JP7625936B2 - Magnetic beads and method for producing magnetic beads - Google Patents

Description

The present invention relates to magnetic beads and a method for producing magnetic beads.

In recent years, magnetic separation methods using tiny magnetic beads have been widely used in diagnostics and various tests in the medical field. For example, magnetic separation methods are used in PCR methods for extracting biological materials and cell tests.

The PCR method is a method of extracting nucleic acid, amplifying it specifically, and detecting it. In order to efficiently extract this nucleic acid, recent PCR methods use magnetic beads that can hold nucleic acid and extract the nucleic acid by applying a magnetic field. Specifically, the target substance is extracted by repeatedly turning the magnetic field on and off multiple times. A similar method is also used in the field of cell testing, such as the extraction of cancer cells.
Various types of magnetic beads have been investigated so far.

For example, Patent Document 1 describes magnetic beads with an average particle size of 0.5 μm to 10 μm, in which amorphous magnetic powder is coated with a silica film.

Patent document 2 also discloses magnetic silica particles that have a structure in which metal particles and ceramic particles with an average particle size of 0.2 to 10 μm are mixed.

Patent document 3 discloses metal microparticles and magnetic beads in which the 50% particle size (volume-based median diameter (d50)) of the metal microparticles is 0.1 μm to 10 μm, and the 90% particle size (volume-based 90% cumulative particle size (d90)) of the metal microparticles is 0.15 to 15 μm.

JP 2017-176023 A JP 2007-224323 A International Publication No. 2007/148734

Coupled with the recent increase in demand for PCR testing, there is a demand for shorter testing times and improved extraction efficiency for test subjects in diagnostics and various tests in the medical field.

However, the magnetic beads described in Patent Documents 1 to 3 sometimes failed to extract a sufficient amount of the target substance. As a result, the testing time was long, which could lead to a decrease in testing efficiency.

In addition, conventional magnetic beads are often classified using dry classifiers such as rotary classifiers that use centrifugal force, and the particle size distribution of the resulting magnetic beads often contains coarse particles that are very large compared to the average particle size. Such coarse particles are highly magnetized in a magnetic field, so they attract surrounding particles and aggregate, and even when the magnetic field is turned off, dispersibility is lost, causing significant aggregation. Furthermore, when the particles aggregate, they may settle to the bottom of the reagent due to their own weight. As a result, the extraction efficiency of the test target substance is reduced, and the test time is extended accordingly.

In order to solve the above problems, one embodiment of the magnetic beads of the present invention is a magnetic bead having a magnetic metal powder and a coating layer that covers the surface of the magnetic metal powder, in which the 50% particle diameter D50 of the magnetic metal powder in the particle size distribution is 0.1 to 1.5 μm, and the ratio D90/D50 of the 90% particle diameter D90 of the magnetic metal powder in the particle size distribution to the 50% particle diameter D50 is 2.00 or less.

The method for producing magnetic beads according to one embodiment of the present invention includes a magnetic metal powder preparation step for obtaining magnetic metal powder, a liquid classification step for classifying the magnetic metal powder in a liquid to extract magnetic metal powder having a 50% particle diameter D50 of 0.1 to 1.5 μm and a D90/D50 ratio of the 90% particle diameter D90 of the magnetic metal powder to the 50% particle diameter D50 in the particle size distribution of 2.00 or less, and a coating step for forming a coating layer on the magnetic metal powder.

The following describes magnetic beads and a method for producing them according to one embodiment of the present invention.

[Magnetic beads]
The magnetic beads according to this embodiment are magnetic beads having a magnetic metal powder and a coating layer that coats the surface of the magnetic metal powder, wherein the 50% particle diameter D50 of the magnetic metal powder in the particle size distribution is 0.1 to 1.5 μm, and the ratio D90/D50 of the 90% particle diameter D90 of the magnetic metal powder in the particle size distribution to the 50% particle diameter D50 is 2.00 or less.

The magnetic beads according to this embodiment are a particle group capable of adsorbing biological materials such as nucleic acids such as DNA and cells, and have magnetic metal powder and a coating layer that covers the surface of the magnetic metal powder. For example, when extracting nucleic acids, powdered magnetic beads are used.

