Magnetic bead dispersion kit and magnetic beads

Abstract

To provide a magnetic bead dispersion kit and a magnetic bead that can achieve both a reduction in the time required for magnetic separation and the amount of adsorption of a biological substance.SOLUTION: A magnetic bead dispersion kit comprises: a first dispersion that has first beads with an average particle diameter of 1 μm or more and 15 μm or less each including first magnetic metal powder and a first coating layer covering a particle surface of the first magnetic metal powder and having affinity with a biological substance, and a first dispersion medium to disperse the first beads; and a second dispersion that has second beads with an average particle diameter of larger than the first beads by 5 μm or more and of 100 μm or less each having second magnetic metal powder including an amorphous structure or a nano-crystalline structure and a second coating layer covering a particle surface of the second magnetic metal powder and having affinity with the biological substance, and a second dispersion medium to disperse the second beads.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a magnetic bead dispersion kit and magnetic beads.

Background Art

[0002] In recent years, in the medical field of diagnosis and the field of life sciences, the demand for the examination of biological substances has been increasing. Among the biological substance examination methods, the PCR (Polymerase chain reaction) method is a method of extracting nucleic acids such as DNA and RNA and specifically amplifying and detecting the nucleic acids. In the process of examining such biological substances, first, it is necessary to extract the substance to be examined from the specimen. For this extraction of biological substances, a magnetic separation method using magnetic beads is widely used. In the magnetic separation method, magnetic beads having a function of carrying the biological substance to be extracted are used, and the biological substance is extracted by applying a magnetic field. Specifically, after magnetic beads having the ability to carry the substance to be examined on the surface are dispersed in a dispersion medium, the obtained dispersion is attached to a magnetic field generating device such as a magnetic stand, and the application of the magnetic field is repeated ON / OFF a plurality of times. Thereby, the substance to be examined is extracted. Since such a magnetic separation method is a method of separating and collecting magnetic beads by magnetic force, a rapid separation operation is possible. Moreover, not only in the extraction in the PCR method, but also in the fields of protein purification, exosome, cell separation, extraction, etc., the same magnetic separation method is used.

[0003] For example, Patent Document 1 discloses rapid capture magnetic beads that are blended with magnetic beads and magnetic particles having a saturation magnetization higher than that of the magnetic beads and used for magnetic separation. In such a mixed powder in which particles having different saturation magnetizations are blended, when an external magnetic field is applied, even magnetic beads with a low saturation magnetization can obtain a capture speed close to that of magnetic particles having a high saturation magnetization. Therefore, the entire rapid capture magnetic beads, including magnetic beads with a low saturation magnetization, can be captured in a short time.

Prior Art Documents

Patent Documents

[0004] [Patent Document 1] Japanese Patent Publication No. 2010-214257 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Patent Document 1 states that it is preferable for the particle size of the magnetic particles to be less than or equal to the particle size of the magnetic beads. However, since the difference in particle size between the two is very small, 0.2 μm, the above-mentioned effect is limited. Furthermore, if the particle size of the magnetic beads or magnetic particles is increased, the saturation magnetization increases, while the specific surface area decreases, thus reducing the amount of biomolecules adsorbed. On the other hand, if the particle size of the magnetic beads or magnetic particles is decreased, the saturation magnetization decreases, while the specific surface area increases, thus increasing the amount of biomolecules adsorbed. Therefore, the challenge is to achieve both the separation speed in magnetic separation and the amount of biomolecules adsorbed. [Means for solving the problem]

[0006] A magnetic bead dispersion kit according to an application example of the present invention is A first dispersion comprising a first magnetic metal powder, a first coating layer that coats the particle surface of the first magnetic metal powder and has the ability to bind to biomaterials, first beads having an average particle size of 1 μm or more and 15 μm or less, and a first dispersion medium for dispersing the first beads, A second magnetic metal powder comprising an amorphous or nanocrystalline structure, and a second coating layer coating the particle surface of the second magnetic metal powder and having bonding properties with the biomaterial, wherein the average particle size is smaller than that of the first beads. 10 A second dispersion comprising a second bead having a size greater than or equal to 100 μm and a size less than or equal to 100 μm, and a second dispersion medium for dispersing the second bead, Equipped with 、 The first coating layer and the second coating layer each contain silicon oxide, or a composite oxide of silicon and one or more elements selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr. The aforementioned When the volume fraction of the second bead is set to 1, the first dispersion and the second dispersion are mixed such that the volume fraction of the first bead is between 5 and 30. It is characterized by the following:

[0007] The magnetic beads according to the application example of the present invention are A first magnetic metal powder comprises a first magnetic metal powder and a first coating layer that covers the particle surface of the first magnetic metal powder and has the ability to bind to biomaterials, wherein the first powder has an average particle size of 1 μm or more and 15 μm or less. A second magnetic metal powder comprising an amorphous or nanocrystalline structure, and a second coating layer that covers the particle surface of the second magnetic metal powder and has bonding properties with the biomaterial, wherein the average particle size is greater than that of the first powder. 10 A second powder having a size greater than or equal to 100 μm and less than or equal to 100 μm, Includes, The first coating layer and the second coating layer each contain silicon oxide, or a composite oxide of silicon and one or more elements selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr. When the volume fraction of the second powder is set to 1, The volume fraction of the first powder is It is between 5 and 30. It is characterized by the following: [Brief explanation of the drawing]

[0008] [Figure 1] This is a schematic diagram showing a magnetic bead dispersion kit according to an embodiment. [Figure 2] Figure 1 is a schematic diagram illustrating magnetic separation using the magnetic bead dispersion kit shown, illustrating how an external magnetic field acts on a mixed dispersion containing both first and second beads, causing the first and second beads to form aggregates and move. [Figure 3] This is a schematic diagram illustrating the prior art, showing an example in which only a first dispersion containing first beads and a first dispersion medium is used, and each first bead has one of two types of binding sites S1 or S2. [Figure 4] This is a schematic diagram illustrating the prior art, showing a state in which binding site S1 specifically binds to antigen A1 and binding site S2 specifically binds to antigen A2, and cells are adsorbed onto the first bead. [Figure 5] This is a schematic diagram illustrating magnetic separation using a magnetic bead dispersion kit according to an embodiment, in which each first bead has either a binding site S1 or S2, and the second bead has both binding sites S1 and S2. [Figure 6] It is a figure showing the particle size distribution of magnetic beads according to an embodiment, and an example of the result of fitting a lognormal distribution to this particle size distribution.

Mode for Carrying Out the Invention

[0009] Hereinafter, a preferred embodiment of the magnetic bead dispersion kit and magnetic beads of the present invention will be described in detail based on the accompanying drawings.

[0010] FIG. 1 is a schematic diagram showing a magnetic bead dispersion kit 3 according to an embodiment. FIG. 2 is a schematic diagram for explaining magnetic separation using the magnetic bead dispersion kit 3 shown in FIG. 1, in which an external magnetic field acts on a mixed dispersion liquid 300 containing both the first beads 1 and the second beads 2, and the first beads 1 and the second beads 2 form an aggregate 41 and move.

[0011] 1. Magnetic bead dispersion kit The magnetic bead dispersion kit 3 shown in FIG. 1 includes a first dispersion liquid 100 and a second dispersion liquid 200, and is a reagent kit for extracting biological substances.

[0012] Biological substances refer to substances such as nucleic acids such as DNA and RNA, proteins, saccharides, various cells such as cancer cells, peptides, bacteria, viruses, etc. The nucleic acid may be present in a biological sample such as a cell or a biological tissue, a virus, a bacterium, etc. The magnetic bead dispersion kit is used for magnetic separation between a solid phase containing magnetic beads and a liquid phase when extracting such biological substances through each step of dissolution / adsorption, separation, washing, and elution.

