims

1. magnetic metal particles; an inorganic oxide layer that coats the surfaces of the magnetic metal particles and contains an inorganic oxide; a base layer provided on the opposite side of the inorganic oxide layer from the magnetic metal particles, the base layer containing a metal element that constitutes a reducing agent for gold ions or a catalyst for a reduction reaction of gold ions; a gold layer that covers a surface of the underlayer and contains gold; an immobilization layer comprising a ligand or a ligand-reactive group bonded to the gold layer via an Au—S bond; and Magnetic beads for extracting biological substances, characterized in that the mass ratio M / Au of the content of said metal element to the content of said gold is 0.03 or more and 19.0 or less.

2. 2. The magnetic beads for extracting biological substances according to claim 1, wherein the immobilization layer contains a reaction product of a thiol derivative represented by the following formula (1) or a salt thereof with the gold layer: R １ -(CH ２ ) ｘ -(C ２ H ４ O) ｙ -R ２ … (1) [In the above formula (1), x is an integer of 2 or more and 18 or less, and y is an integer of 0 or more and 100 or less. １ is a thiol group or a disulfide bond, and R ２ is the ligand or the ligand-reactive group.

3. R in the above formula (1) ２ is the ligand nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA), or the ligand reactive group is an aldehyde group, a carboxy group, an amide group, or N-hydroxysuccinimide (NHS).

4. 4. The magnetic beads for extracting biological substances according to claim 1, wherein the inorganic oxide is silicon oxide or titanium oxide.

5. 5. The magnetic beads for extracting biological substances according to claim 4, wherein the mass ratio Z / M of the content of said inorganic oxide to the content of said metal element is 0.030 or more and 0.300 or less.

6. 4. The magnetic beads for extracting biological substances according to claim 1, wherein the metal element is nickel.

7. 4. The magnetic beads for extracting biological substances according to claim 1, wherein the total average thickness of the inorganic oxide layer, the underlayer, and the gold layer is 10 nm or more and 400 nm or less.

8. 4. The magnetic beads for extracting biological substances according to claim 1, wherein the average thickness of the underlayer is 1.2 times or more and 5.0 times or less the average thickness of the inorganic oxide layer.

9. 4. The magnetic beads for extracting biological substances according to claim 1, wherein the average particle size is 0.5 μm or more and 30 μm or less.

10. the saturation magnetization of the magnetic metal particles is 50 emu / g or more; 4. The magnetic beads for extracting biological substances according to claim 1, wherein the coercive force of the magnetic metal particles is 100 A / m or less.

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