<Magnetic metal powder>
The magnetic metal powder is a particle having magnetism. The magnetic metal powder is preferably a magnetic metal particle containing at least one of Fe, Co, and Ni. In particular, from the viewpoint of obtaining high saturation magnetization, it is preferable to increase the Fe content in the composition of the magnetic metal powder, and more preferably to make the composition mainly composed of Fe. More preferably, Fe is 70% or more in atomic ratio. The magnetic metal powder may be pure Fe consisting of only Fe. The composition of the magnetic metal powder may also be an alloy mainly composed of Fe (Fe-based alloy), for example, an Fe-Co-based alloy, an Fe-Ni-based alloy, an Fe-Co-Ni-based alloy, or a compound containing Fe, Co, and Ni.

The Fe-based alloy may contain, in addition to the above-mentioned elements such as Co and Ni that exhibit ferromagnetic properties by themselves, 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. Note that the Fe-based alloy may contain unavoidable impurities to the extent that they do not impair the effects of the present invention.

In this embodiment, unavoidable elements are elements (impurities) that are unintentionally mixed into the raw materials of magnetic metal powder or during the production of magnetic beads. Examples of unavoidable elements include, but are not limited to, O, N, S, Na, Mg, K, etc.

The structure constituting the magnetic metal powder is not particularly limited, and can take various forms such as a crystalline structure, an amorphous structure, and a nanocrystalline structure. From the viewpoint of obtaining high magnetization, the magnetic metal powder is preferably a carbonyl iron powder consisting essentially of 100% Fe by mass, an Fe-Si alloy powder, or an Fe-Si-Cr alloy powder.

On the other hand, the magnetic beads of this embodiment are required to exhibit high dispersibility in the extraction process of nucleic acids, cells, etc. In order to obtain this high dispersibility, it is preferable that the magnetic metal powder has a low coercive force (Hc). As a structure of magnetic metal powder that can achieve low coercive force, an amorphous structure or a nanocrystalline structure is preferable. In particular, an amorphous structure has a relatively high electrical resistance value and a small coercive force because the atomic arrangement is irregular. Therefore, magnetic beads made of magnetic metal powder with an amorphous structure are less likely to aggregate when a magnetic field (magnetic field) is not applied, and can be uniformly dispersed in the test solution. Furthermore, even if the application of a magnetic field is repeatedly turned on and off, the redispersibility is excellent, so aggregation of the beads can be further suppressed.

To obtain amorphous and nanocrystalline structures, it is effective to increase the rapid cooling rate during solidification when manufacturing magnetic metal powder. In addition, the ease of forming amorphous and nanocrystalline structures also depends on the alloy composition.

A specific alloy system suitable for forming an amorphous structure or nanocrystalline structure is preferably a composition containing Fe and one or more elements selected from the group consisting of Cr, Si, B, C, P, Nb, and Cu.

The composition ratio of the magnetic metal powder can be determined, for example, by ICP emission spectrometry as specified in JIS G 1258:2014 or spark emission spectrometry as specified in JIS G 1253:2002. Examples of the analysis device include a solid-state optical emission spectrometer (spark optical emission spectrometer, model: SPECTROLAB, type: LAVMB08A) manufactured by SPECTRO and an ICP device (CIROS120 type) manufactured by Rigaku Corporation. In addition, when quantifying the C content, the oxygen gas flow combustion (high-frequency induction heating furnace combustion)-infrared absorption method as specified in JIS G 1211:2018 can be particularly applied. Examples of the carbon content analysis device include a carbon/sulfur analyzer (CS200 type) manufactured by LECO.

In addition, X-ray diffraction can be used to determine whether the structure of magnetic metal powder is amorphous. If no clear diffraction peaks are observed using X-ray diffraction, the structure can be determined to be amorphous.