[0013] The first dispersion liquid 100 has the first beads 1 and the first dispersion medium 10. The first beads 1 include a first magnetic metal powder and a first coating layer covering the particle surface of the first magnetic metal powder, and have an average particle size of 1 μm or more and 15 μm or less. The first coating layer has binding properties with biological substances. [[ID=二十九]]

[0014] The second dispersion liquid 200 has second beads 2 and a second dispersion medium 20. The second beads 2 include a second magnetic metal powder containing an amorphous structure or a nanocrystalline structure, and a second coating layer covering the particle surface of the second magnetic metal powder, and have an average particle size that is 5 μm or more larger than that of the first beads 1 and 100 μm or less. The second coating layer has binding properties with a biological substance.

[0015] The first dispersion liquid 100 and the second dispersion liquid 200 are subjected to a step of extracting a biological substance by magnetic separation after being brought into contact with the biological substance in a mixed state. The first beads 1 contained in the first dispersion liquid 100 and the second beads 2 contained in the second dispersion liquid 200 each have binding properties with the biological substance, while having different average particle sizes from each other. The first beads 1 with a small average particle size contribute to increasing the adsorption amount of the biological substance by taking advantage of a large specific surface area. The second beads 2 with a large average particle size capture the surrounding first beads 1 by taking advantage of a large saturation magnetization and move together with the first beads 1, thus contributing to an improvement in the separation speed in magnetic separation. As a result, by using the magnetic bead dispersion liquid kit 3, it is possible to achieve both the separation speed in magnetic separation and the adsorption amount of the biological substance.

[0016] 1.1. The first dispersion liquid The first dispersion liquid 100 is put in a container or the like and constitutes a part of the magnetic bead dispersion liquid kit 3. As described above, the first dispersion liquid 100 has the first beads 1 and the first dispersion medium 10.

[0017] 1.1.1. The first beads The first beads 1 include a first magnetic metal powder having magnetism and a first coating layer covering the particle surface thereof and having binding properties with a biological substance. The first beads 1 in this specification refer to single particles or aggregates of particles.

[0018] The average particle size of the first bead 1 is set to be between 1 μm and 15 μm, preferably between 1 μm and 10 μm. If the average particle size of the first bead 1 is within the above range, the specific surface area of ​​the first bead 1 can be sufficiently large, and the saturation magnetization of the first bead 1 can be sufficiently high. Furthermore, aggregation of the first bead 1 can be suppressed, and dispersibility can be improved. If the average particle size of the first bead 1 falls below the lower limit, the magnetization value of the first bead 1 will decrease, and aggregation will become more likely, which may result in a decrease in the extraction efficiency of biomaterials. In addition, the migration speed of the first bead 1 may decrease, which may increase the time required for magnetic separation. On the other hand, if the average particle size of the first bead 1 exceeds the upper limit, the specific surface area of ​​the first bead 1 will decrease, so it may not be able to adsorb a sufficient amount of biomaterials, which may result in a decrease in the amount of biomaterials extracted.

[0019] The average particle size of the first bead 1 can be determined by measuring the volume-based particle size distribution using laser diffraction-dispersion and then obtaining the cumulative distribution curve from this distribution. Specifically, in the cumulative distribution curve, the particle size D50 (median diameter), where the cumulative value from the smallest diameter side reaches 50%, is the average particle size of the first bead 1. Examples of devices for measuring particle size distribution using laser diffraction-dispersion include the MT3300 series manufactured by Microtrac-Bell. Note that methods other than laser diffraction-dispersion, such as image analysis, may also be used.

[0020] Furthermore, when the average thickness of the first coating layer is t and the average particle size of the first beads 1 is D50, the ratio of t to D50, t / D50, is preferably 0.0001 or more and 0.05 or less, and more preferably 0.001 or more and 0.01 or less. If t / D50 falls below the lower limit, the ratio of the thickness of the first coating layer to the size of the first magnetic metal powder becomes too small, which may cause the first coating layer to break or peel off when the first beads 1 collide with each other or with the inner wall of the container, etc. As a result, the amount of biomolecules that are normally adsorbed and extracted from the surface of the first coating layer decreases, which may reduce the extraction efficiency. In addition, fragments of the peeled first coating layer and first magnetic metal powder will be present in the dispersion, which may be introduced as contaminants when the biomolecules are extracted. Furthermore, if the first coating layer is damaged or peeled off, the first magnetic metal powder may be exposed, and if it comes into contact with an acidic solution, for example, iron ions and other substances may be leached out, which may result in a decrease in the extraction efficiency of biological substances. On the other hand, if t / D50 exceeds the upper limit, the volume ratio of the first coating layer to the total volume of the first bead 1 will increase, which may reduce the magnetization per unit volume of the first bead 1. As a result, the movement speed when an external magnetic field acts on the first bead 1 during magnetic separation will decrease, which may increase the time required for magnetic separation.

[0021] The saturation magnetization of the first bead 1 is preferably 50 emu / g or higher, and more preferably 100 emu / g or higher. Saturation magnetization is the magnetization value exhibited by a magnetic material when a sufficiently large magnetic field is applied from the outside. The higher the saturation magnetization of the first bead 1, the more fully it can function as a magnetic material. Specifically, the movement speed of the first bead 1 in the magnetic field can be improved, thereby shortening the time required for magnetic separation. Furthermore, the saturation magnetization of the first bead 1 affects the adsorption force when it is fixed by an external magnetic field. If the saturation magnetization is within the above range, a sufficiently high adsorption force can be obtained, so when the liquid is discharged after magnetic separation, the discharge of the first bead 1 together with the liquid can be suppressed. This makes it possible to suppress the decrease in the yield of biomaterials that occurs with the decrease in the first bead 1.

[0022] The upper limit of the saturation magnetization of the first bead 1 is not particularly limited, but from the viewpoint of ease of material selection that is suitable for balancing performance and cost, it is preferable to set it to 220 emu / g or less.

[0023] The saturation magnetization of the first bead 1 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 applied magnetic field when measuring saturation magnetization is, for example, 0.5T or higher.

[0024] Furthermore, the coercivity Hc of the first bead 1 is preferably 1500 A / m or less, more preferably 800 A / m or less, and even more preferably 300 A / m or less. Coercivity Hc refers to the value of the opposing external magnetic field required to return a magnetized magnetic material to an unmagnetized state. In other words, coercivity Hc represents the resistance to an external magnetic field. The smaller the coercivity Hc of the first bead 1, the less likely the first beads 1 are to aggregate when switching from a magnetic field applied to an unapplied state, and the more uniformly the first beads 1 can be dispersed in the first dispersion 100. Moreover, even when switching the magnetic field application is repeated, the smaller the coercivity Hc, the better the redispersibility of the first beads 1, and therefore the more effectively aggregation of the first beads 1 can be suppressed. The lower limit of the coercivity Hc of the first bead 1 is not particularly limited, but from the viewpoint of ease of material selection that is suitable for balancing performance and cost, it is preferably 5 A / m or more. The coercivity Hc of the first bead 1 can be measured using a vibrating sample magnetometer or the like, similar to the saturation magnetization described above.

[0025] Furthermore, the relative permeability of the first bead 1 is preferably 5 or higher. If the relative permeability of the first bead 1 falls below the lower limit, the movement speed of the first bead 1 will decrease, and the time required for magnetic separation may increase. The upper limit of the relative permeability of the first bead 1 is not particularly limited, but since the first bead 1 is in powder form, the relative permeability is often substantially 100 or less due to the effect of the demagnetizing field.

[0026] 1.1.1.1.First magnetic metal powder The first magnetic metal powder is a magnetic particle and preferably contains at least one of Fe, Co, and Ni as a constituent element. In particular, from the viewpoint of obtaining high saturation magnetization, it is preferable to increase the Fe content in the composition of the first magnetic metal powder, and more preferably to have a composition in which Fe is the main component. Specifically, it is more preferable to have Fe as 50% or more by atomic ratio, and even more preferably 70% or more by atomic ratio. The composition of the first magnetic metal powder may also be an alloy in which Fe is the main component (Fe-based alloy), for example, Fe-Co alloy, Fe-Ni alloy, Fe-Co-Ni alloy, or a compound containing Fe, Co, and Ni can be cited. Furthermore, from the viewpoint of obtaining high magnetization, carbonyl iron powder consisting of substantially 100% by mass of Fe, Fe-Si alloy powder, Fe-Si-Cr alloy powder, etc., are preferably used as the first magnetic metal powder.

[0027] Fe-based alloys, in addition to Co or Ni which exhibit ferromagnetism on their own as mentioned above, may 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 in alloy powders, but it is also an element that promotes amorphous formation.

[0028] Furthermore, the Fe-based alloy may contain impurities to the extent that they do not impair the effects of the first magnetic metal powder. In this embodiment, impurities refer to elements that are unintentionally mixed in during the production of the first magnetic metal powder or as raw materials for the first magnetic metal powder. The impurities are not particularly limited, but examples include O, N, S, Na, Mg, K, etc.

[0029] An example of an Fe-based alloy is an alloy in which the Si content is preferably 1.0 atomic% to 30.0 atomic%, more preferably 1.5 atomic% to 13.0 atomic%, and even more preferably 2.0 atomic% to 7.0 atomic%. Such alloys tend to have high saturation magnetization due to their high magnetic permeability.

[0030] Furthermore, the Fe-based alloy may contain at least one of the following: boron (B) in a content of 5.0 atomic% to 16.0 atomic% and carbon (C) in a content of 0.5 atomic% to 5.0 atomic%. These are elements that promote amorphous formation and contribute to the formation of a stable amorphous or nanocrystalline structure in the first magnetic metal powder.

[0031] Furthermore, it is preferable that the Fe-based alloy contains 1.0 atomic% to 8.0 atomic% of Cr (chromium). This can improve the corrosion resistance of the first magnetic metal powder.

[0032] Furthermore, it is preferable that the impurity content is 1.0 atomic percent or less in total for all elements. At this level, the effect of the first magnetic metal powder will not be impaired even if impurities are present.

[0033] A particularly preferred example of an Fe-based alloy is one in which Fe is the main component, Si content is 2.0% to 9.0% by mass, B content is 1.0% to 5.0% by mass, and Cr content is 1.0% to 3.0% by mass. Such Fe-based alloys have a stable amorphous structure, resulting in low coercivity, and high saturation magnetization due to their high Fe content. Furthermore, the inclusion of Cr enhances corrosion resistance, thereby suppressing the elution of iron ions. Since iron ions can adversely affect the detection of biological materials, it is preferable to suppress their elution.

[0034] The constituent elements and composition of the first magnetic metal powder can be determined by methods such as ICP emission spectrometry as specified in JIS G 1258:2014 or spark emission spectrometry as specified in JIS G 1253:2002. If the first magnetic metal powder has a first coating layer, it can be removed by chemical or physical means before measurement using the above methods. If removing the first coating layer is difficult, for example, the first bead 1 can be cut, and the core portion containing the first magnetic metal powder can be analyzed using analytical instruments such as EPMA (Electron Probe Micro Analyzer) or EDX (Energy Dispersive X-ray spectroscopy).

[0035] The Vickers hardness of the first magnetic metal powder is preferably 100 or higher, more preferably 300 or higher, and even more preferably 800 or higher. A method for measuring the hardness of the first magnetic metal powder is as follows: Multiple particles of the first magnetic metal powder are taken out and embedded in resin to prepare a resin-embedded sample. Then, the cross-section of the first magnetic metal powder is exposed on the surface of the resin-embedded sample by grinding and polishing. 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. Furthermore, if the Vickers hardness of the first magnetic metal powder is below the lower limit, the impact of the first bead 1 colliding with it may cause plastic deformation of the first magnetic metal powder. If plastic deformation occurs, the first coating layer may peel off or fall off. The upper limit of the Vickers hardness is not particularly limited, but it is preferably 3000 or less from the viewpoint of ease of material selection that is suitable for a balance of performance and cost.

[0036] The main metal structure constituting the first magnetic metal powder can take various forms, such as crystalline, amorphous, and nanocrystalline structures. An amorphous structure is an amorphous structure in which no crystals are present, and a nanocrystalline structure refers to a structure mainly composed of fine crystals with a grain size of 100 nm or less. The first magnetic metal powder preferably contains an amorphous or nanocrystalline structure. The amorphous and nanocrystalline structures give the first magnetic metal powder high hardness. Furthermore, by using an amorphous or nanocrystalline structure, the coercivity Hc of the first bead 1 becomes a low value, contributing to improved dispersibility of the first bead 1. The volume fraction of the amorphous or nanocrystalline structure in the first magnetic metal powder is preferably 40% or more, and more preferably 60% or more. This volume fraction can be determined from the results of crystal structure analysis by X-ray diffraction. In addition, the crystalline, amorphous, and nanocrystalline structures may exist individually, or two or more of them may be mixed together.

[0037] The microstructure of the first magnetic metal powder can be identified by performing crystal structure analysis on the first magnetic metal powder using X-ray diffraction. Furthermore, it can be identified by analyzing the microstructure image or diffraction pattern of a cut sample using a transmission electron microscope (TEM). More specifically, in the case of an amorphous microstructure, no diffraction peaks originating from metal crystals such as the α-Fe phase are observed in the peak analysis by X-ray diffraction. Also, in the case of an amorphous microstructure, a so-called halo pattern is formed in the electron diffraction pattern by TEM, and the formation of spots by crystals is not observed. A nanocrystalline microstructure consists of crystals with a particle size of, for example, 100 nm or less, and can be confirmed from TEM observation images. Furthermore, the average particle size can be calculated from multiple TEM microstructure observation images containing multiple crystals by image processing. In addition, the crystal grain size can be estimated from the diffraction peak of the target crystal phase by the Sherer method using X-ray diffraction. Furthermore, for crystal microstructures with large particle sizes, the crystal grain size can be measured by methods such as observing the cross-section with an optical microscope or scanning electron microscope (SEM).

[0038] To obtain amorphous and nanocrystalline structures, it is effective to increase the cooling rate when manufacturing the first magnetic metal powder after pulverizing the molten raw material. Furthermore, the ease of forming amorphous and nanocrystalline structures also depends on the alloy composition. A specific alloy system suitable for forming amorphous and nanocrystalline structures is preferably a composition in which one or more elements selected from the group consisting of Cr, Si, B, C, P, Nb, and Cu are added to Fe.

[0039] 1.1.1.2.First coating layer The first coating layer covers the particle surface of the first magnetic metal powder. The first coating layer only needs to cover at least a portion of the particle surface of the first magnetic metal powder, but it is preferable that it covers the entire particle surface.