Regarding the average particle size of the magnetic metal powder in this embodiment, the 50% particle diameter D50 in the particle size distribution is 0.1 μm to 1.5 μm. If the 50% particle diameter D50 is less than 0.1 μm, the magnetic properties per particle become excessively small, and the extraction efficiency of the magnetic beads decreases. Therefore, the 50% particle diameter D50 of the magnetic metal powder is set to 0.1 μm or more. In this way, by setting the 50% particle diameter D50 of the magnetic metal powder to 0.1 μm or more, it is possible to obtain magnetic properties sufficient for performing the extraction and recovery operation of the test target substance using a magnetic field at high speed. For these reasons, the 50% particle diameter D50 is more preferably 0.3 μm or more, and even more preferably 0.5 μm or more.

On the other hand, if the 50% particle diameter D50 of the magnetic metal powder exceeds 1.5 μm and becomes coarse, it is not possible to secure a sufficient specific surface area, and there is a risk that the test target substance such as nucleic acid cannot be sufficiently extracted. In addition, if the 50% particle diameter D50 of the magnetic metal powder becomes excessively large, the weight per particle increases, so that it precipitates at an early stage of the test. As a result, the magnetic metal powder that contributes to the extraction of the test target substance decreases, and the extraction efficiency decreases. Therefore, the 50% particle diameter D50 of the magnetic metal powder is set to 1.5 μm or less. In this way, by setting the 50% particle diameter D50 of the magnetic metal powder to 1.5 μm or less, the amount of extraction of the test target substance can be increased, and for example, the Ct (Threshold Cycle) value during PCR testing can be reduced. The "Ct value" is the cycle number (thermal cycle number) when the amplified product by PCR testing reaches a certain amount and the fluorescent brightness reaches a certain value or more. For these reasons, it is more preferable that the 50% particle size D50 of the magnetic metal powder be 1.2 μm or less, and even more preferable that it be 1.0 μm or less.

In addition, the magnetic metal powder of this embodiment has a ratio D90/D50 of 2.00 or less, which is the ratio of the 90% particle diameter D90 to the 50% particle diameter D50 in the particle size distribution. The 90% particle diameter D90 is the particle diameter at which the integrated value is 90% in the integrated distribution curve obtained from the measurement results of the particle size distribution. In other words, the 90% particle diameter D90 is an index that represents the size of coarse particles mixed in the magnetic metal powder.
In this embodiment, when defining the particle size distribution of the magnetic metal powder, D90/D50, which is the ratio of the particle size of the coarse particles to the 50% particle size D50, which is the average particle size, is used, and this D90/D50 is set to 2.00 or less. When D90/D50 is greater than 2.00, the particle size distribution is one in which a large amount of coarse particles are mixed with respect to the average particle size (D50). Since coarse particles have high magnetism in a magnetic field, when a large amount of coarse particles are mixed, they will aggregate while attracting surrounding particles, and even if the magnetic field is turned off, dispersibility will be lost and significant aggregation will occur. Furthermore, when the particles aggregate, they will settle to the bottom of the reagent due to their own weight, which may lead to a decrease in extraction efficiency and an increase in the length of the test time. Therefore, D90/D50 is set to 2.00 or less, more preferably 1.75 or less, and even more preferably 1.55 or less.

The 50% particle diameter D50 and the 90% particle diameter D90 can be determined, for example, by measuring the volumetric particle size distribution using a laser diffraction/dispersion method and then from the cumulative distribution curve obtained from this particle size distribution. Specifically, in the cumulative distribution curve, the particle diameter at which the cumulative value is 50% from the small diameter side is the 50% particle diameter D50 (median diameter), and the particle diameter at which the cumulative value is 90% from the small diameter side is the 90% particle diameter D90.

The shape of the magnetic metal powder in this embodiment is not particularly limited, and may be a circular, elliptical or polygonal cross-sectional shape. From the viewpoint of suppressing powder aggregation and improving the mobility of the magnetic metal powder, it is preferable that the ratio of particles with a circularity of 0.60 or less in the magnetic metal powder is 2.0% or less. If the particles with a circularity of 0.60 or less exceed 2.0%, the density of the magnetic field lines formed by the particles becomes non-uniform due to the contribution of shape magnetic anisotropy in the magnetized particles, and as a result, the aggregation of the magnetic metal powder becomes significant. Furthermore, such aggregation reduces the mobility of the magnetic metal powder.
The circularity of the magnetic metal powder is defined by the following formula.