[0040] The primary function of the first coating layer is to capture the biomolecule to be extracted on its surface. From this perspective, the first coating layer has a substance or chemical structure that has the ability to bind to biomolecules. Examples of such substances or chemical structures include the following:

[0041] The first preferred material for the first coating layer is an oxide film such as silicon oxide. Silicon oxide is a substance particularly suitable for extracting nucleic acids such as DNA and RNA, and its chemical formula is, for example, SiO₂ x(0 < x ≤ 2) is preferable, specifically SiO2 is preferable. Silicon oxide enables the extraction and recovery of nucleic acids by specifically adsorbing nucleic acids in an aqueous solution in which a chaotropic substance is present. A "chaotropic substance" has the effect of increasing the water solubility of hydrophobic molecules and is a substance that contributes to nucleic acid adsorption. Specific chaotropic substances include guanidine hydrochloride, sodium iodide, sodium perchlorate, etc. Further, it may contain silicon and a composite oxide or composite of one oxide selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr or two or more thereof. Al, Ti, V, Nb, Cr, Mn, Sn, and Zr are elements that are excellent in so-called elution resistance that suppresses ion elution from the first magnetic metal powder to be coated. Therefore, by using oxides or composite oxides or composites of these elements as the first coating layer, the extraction performance of biological substances can be improved while ensuring elution resistance. Also, the first coating layer may form a plurality of layers with oxides of different elements, etc.

[0042] The second preferable substance that the first coating layer has is a substance having a functional group having binding property with the biological substance to be extracted. Examples of the functional group having binding property include, although it depends on the biological substance, OH group, COOH group, NH2 group, epoxy group, trimethylsilyl group, NHS group, etc.

[0043] Other preferable substances that the first coating layer has include proteins such as streptavidin, protein A, protein G, and carbon. Also, when nucleic acids are the extraction target, nucleic acids having properties complementary to the target nucleic acids, specifically oligo(dT) primer cDNA, etc. are also mentioned as preferable substances. Further, when separating cells or exosomes, antibodies such as CD3, CD4, CD8, CD9, CD63, CD81, etc. are mentioned.

[0044] It is desirable that the first coating layer does not capture substances that are not the target of extraction, such as impurities. From this viewpoint, it is preferable that the first coating layer contains a substance called a blocking substance together with the preferred substance contained in the first coating layer. Examples of blocking substances include polyethylene glycol, albumin, and dextrin.

[0045] The first coating layer may contain impurities, as long as they do not impair its effectiveness. For example, when silicon oxide is used as the coating layer, examples of impurities in the silicon oxide include C, N, P, etc. The materials and composition of the first coating layer can be confirmed, for example, by EDX analysis, Auger electron spectroscopy, etc.

[0046] The average thickness t of the first coating layer is preferably 1 nm to 100 nm, and more preferably 10 nm to 50 nm. This prevents the first coating layer from being damaged or peeled off even if it collides with the first bead 1 or the second bead 2, or with the inner wall of the container, etc. As a result, it is possible to suppress a decrease in the extraction efficiency of biomaterials and the generation of impurities when extracting biomaterials. In addition, it is possible to suppress the elution of iron ions and the like that occurs when the first magnetic metal powder is exposed. Furthermore, it is possible to suppress a decrease in the magnetization per unit volume of the first bead 1 and suppress a decrease in the movement speed of the first bead 1.

[0047] The thickness of the first coating layer can be measured, for example, from a cross-sectional image of the first bead 1 using a transmission electron microscope or scanning electron microscope. The average thickness t can be calculated by acquiring multiple observation images and averaging the measured values ​​obtained through image processing. For example, the average thickness t is obtained by measuring the thickness of the first coating layer at five or more locations on one first bead 1, calculating the average value, and then averaging those average values ​​over ten or more first beads 1.

[0048] 1.1.2.First dispersion medium Examples of the first dispersion medium 10 include water, saline solution, polar organic solvents such as alcohols, or aqueous solutions thereof. Examples of water include sterile water and purified water. Examples of alcohols include ethanol and isopropyl alcohol.

[0049] The concentration of the first beads 1 in the first dispersion is preferably 30% by mass or more and 80% by mass or less. If the concentration of the first beads 1 falls below the lower limit, it may not be possible to ensure sufficient adsorption of biomaterials, which may hinder the analysis of the biomaterials extracted in the end. On the other hand, if the concentration of the first beads 1 exceeds the upper limit, the uniformity of the first dispersion 100 may decrease.

[0050] Furthermore, a surfactant may be added to improve the dispersibility of the first beads 1 in the first dispersion 100. Examples of surfactants include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants.

[0051] Examples of nonionic surfactants include Triton-type surfactants such as Triton®-X, Tween-type surfactants such as Tween®-20, and acylsorbitan. Examples of cationic surfactants include dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, and cetyltrimethylammonium bromide. Examples of anionic surfactants include sodium dodecyl sulfate, sodium N-lauroyl sarcosinate (SDS), sodium cholate, sodium lauryl sulfate, and sarcosine. Examples of amphoteric surfactants include phosphatidylethanolamine. These surfactants can be used individually or in combination of two or more.

[0052] The surfactant content in the first dispersion 100 is preferably equal to or greater than the critical micelle concentration of the surfactant. The critical micelle concentration, also known as CMC, is the concentration at which surfactant molecules dispersed in a liquid aggregate to form micelles. By having a surfactant content equal to or greater than the critical micelle concentration, the surfactant is more likely to form a layer around the first bead 1. This further enhances the effect of suppressing the aggregation of the first bead 1.

[0053] Furthermore, the surfactant content is not limited to being above the critical micelle concentration, but may be below the critical micelle concentration. For example, the surfactant content in the first dispersion 100 is preferably 0.05% by mass or more and 3.0% by mass or less, regardless of the critical micelle concentration.

[0054] Furthermore, it is preferable to add a preservative to the first dispersion 100 in order to provide long-term storage and preservative effects. Examples of preservatives include sodium azide. The concentration of the preservative added to the first dispersion 100 is preferably 0.02% by mass or more and less than 0.1% by mass. If the concentration of the preservative added falls below the lower limit, long-term storage and preservation may be insufficient. On the other hand, if the concentration of the preservative added exceeds the upper limit, the extraction efficiency of biomolecules may decrease. Furthermore, a buffer solution for pH adjustment may be added to the first dispersion 100. Examples of buffer solutions include Tris buffer.

[0055] 1.2.Second dispersion liquid The second dispersion 200 is placed in a container or the like and constitutes part of the magnetic bead dispersion kit 3. As described above, the second dispersion 200 has the second beads 2 and the second dispersion medium 20.

[0056] 1.2.1. Second Bead The second bead 2 comprises a second magnetic metal powder having magnetism and a second coating layer that covers the surface of the particles and has the ability to bond with biomaterials. In this specification, the second bead 2 refers to a single particle or an aggregate of particles.

[0057] When a magnet is brought near a single first dispersion 100, an external magnetic field acts on multiple first beads 1. As a result, each first bead 1 is attracted to the magnet individually. However, because the first beads 1 have a small particle size, their saturation magnetization is low. Consequently, the magnetic attraction force is also small, resulting in a low migration speed and a long time required for magnetic separation.

[0058] In contrast, when the first dispersion 100 and the second dispersion 200 shown in Figure 1 are mixed, as shown in Figure 2, both the first bead 1 and the second bead 2 are dispersed in the mixing dispersion medium 30, forming the mixed dispersion 300. The average particle size of the second bead 2 is 5 μm or more larger than the average particle size of the first bead 1, and 100 μm or less. If the average particle size of the second bead 2 is within this range, when a magnet 5 is placed near the mixed dispersion 300 and an external magnetic field acts on the first bead 1 and the second bead 2, the magnetization of the second bead 2 becomes greater than that of the first bead 1. As a result, as shown in Figure 2, the first bead 1 is attracted to the nearby second bead 2 and forms an aggregate 41. When an external magnetic field acts on this aggregate 41, the entire aggregate 41 is attracted to the magnet 5. Consequently, the movement speed of the aggregate 41 becomes greater than that of the first bead 1 alone. Furthermore, because the aggregate 41 contains the first bead 1, its specific surface area is larger than that of the second bead 2 alone. Therefore, the formation of aggregates 41 allows for a reduction in the time required for magnetic separation while increasing the amount of biomolecules adsorbed. As a result, the analysis of biomolecules can be accelerated and made more accurate.

[0059] The average particle size of the second bead 2 should be at least 5 μm larger than the average particle size of the first bead 1, but it is preferably at least 10 μm larger, more preferably at least 20 μm larger, and even more preferably at least 30 μm larger. Furthermore, the average particle size of the second bead 2 is set to be 100 μm or less, but it is preferably 80 μm or less, and more preferably 60 μm or less.

[0060] Furthermore, if the average particle size of the second bead 2 falls below the lower limit, the particle size difference between the first bead 1 and the second bead 2 becomes almost negligible, and the above effect cannot be fully obtained. On the other hand, if the average particle size of the second bead 2 exceeds the upper limit, the particle size of the second bead 2 becomes too large, leading to problems such as a decrease in the specific surface area of ​​the second bead 2 and an increased tendency for the second bead 2 to settle. Furthermore, the difference between the average particle size of the first bead 1 and the average particle size of the second bead 2 is preferably 60 μm or less, and more preferably 50 μm or less. This ensures a particle size difference between the first bead 1 and the second bead 2, thereby fully achieving the above-mentioned effects. The average particle size of the second bead 2 is determined using the same method as the average particle size of the first bead 1.

[0061] Furthermore, it is preferable that the relationship between the average thickness of the second coating layer and the average particle size of the second bead 2 satisfies appropriate conditions, similar to the relationship between the average thickness of the first coating layer and the average particle size of the first bead 1. This ensures that the same effect as in the case of the first bead 1 is obtained in the second bead 2 as well.

[0062] Furthermore, it is preferable that the saturation magnetization, coercivity, and relative permeability of the second bead 2 also fall within the same appropriate range as those of the first bead 1. This ensures that the same effects as those of the first bead 1 are obtained with the second bead 2.

[0063] 1.2.1.1.Second magnetic metal powder The second magnetic metal powder consists of magnetic particles, and its composition is appropriately selected from the alloys and the like listed as the composition of the first magnetic metal powder. Therefore, the composition of the second magnetic metal powder may be the same as or different from the composition of the first magnetic metal powder.

[0064] The Vickers hardness of the second magnetic metal powder is preferably within the same appropriate range as that of the first magnetic metal powder. This ensures that the same effect as in the first bead 1 is obtained in the second bead 2 as well.

[0065] The main metal structure constituting the second magnetic metal powder is amorphous or nanocrystalline. This allows for a lower coercivity of the second beads 2. As a result, when no external magnetic field is applied, the remanent magnetization of the second beads 2 is suppressed, thus suppressing aggregation of the second beads 2. This prevents problems caused by aggregation of the large-particle second beads 2, such as a large amount of liquid being retained in the gaps between the second beads 2, which reduces magnetic separation performance. Furthermore, by suppressing aggregation of the second beads 2, the movement to form aggregates 41 is less likely to be hindered. This makes it possible to more reliably obtain the effect of increasing the separation speed in magnetic separation while also increasing the amount of biomolecules adsorbed.

[0066] The description of the microstructure constituting the second magnetic metal powder is the same as the description of the microstructure constituting the first magnetic metal powder.

[0067] 1.2.1.2.Second coating layer The second coating layer covers the particle surface of the second magnetic metal powder. The second coating layer only needs to cover at least a portion of the particle surface of the second magnetic metal powder, but it is preferable that it covers the entire particle surface.

[0068] The primary function of the second coating layer is to capture the biomolecule to be extracted on its surface. From this perspective, the second coating layer has a substance or chemical structure that has the ability to bind to biomolecules. The description of the substance or chemical structure of the second coating layer that has the ability to bind to biomolecules is the same as the description of the first coating layer.

[0069] The substances in the second coating layer may be the same as or different from those in the first coating layer. The types of these substances are appropriately selected according to the type of biomolecule to be extracted. For example, if the biomolecule to be extracted has one type of target molecule (antigen, etc.), the substances in the first coating layer and the second coating layer may both be substances that bind to that target molecule (antibodies, etc.). On the other hand, if the biomolecule to be extracted has two or more types of target molecules, the substances in the first coating layer and the second coating layer may be of different types. This allows the substances in the first and second coating layers to work together to contribute to the adsorption of the biomolecule. Examples of biomolecules with two or more types of target molecules include cells and exosomes.

[0070] Figure 3 is a schematic diagram illustrating the prior art, showing an example in which only the first dispersion 100 containing the first bead 1 and the first dispersion medium 10 is used, and each first bead 1 has one of two types of binding sites S1 and S2. The use of first beads 1 having different types of binding sites S1 and S2 in this manner has been practiced conventionally. When cells 91 are dispersed as biomaterials in the first dispersion 100, two or more types of antigens A1 and A2 may be present on the surface of the cells 91.

[0071] Figure 4 is a schematic diagram illustrating the prior art, showing a state in which binding site S1 specifically binds to antigen A1 shown in Figure 3, binding site S2 specifically binds to antigen A2, and cell 91 is adsorbed onto the first bead 1. In this case, binding sites S1 and S2 are, for example, antibodies. When cell 91 and the first bead 1 come into contact, as shown in Figure 4, one cell 91 may be adsorbed onto both the first bead 1 having binding site S1 and the first bead 1 having binding site S2. In this case, an aggregate 92 is formed in which one cell 91 and multiple first beads 1 are aggregated. However, depending on whether or not there is an opportunity for contact, as shown in Figure 4, in some cases only one first bead 1 may be adsorbed onto one cell 91.

[0072] When a magnet 5 is brought close to the first dispersion 100 shown in Figure 4 and an external magnetic field is applied, the first beads 1 are attracted to the magnet 5. However, because the average particle size of the first beads 1 is small, the saturation magnetization per bead is small. Therefore, even if aggregates 92 are formed, the separation rate in magnetic separation is small, and magnetic separation takes a long time. Also, because the average particle size of the first beads 1 is small, it is difficult for a single first bead 1 to have two or more types of bonding sites.

[0073] In contrast, in this embodiment, as described above, a first bead 1 and a second bead 2, which have different average particle sizes, are used in combination. Since the average particle size of the second bead 2 is larger than that of the first bead 1, one second bead 2 can have two or more types of bonding sites.

[0074] Figure 5 is a schematic diagram illustrating magnetic separation using the magnetic bead dispersion kit 3 according to the embodiment, showing an example where each first bead 1 has either binding site S1 or S2, and the second bead 2 has both binding sites S1 and S2. The binding sites S1 and S2 are appropriately selected from, for example, the substances contained in the first coating layer described above. In this case, as shown in Figure 5, one second bead 2 can adsorb multiple cells 91. In other words, by having both binding sites S1 and S2 in one second bead 2, the probability and amount of cells 91 adsorbed onto the second bead 2 can be increased. In Figure 5, one of the multiple cells 91 is referred to as "cell 91a" and the other as "cell 91b". In this example shown in Figure 5, antigen A1 on cell 91a binds to binding site S1 on the second bead 2, and antigen A2 on cell 91b binds to binding site S2 on the second bead 2. As a result, one second bead 2 adsorbs two cells 91a and 91b.

[0075] Furthermore, antigen A2 present in cell 91a is bound to binding site S2 on the first bead 1. In addition, antigen A1 present in cell 91b is bound to binding site S1 on another first bead 1.