Circularity=4πS/ ^L2 (1)
In the above formula, S represents the projected area of the particle, and L represents the circumferential length of the particle.

The circularity of the magnetic metal powder can be measured by the following image processing.
By processing images of multiple magnetic metal powder particles taken with a scanning electron microscope (SEM) or optical microscope, the area and perimeter of each powder particle can be calculated. Furthermore, the ratio of powder particles having a specific circularity among multiple powder particles can be calculated. Specifically, for example, the projected area, perimeter, and ratio can be measured using the image processing system "Image-J" (National Institutes of Health, USA).

In addition, the saturation magnetization of the magnetic metal powder in this embodiment is preferably 1.1 T or more. "Saturation magnetization" is the value of magnetization that becomes saturated when the applied magnetic field is strong. Therefore, the greater the saturation magnetization of the magnetic metal powder, the more fully it can function as a magnetic material, and the movement speed (recovery speed) after extraction in a magnetic field can be improved. As a result, the inspection time can be shortened. In order to achieve this effect, the saturation magnetization of the magnetic metal powder is preferably 1.1 T or more, and more preferably 1.2 T or more. The upper limit of the saturation magnetization of the magnetic metal powder is not particularly limited, and may be 2.2 T or less from the viewpoint of ease of material selection suitable for the balance between performance and cost.

The saturation magnetization of the magnetic metal powder can be measured using a vibrating sample magnetometer (VSM). Specifically, it can be measured using a model such as "TM-VSM1230-MHHL" manufactured by Tamagawa Seisakusho Co., Ltd.

In addition, the coercive force Hc of the magnetic metal powder in this embodiment is preferably 1500 A/m or less. "Coercive force Hc" refers to the value of the external magnetic field in the opposite direction required to return a magnetized magnetic body to an unmagnetized state. In other words, coercive force Hc means resistance to external magnetization. The smaller the coercive force Hc of the magnetic metal powder, the less likely the beads are to aggregate even when switching from a state in which a magnetic field (magnetic field) is applied to a state in which it is not applied, and the beads can be uniformly dispersed in the test solution. Furthermore, even when the application of a magnetic field is repeatedly switched, the smaller the coercive force Hc, the better the redispersibility of the magnetic metal powder is, so that the aggregation of the beads can be further suppressed. In order to obtain such an effect, the coercive force Hc of the magnetic metal powder is preferably 1500 A/m or less, and more preferably 800 A/m or less. The lower limit of the coercive force Hc of the magnetic metal powder is not particularly limited, and may be 5 A/m or more from the viewpoint of ease of material selection suitable for the balance between performance and cost.

Although the magnetic metal powder has been described above, the manufacturing method of the magnetic metal powder constituting the magnetic beads of this embodiment is not particularly limited, and the manufacturing method may be selected according to the desired composition and shape. For example, the magnetic metal powder is preferably an Fe-based metal alloy powder produced by an atomization method, which is easy to obtain spherical powder.

<Coating Layer>
The coating layer is formed on the surface of the magnetic metal powder. The coating layer may be formed on at least a part of the surface of the magnetic metal powder, but is preferably formed so as to cover the entire surface.

The coating layer of this embodiment may be made of any material that can provide the extraction performance (adsorption performance) of the test target substance such as nucleic acid. Silicon oxide is, for example, SiOx (0<x≦2), and specifically, _SiO2 is preferable. Silicon oxide specifically adsorbs nucleic acid in an aqueous solution containing a chaotropic substance, thereby enabling the extraction and recovery of the nucleic acid. A "chaotropic substance" is a substance that has the effect of increasing the water solubility of hydrophobic molecules by generating chaotropic ions (monovalent anions with a large ionic radius) in an aqueous solution, and contributes to nucleic acid adsorption. Specific chaotropic substances include guanidine hydrochloride, sodium iodide, sodium perchlorate, etc.