[0076] As a result, aggregates 42 are formed by the aggregation of two first beads 1 and one second bead 2 via two cells 91a and 91b. When an external magnetic field acts on the aggregate 42, the entire aggregate 42 is attracted to the magnet 5. As a result, the movement speed of the aggregate 42 becomes greater than that of the first bead 1 alone. Furthermore, because the aggregate 42 contains the first bead 1, its specific surface area is larger than that of the second bead 2 alone. Therefore, the formation of the aggregate 42 makes it possible to increase the amount of biomaterial adsorbed while shortening the time required for magnetic separation. As a result, it is possible to speed up and improve the accuracy of biomaterial analysis.

[0077] The average thickness t of the second coating layer is preferably 1 nm to 100 nm, and more preferably 10 nm to 50 nm. This prevents the second coating layer from being damaged or peeling off even if it collides with the first bead 1 or the second bead 2, or with the inner wall of the container, etc. As a result, it is possible to suppress a decrease in the extraction efficiency of biomaterials and the generation of impurities when extracting biomaterials. In addition, it is possible to suppress the elution of iron ions and the like that occurs when the second magnetic metal powder is exposed. Furthermore, it is possible to suppress a decrease in the magnetization per unit volume of the second bead 2 and a decrease in the movement speed of the second bead 2. The thickness of the second coating layer is measured in the same manner as the thickness of the first coating layer.

[0078] 1.2.2.Second dispersion medium The second dispersion medium 20 can be, like the first dispersion medium 10, for example, water, saline solution, polar organic solvents such as alcohols, or aqueous solutions thereof. The description of the second dispersion medium 20 is the same as the description of the first dispersion medium 10.

[0079] 1.3.How to use Next, we will describe an example of how to use the magnetic bead dispersion kit 3, namely, a magnetic separation method.

[0080] First, the first dispersion 100 and the second dispersion 200 are placed in a container along with the biomaterial. If necessary, a dissolving adsorbent is also added to the container. Then, the contents of the container are mixed. This disperses the first bead 1 and the second bead 2 in the liquid. The dissolving adsorbent is used to dissolve and extract the biomaterial if it is encapsulated within, for example, a cell membrane or nucleus. Furthermore, the biomaterial is adsorbed onto the first bead 1 and the second bead 2 by the adsorption action of the dissolving adsorbent.

[0081] The mixing ratio of the first dispersion 100 and the second dispersion 200 varies depending on the concentration of each dispersion, but it is preferable that the volume of the first bead 1 is set to be larger than the volume of the second bead 2. This optimizes the ratio of the first bead 1 to the second bead 2 when the first bead 1 is attracted to the second bead 2 to form aggregates 41. As a result, the specific surface area of ​​the aggregates 41 can be sufficiently increased without reducing the separation rate of the aggregates 41 in magnetic separation.

[0082] When mixing the first dispersion 100 and the second dispersion 200, the volume of the first bead 1 should be greater than the volume of the second bead 2. However, when the volume of the second bead 2 is set to 1, the first bead 1 is preferably greater than 1 and 30 or less, more preferably between 2 and 20, and even more preferably between 5 and 15. This optimizes the balance between the two, increasing the separation rate of the aggregates 41 while sufficiently increasing the specific surface area of ​​the aggregates 41.

[0083] Next, an external magnetic field is applied to the contents of the container to magnetically separate the first bead 1 and the second bead 2 from the liquid phase. Subsequently, the magnetically separated liquid phase is discharged using a pipette or similar tool. Next, the washing solution is placed in a container and stirred with the first bead 1 and the second bead 2. Then, the first bead 1 and the second bead 2 are magnetically separated from the washing solution. Finally, the magnetically separated washing solution is discharged using a pipette or similar tool.

[0084] Next, the eluate is placed in a container and the first bead 1 and second bead 2 are stirred with the eluate. The biomaterial adsorbed on the first bead 1 and second bead 2 is eluted into the eluate. Subsequently, the first bead 1 and second bead 2 are magnetically separated from the eluate. Then, the magnetically separated eluate is collected using a pipette or similar tool. This allows for the extraction of the biomaterial.

[0085] 1.4. Effects of the Magnetic Bead Dispersion Kit According to the Embodiment As described above, the magnetic bead dispersion kit 3 according to the embodiment comprises a first dispersion 100 and a second dispersion 200. The first dispersion 100 comprises first beads 1 comprising a first magnetic metal powder and a first coating layer, and a first dispersion medium 10 for dispersing the first beads 1. The first coating layer coats the particle surface of the first magnetic metal powder and has the ability to bind to biomolecules. The second dispersion 200 comprises second beads 2 comprising a second magnetic metal powder and a second coating layer, and a second dispersion medium 20 for dispersing the second beads 2. The second magnetic metal powder includes an amorphous structure or a nanocrystalline structure. The second coating layer coats the particle surface of the second magnetic metal powder and has the ability to bind to biomolecules. The average particle size of the first bead 1 is between 1 μm and 15 μm. On the other hand, the average particle size of the second bead 2 is at least 5 μm larger than that of the first bead 1, and is 100 μm or less.

[0086] With this configuration, mixing the first dispersion 100 and the second dispersion 200 yields a mixed dispersion 300 containing both the first beads 1 and the second beads 2, which have different average particle sizes. When an external magnetic field acts on this mixed dispersion 300, the magnetization of the second beads 2 is greater than that of the first beads 1, causing the first beads 1 to be attracted to the second beads 2 and form aggregates 41. The migration speed of the aggregates 41 is greater than that of the first beads 1 alone. Also, the specific surface area of ​​the aggregates 41 is larger than that of the second beads 2 alone. Therefore, the formation of aggregates 41 shortens the time required for magnetic separation while increasing the amount of biomolecules adsorbed. Furthermore, because the second magnetic metal powder contains the above structure, it has a low coercivity Hc. Therefore, magnetized second beads 2 are less likely to aggregate with each other, improving the dispersibility of the second beads 2 in the liquid and reducing the inhibition of aggregate formation 41. As a result, the analysis of biomolecules can be accelerated and the accuracy improved.

[0087] Furthermore, the second coating layer may have binding properties for biomolecules and may have two or more binding sites of different types, for example, two binding sites S1 and S2 as shown in Figure 5. With such a configuration, for example, if the biomolecule has two or more target molecules, specifically if the cells 91 shown in Figure 5 have antigens A1 and A2, one second bead 2 can adsorb multiple cells 91. This increases the probability of adsorbing biomolecules and increases the amount of adsorption. As a result, the accuracy and reliability of biomolecule analysis can be improved.

[0088] Furthermore, the binding sites on the second coating layer are, for example, antibodies. As shown in Figure 5, when the second bead 2 has binding sites S1 and S2 which are antibodies, these binding sites S1 and S2 specifically bind to antigens A1 and A2 present on the cell 91. This makes it possible to realize a magnetic bead dispersion kit 3 that can accurately extract the cell 91.

[0089] Furthermore, when the first dispersion 100 and the second dispersion 200 are mixed together with the biomaterial, the first bead 1 and the second bead 2 may aggregate via the biomaterial. For example, as shown in Figure 5, the first bead 1 and the second bead 2 may aggregate via the cell 91, forming the aggregate 42 shown in Figure 5. In other words, aggregation may occur not only due to magnetization but also via the biomaterial in the first bead 1 and the second bead 2. The aggregate 42 has a greater migration speed than the first bead 1 alone, and a greater specific surface area than the second bead 2 alone. Therefore, the formation of the aggregate 42 can shorten the time required for magnetic separation while increasing the amount of biomaterial adsorbed.

[0090] Furthermore, the first magnetic metal powder preferably contains an amorphous or nanocrystalline structure. This results in a lower coercivity Hc not only for the second beads 2 but also for the first beads 1. As a result, the dispersibility of the first beads 1 in liquid can be improved, and the formation of aggregates 41 and 42 is promoted.