The coating layer may also include a composite oxide or compound of silicon and one or more elements selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, and Zr. These elements Al, Ti, V, Nb, Cr, Mn, and Zr are elements with excellent resistance to elution, which suppresses ion elution from the magnetic metal powder to be coated. Therefore, by using a composite oxide or compound of at least one of these elements and silicon as the coating layer, it is possible to improve the extraction performance of the test substance while ensuring elution resistance. The coating layer may also be formed of multiple layers made of oxides of different elements. Furthermore, a coating layer that can achieve the same effect may be formed of an inorganic substance such as silicate, or an organic substance such as organosiloxane and phosphonic acid.

The coating layer may contain unavoidable impurities as long as the effects of the present invention are not impaired. For example, when silicon oxide is used as the coating layer, examples of unavoidable impurities in the silicon oxide include C, N, and P.

The average thickness of the coating layer is preferably 1 nm to 100 nm. If the average thickness of the coating layer is less than 1 nm, the elution of ions from the magnetic metal powder to be coated cannot be sufficiently suppressed, and the extraction performance of the magnetic metal powder is impaired. On the other hand, if the average thickness of the coating layer exceeds 100 nm, the extraction performance of the test target substance will saturate and the manufacturing costs will increase. Therefore, it is more preferable that the average thickness of the coating layer is 3 nm to 50 nm.

The thickness of the coating layer can be measured, for example, from a cross-sectional observation image obtained by a transmission electron microscope (TEM). In this embodiment, the thickness of each coating layer is measured for 10 or more particles, and the average value is calculated. The thickness of the coating layer of each particle is measured at 5 or more points per particle, and the average value is calculated.

The composition of the coating layer can be confirmed, for example, by EDX analysis, Auger electron spectroscopy, etc. For example, the composition of the coating layer can be confirmed by measuring the radial composition distribution of the particles through EDX analysis of the formed coating layer.

In the magnetic beads of this embodiment, the coating layer is formed on at least a portion of the surface of the magnetic metal powder. This configuration improves the amount and efficiency of extraction of the test target substance, and thereby shortens the test time.

According to the magnetic beads described above, by setting the average particle size (D50) and particle size distribution (D90/D50) of the magnetic metal powder within an appropriate range, it is possible to improve the amount and efficiency of extraction of the test target substance, and also to reduce the Ct value during PCR testing. Furthermore, by setting a particle size distribution that reduces the proportion of coarse particles, it is possible to significantly suppress particle aggregation and sedimentation, and further improve the redispersibility of the particles.

[Method of manufacturing magnetic beads]
Next, a method for producing the above magnetic beads will be described.
The method for producing magnetic beads according to this embodiment includes a magnetic metal powder production step of obtaining magnetic metal powder, an in-liquid classification step of classifying the magnetic metal powder in liquid to extract magnetic metal powder having a 50% particle diameter D50 of 0.1 to 1.5 μm and a D90/D50 ratio, which is the ratio of the 90% particle diameter D90 of the magnetic metal powder to the 50% particle diameter D50 in the particle size distribution, of 2.00 or less, and a coating step of forming a coating layer on the magnetic metal powder.

<Magnetic metal powder production process>
First, magnetic metal powder is prepared. The magnetic metal powder is formed by using the above-mentioned magnetic metal powder material, for example, by various powdering methods such as atomization (for example, water atomization, gas atomization, high-speed rotating water flow atomization, etc.) and pulverization. Among them, it is preferable to use the atomization method because it is easy to obtain spherical powder.

Depending on the type of coolant and the configuration of the equipment, atomization methods can be divided into water atomization, high-pressure water atomization, high-speed rotating water flow atomization, gas atomization, etc. The atomization method is a method for producing metal powder (amorphous alloy powder) by colliding molten metal (molten metal) with a fluid (liquid or gas) sprayed at high speed to pulverize and cool it. By producing amorphous alloy powder using this type of atomization method, extremely fine powder can be produced efficiently. Furthermore, the particle shape of the amorphous alloy powder becomes close to spherical due to the action of surface tension.

In the high-pressure water atomization method, the molten metal is first split in the air by a large negative pressure, forming fine molten metal droplets. These fine molten metal droplets are then rapidly cooled and solidified by a high-speed water jet, resulting in a rapidly cooled, fine powder that is nearly spherical. For this reason, the high-pressure water atomization method is the most suitable method for producing magnetic metal powder. In addition, since the molten metal can be cooled extremely quickly, the disordered atomic arrangement of the molten metal can be maintained to a high degree during solidification. This makes it possible to efficiently produce amorphous alloy powder with a particularly high degree of amorphization (alloy powder that is more reliably amorphous). Therefore, the high-pressure water atomization method is preferred as a method for forming magnetic metal powder.

After the magnetic metal powder production process, a submerged classification process or a coating process is performed. That is, in this embodiment, the order of the submerged classification process and the coating process does not matter, and after the magnetic metal powder production process, the submerged classification process may be performed before the coating process, or the coating process may be performed before the submerged classification process.
The following describes a case where the coating step is carried out after the magnetic metal powder production step and then the submerged classification step.

<Submerged classification process>
In this embodiment, by classifying the magnetic metal powder in a liquid, a magnetic metal powder having a 50% particle size D50 of 0.1 to 1.5 μm and a D90/D50 of 2.00 or less can be obtained. In order to obtain a magnetic metal powder having such a desired particle size and particle size distribution, the classification is performed in a liquid, that is, a so-called wet classification. While dry classification classifies particles by utilizing differences in mechanical behavior in an air stream, wet classification performed in a liquid classifies particles by centrifugal force, gravity, or the like in the liquid. Therefore, wet classification performed in a liquid can classify particles with high accuracy even at sizes on the order of submicrons, and can also suppress the aggregation of particles during classification more than dry classification.

Specifically, in the submerged classification process, classification is preferably performed by centrifugal force or gravity. High classification accuracy can be obtained in either a centrifugal field or a gravitational field. From the viewpoint of more precise classification, classification by gravity is preferred.

In the case of classification by gravity, the settling speed in liquid varies depending on the particle size (particle diameter), and for example, an upright cylindrical wet classifier can be used. In addition, the settling speed for each particle size (particle diameter) is calculated in advance, and powder can be collected from the classifier according to the settling time to obtain metal powder with the desired particle diameter. Note that a dispersant such as a carboxylic acid may be used to suppress aggregation of particles in the liquid.

<Coating process>
After the submerged classification process, a coating layer is formed on the surface of the magnetic metal powder. The method of forming the coating layer is not particularly limited as long as it is a means that realizes the above-mentioned material and average thickness of the coating layer. For example, a wet formation method such as a sol-gel method, an ALD (Atomic Layer Deposition), a CVD (Chemical Vapor Deposition), a dry formation method such as ion plating, etc. can be mentioned. When the coating layer is a silicon oxide film, among these, the Stöber method, which is a kind of sol-gel method, can be used.

The Stober process is a method for forming monodisperse particles by hydrolysis of metal alkoxides. When the coating layer is made of silicon oxide, it is obtained by the hydrolysis reaction of silicon alkoxide using the Stober process.

Specifically, first, the magnetic metal powder is dispersed in an alcohol solution containing silicon alkoxide. Examples of the alcohol solution include lower alcohols such as ethanol and methanol. The ratio of silicon alkoxide to alcohol is, for example, 10 to 50 parts by weight of alcohol mixed with 1 part by weight of tetraethoxysilane. In order to achieve a uniform coating on the particle surface, the ratio of the magnetic metal powder to the silicon alkoxide is preferably 0.01 to 0.1 parts by weight of silicon alkoxide mixed with 1 part by weight of magnetic metal powder. In addition, it is preferable to use TEOS (tetraethoxysilane, Si(OC ₂ H ₅ ) ₄ ) as the silicon alkoxide.

Next, ammonia water is supplied as a catalyst to promote the reaction, causing hydrolysis. This causes a dehydration condensation reaction between the hydrolyzates themselves and between the hydrolyzates and the silicon alkoxide, forming -Si-O-Si- bonds on the particle surface, resulting in the formation of a silicon oxide film.