[0091] Furthermore, the average thickness of the first coating layer and the average thickness of the second coating layer are preferably 1 nm to 100 nm, respectively. This helps to prevent the first and second coating layers from breaking or peeling off. As a result, it is possible to suppress a decrease in the extraction efficiency of biomaterials and the generation of impurities when extracting biomaterials. In addition, by suppressing the peeling of the first and second coating layers, the elution of iron ions and the like can be suppressed. Furthermore, it is possible to suppress a decrease in the magnetization per unit volume of the first bead 1 and the second bead 2, and a decrease in the movement speed of the first bead 1 and the second bead 2.

[0092] 2. Magnetic beads The aforementioned magnetic bead dispersion kit 3 is a kit that contains first beads 1 and second beads 2 having different average particle sizes as separate dispersions, but the first beads 1 and second beads 2 may also be provided as a mixed powder. The magnetic beads according to the embodiment correspond to this mixed powder.

[0093] The magnetic beads according to this embodiment are used for magnetic separation, similar to the magnetic bead dispersion kit 3 described above. Magnetic separation is a method of separating a solid phase containing magnetic beads and a liquid phase containing liquid by applying an external magnetic field to a container containing both the solid phase and the liquid phase, thereby magnetically attracting the solid phase.

[0094] The magnetic beads according to this embodiment are broadly classified into a first powder and a second powder, which have different average particle sizes, based on the particle size distribution described later. In other words, while the particle size distribution of metal powders such as magnetic beads is generally close to a log-normal distribution, the particle size distribution of the magnetic beads according to this embodiment is a distribution that can be fitted by two log-normal distributions whose modes are separated by 5 μm or more. Therefore, the magnetic beads according to this embodiment are a mixed powder obtained by mixing two types of powders (first powder and second powder) that are represented by log-normal distributions with different modes.

[0095] Figure 6 shows the particle size distribution of the magnetic beads according to the embodiment, and an example of the result of fitting a log-normal distribution to this particle size distribution. In Figure 6, the particle size distribution of the magnetic beads according to the embodiment is denoted as D. The log-normal distribution obtained by fitting, where the mode is on the smaller diameter side, is called the "first log-normal distribution A," and the log-normal distribution where the mode is on the larger diameter side is called the "second log-normal distribution B." The first log-normal distribution A and the second log-normal distribution B are obtained as follows.

[0096] First, the particle size distribution D of the magnetic beads is obtained. The particle size distribution D is a frequency distribution curve obtained by measuring the volume-based particle size distribution of the magnetic beads and analyzing the resulting particle size distribution. Next, the particle size corresponding to the peak of the particle size distribution D is defined as the most frequent particle size M of the magnetic beads. Then, a fitting is performed on the distribution of particle size distribution D that is smaller than the most frequent particle size M to obtain the first log-normal distribution A. Specifically, a line passing through the peak of the particle size distribution D and parallel to the vertical axis is defined as the central axis L, and the fitting is performed on the distribution of particle size distribution D that is smaller than the central axis L. This gives the first log-normal distribution A centered on the most frequent particle size M.

[0097] Next, we calculate the difference between the particle size distribution D and the first log-normal distribution A. This difference becomes the second log-normal distribution B. The starting point on the smaller diameter side of the second log-normal distribution B is defined as the point where the frequency becomes 0% on the larger diameter side than the most frequent particle size M.

[0098] As a result of fitting using the method described above, a first log-normal distribution A and a second log-normal distribution B are obtained. When the mode of the first log-normal distribution A is Am and the mode of the second log-normal distribution B is Bm, then Bm-Am is between 5 μm and 100 μm.

[0099] Furthermore, the powder represented by the first log-normal distribution A is referred to as the first powder, and the powder represented by the second log-normal distribution B is referred to as the second powder. In this embodiment, the magnetic beads are configured such that the volume fraction of the first powder is greater than the volume fraction of the second powder. The volume fractions of the first powder and the second powder are determined from the area enclosed by the curve of the first log-normal distribution A and the area enclosed by the curve of the second log-normal distribution B.

[0100] Having the above-described configuration, the magnetic beads according to the embodiment exhibit the same effects as those achieved by the magnetic bead dispersion kit described above. Specifically, the magnetic beads according to the embodiment include a first powder and a second powder, where the first powder corresponds to the first bead 1 described above, and the second powder corresponds to the second bead 2 described above. Therefore, the first powder is, for example, a powder comprising the first magnetic metal powder described above, and a first coating layer that covers the particle surface and has the ability to bind to biomolecules. The second powder is, for example, a powder comprising the second magnetic metal powder described above, and a second coating layer that covers the particle surface and has the ability to bind to biomolecules. In the magnetic beads according to the embodiment, as shown in Figure 6, the mixing ratio is set such that the volume fraction of the first powder is greater than the volume fraction of the second powder.

[0101] By using such magnetic beads for the extraction of biomaterials, it is possible to shorten the time required for magnetic separation while increasing the amount of biomaterial adsorbed.

[0102] The volume fraction of the first powder should be greater than the volume fraction of the second powder, but preferably it is greater than 1 and 30 or less, more preferably 2 or more and 20 or less, and even more preferably 5 or more and 15 or less, when the volume fraction of the second powder is set to 1. This optimizes the balance between the two, making it possible to increase the separation rate of the aggregates mentioned above while sufficiently increasing the specific surface area of ​​the aggregates.

[0103] As described above, the magnetic beads according to this embodiment include a first powder and a second powder. The first powder comprises a first magnetic metal powder and a first coating layer that covers the particle surface of the first magnetic metal powder and has the ability to bond with biomolecules, and has an average particle size of 1 μm or more and 15 μm or less. The second powder comprises a second magnetic metal powder containing an amorphous or nanocrystalline structure and a second coating layer that covers the particle surface of the second magnetic metal powder and has the ability to bond with biomolecules, and has an average particle size of 5 μm or more larger than that of the first powder and 100 μm or less. Furthermore, the volume fraction of the first powder is greater than the volume fraction of the second powder.

[0104] Such magnetic beads are a mixed powder of a first powder and a second powder, each having different average particle sizes. When an external magnetic field acts on a dispersion containing this mixed powder, the magnetization of the second powder is greater than that of the first powder, causing the first powder to be attracted to the second powder and form aggregates. The migration speed of these aggregates is greater than that of the first powder alone. Furthermore, the specific surface area of ​​the aggregates is larger than that of the second powder alone. Moreover, since the volume fraction of the first powder in the mixed powder is greater than that of the second powder, the specific surface area of ​​the aggregates becomes sufficiently large. Therefore, the formation of aggregates shortens the time required for magnetic separation while increasing the amount of biomolecules adsorbed. In addition, because the second magnetic metal powder contains the above structure, it has a low coercivity Hc. Therefore, the magnetized particles of the second powder are less likely to aggregate, improving the dispersibility of the second powder in the liquid and ensuring a sufficient amount of biomolecules adsorbed. As a result, the analysis of biomolecules can be accelerated and made more accurate.

[0105] The magnetic beads according to this embodiment may be used in a pre-mixed state, or the first powder and the second powder may be mixed at the time of use. In other words, the magnetic separation method using the magnetic beads according to this embodiment only needs to include the steps of preparing a mixture of a biomaterial, the first powder, and the second powder, and applying an external magnetic field to the mixture to perform magnetic separation. In such a magnetic separation method, as with the case using magnetic beads, the first powder and the second powder form aggregates, thereby shortening the time required for magnetic separation while increasing the amount of biomaterial adsorbed.

[0106] Although the magnetic bead dispersion kit and magnetic beads of the present invention have been described above based on the illustrated embodiments, the present invention is not limited thereto. For example, the magnetic bead dispersion kit of the present invention may have any components added to the above embodiments. [Examples]

[0107] Next, specific embodiments of the present invention will be described. 3. Preparation of mixed powder A mixed powder was prepared by mixing two types of magnetic powders with different average particle sizes. Experiments were conducted to support the effectiveness of the magnetic beads and magnetic bead dispersion kit according to the above embodiment by evaluating the time required for magnetic separation and the cohesiveness of this mixed powder.

[0108] First, five types of powders, 1-1, 1-2, 1-3, 1-4, and 1-5, shown in Table 1, were prepared as the first magnetic metal powders. The alloy composition, main metal structure, average particle size, and coercivity of the first magnetic metal powders are shown in Table 1. The numbers in the alloy composition shown in Table 1 represent the atomic percentage of each element.

[0109] [Table 1]

[0110] Next, two types of powders, 2-1 and 2-2, shown in Table 2, were prepared as the second magnetic metal powder. The alloy composition, main metal structure, average particle size, and coercivity of the second magnetic metal powder are shown in Table 2. The numbers in the alloy composition shown in Table 2 represent the atomic percentage of each element.

[0111] [Table 2]

[0112] Next, one type of first magnetic metal powder selected from five types and one type of second magnetic metal powder selected from two types were mixed to obtain a mixed powder. The mixing ratio was set so that the mass ratio of the first magnetic metal powder to the second magnetic metal powder was 9:1. The volume ratio was also approximately the same. The types of powders selected are shown in Table 3. In Table 3, the magnetic bead dispersion kit and the mixed powder corresponding to the magnetic beads of the present invention are listed as "Examples," and powders not corresponding to the present invention are listed as "Comparative Examples."

[0113] 4. Evaluation of mixed powders For each example and comparative example, the following two evaluations were performed to assess the separation rate (magnetic separation time) and cohesiveness in magnetic separation of the mixed powders.

[0114] 4.1. Evaluation of the time required for magnetic separation First, 1 mL of mononuclear cell suspension and 25 μL of a mixed powder dispersion were placed in a 1.5 mL microtube (container). Next, the contents were slowly agitated for 20 minutes while maintaining the microtube at 2–8°C. This prepared a suspension of the mixed powder.

[0115] Next, 2.5 mL of phosphate buffer (PBS) and 5 μL of the mixed powder suspension were placed in a cuvette, the lid was closed, and the mixture was inverted and stirred. Then, the cuvette was placed in a spectrophotometer. A Hitachi High-Tech Science U-3900H spectrophotometer was used. The measurement wavelength was set to 550 nm, and the absorbance measurement was started.

[0116] After the measurement began, the permanent magnet was placed in close contact with the side of the cuvette. From that point, the time until the absorbance value became 1% of the initial value was measured. This time can be considered as the time required for magnetic separation to be completed (magnetic separation time). The measurement results are shown in Table 3.

[0117] 4.2. Evaluation of Cohesion First, a dispersion of the mixed powder was prepared by adding phosphate buffer to the mixed powder after magnetic separation in 4.1. The resulting dispersion was placed in a microtube and stirred for 10 seconds using a vortex mixer. This yielded a suspension of the mixed powder.

[0118] Next, the obtained suspension was dropped onto a glass slide, and a coverslip was placed on top to prepare a sample for observation. The resulting sample was then observed under a microscope, and the aggregation state of the magnetic metal powder was evaluated according to the following evaluation criteria. The observation area was limited to a 1 mm square area.

[0119] A: Not aggregated, or the number of aggregates is 10 or less. B: The number of aggregates is greater than 10. The evaluation results are shown in Table 3.

[0120] [Table 3]

[0121] As shown in Table 3, the mixed powders of each example showed shorter magnetic separation times and less aggregation compared to the mixed powders of each comparative example. In contrast, Comparative Example 1, which used only the relatively small-diameter first magnetic metal powder, resulted in a longer magnetic separation time. On the other hand, Comparative Examples 2 and 3 used a mixture of the first and second magnetic metal powders, but because the main metal structure of the second magnetic metal powder was crystalline, aggregation was high. This result is thought to be due to the high coercivity of the second magnetic metal powder. Furthermore, high aggregation reduces the amount of biomolecules adsorbed, which is thought to lead to a decrease in the final yield of biomolecules.

[0122] The results above confirm that by using the magnetic bead dispersion kit and magnetic beads of the present invention for the extraction of biomolecules using magnetic separation, it is possible to achieve both a reduction in the time required for magnetic separation and a sufficient amount of adsorption of biomolecules. [Explanation of Symbols]

[0123] 1…First bead, 2…Second bead, 5…Magnet, 10…First dispersion medium, 30…Mixed dispersion medium, 41…Aggregate, 42…Aggregate, 91…Cell, 91a…Cell, 91b…Cell, 92…Aggregate, A…First log-normal distribution, A1…Antigen, A2…Antigen, B…Second log-normal distribution, D…Particle size distribution, L…Central axis, M…Most frequent particle size, S1…Binding site, S2…Binding site

Claims

1. A first dispersion comprising a first magnetic metal powder, a first coating layer that coats the particle surface of the first magnetic metal powder and has the ability to bind to biomaterials, first beads having an average particle size of 1 μm or more and 15 μm or less, and a first dispersion medium for dispersing the first beads, A second dispersion comprising a second magnetic metal powder containing an amorphous or nanocrystalline structure, a second coating layer coating the particle surface of the second magnetic metal powder and having bonding properties with the biomaterial, second beads having an average particle size 10 μm or more larger than the first beads and 100 μm or less, and a second dispersion medium for dispersing the second beads, Equipped with, The first coating layer and the second coating layer each contain silicon oxide, or a composite oxide of silicon and one or more elements selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr. A magnetic bead dispersion kit characterized in that the first dispersion and the second dispersion are mixed such that, when the volume fraction of the second beads is 1, the volume fraction of the first beads is between 5 and 30.

2. The magnetic bead dispersion kit according to claim 1, wherein the second coating layer has binding properties with the biomaterial and has two or more binding sites of different types.

3. The magnetic bead dispersion kit according to claim 2, wherein the binding site is an antibody.

4. A magnetic bead dispersion kit according to any one of claims 1 to 3, wherein when the biomaterial, the first dispersion, and the second dispersion are mixed, the first beads and the second beads aggregate via the biomaterial.

5. The magnetic bead dispersion kit according to any one of claims 1 to 4, wherein the first magnetic metal powder comprises an amorphous structure or a nanocrystalline structure.

6. The magnetic bead dispersion kit according to any one of claims 1 to 5, wherein the average thickness of the first coating layer and the average thickness of the second coating layer are 1 nm or more and 100 nm or less.

7. The magnetic bead dispersion kit according to any one of claims 1 to 6, wherein when the average thickness of the first coating layer is t and the average particle size of the first beads is D50, t / D50 is 0.0001 or more and 0.05 or less.

8. A first magnetic metal powder comprises a first magnetic metal powder and a first coating layer that covers the particle surface of the first magnetic metal powder and has the ability to bind to biomaterials, wherein the first powder has an average particle size of 1 μm or more and 15 μm or less. A second magnetic metal powder comprising an amorphous or nanocrystalline structure, and a second coating layer that coats the particle surface of the second magnetic metal powder and has bonding properties with the biomaterial, wherein the average particle size of the second powder is 10 μm or more larger than that of the first powder and 100 μm or less, Includes, The first coating layer and the second coating layer each contain silicon oxide, or a composite oxide of silicon and one or more elements selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr. Magnetic beads characterized in that, when the volume fraction of the second powder is set to 1, the volume fraction of the first powder is 5 or more and 30 or less.

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