It is preferable to stir the magnetic metal powder and the alcohol solution using an ultrasonic wave application device before and after supplying the ammonia water. By stirring in each step in this way, it is possible to promote uniform dispersion of the particles and form a uniform silicon oxide film on the particle surface. It is preferable to stir for a time that is sufficient for the hydrolysis reaction of the silicon alkoxide to proceed sufficiently.

In the above, the magnetic metal powder is dispersed in an alcohol solution containing silicon alkoxide, and then ammonia water is supplied, but this is not limited to the above. For example, the alcohol solution in which the magnetic metal powder is dispersed may be mixed with ammonia water, and then the alcohol solution containing silicon alkoxide may be mixed. In such a case, the alcohol solution containing silicon alkoxide may be added in several portions. When adding in several portions, the above-mentioned stirring may be performed after each addition, or the alcohol solution may be added to the solution while stirring.

The thickness of the coating layer is also affected by the ratio of silicon alkoxide in the solution. In other words, if the ratio of silicon alkoxide in the solution is increased, the thickness of the coating layer will increase, but if the ratio is increased too much, there is a risk that excess silica will form alone. Therefore, the ratio of silicon alkoxide in the solution is adjusted so that the desired thickness of the coating layer is obtained.

The magnetic beads of this embodiment can be manufactured by the above steps, but the obtained magnetic beads may be subjected to a heat treatment to further improve their performance. For example, by drying and baking at 60 to 120°C for 10 to 300 minutes, it is possible to remove any hydrates remaining in the beads and improve the strength of the beads.

In addition, in the manufacturing method of magnetic beads of this embodiment, a classification process may be performed between the magnetic metal powder production process and the submerged classification process. In other words, after the magnetic metal powder is produced by the magnetic metal powder production process, most of the coarse particles are classified and removed by a dry or wet method, and then the submerged classification is performed, thereby further improving the classification accuracy.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Alloy powder (magnetic metal powder) was produced by high-pressure water atomization from an alloy having the composition shown in Table 1. The obtained alloy powder was classified by each classification method shown in Table 1.

The conditions for gravity classification in liquid in Example 1 will be described.
First, 30 g of the alloy powder having an average particle size (D50) of about 3 μm was dispersed in 400 mL of pure water by ultrasonic waves to prepare a raw powder dispersion. Next, this raw powder dispersion was slowly poured into 1600 mL of pure water to prepare a slurry, which was then left to stand for 330 minutes and classified. After that, 600 mL of the slurry was collected from the liquid surface using a siphon. The collected slurry was heated and dried at 85° C. for 120 minutes to evaporate the water and obtain a powder.

Next, the conditions for centrifugal classification in liquid in Example 2 will be described.
As in Example 1, the alloy powder having an average particle size of about 3 μm was dispersed in water at a weight content of 7%, and the dispersion was classified using a wet rotary classifier to obtain a dispersion containing powder having the average particle size shown in the table. This was then dried by heating at 85° C. for 120 minutes to obtain a powder.

In the other examples in the table, the submerged gravity classification and submerged centrifugal classification were performed in the same manner as in Examples 1 and 2, respectively.

In addition, in the air classification of Comparative Examples 1 and 2, the alloy powders with average particle sizes of approximately 10 μm and approximately 3 μm, respectively, were classified using a cyclone classifier, and the powders were obtained by adjusting the classification point.

After classification, the alloy powder was measured for D50, D90, D90/D50, and the proportion of beads with a circularity of 0.60 or less using the same method as above. The results are shown in Table 2.

Next, a silicon oxide (SiO ₂ ) film (average thickness: about 20 nm) was formed on the surface of the classified alloy powder by the following method, to obtain magnetic beads.

First, 100 g of the alloy powder sample was dispersed and mixed in 950 mL of ethanol, and the mixture was stirred for 20 minutes using an ultrasonic wave application device. After stirring, a mixed solution of 30 mL of pure water and 180 mL of ammonia water was added and stirred for another 10 minutes. Then, a mixed solution of 3.3 mL of tetraethoxysilane and 100 mL of ethanol was further added and stirred for 120 minutes, forming a silicon oxide film on the surface of the alloy powder and producing magnetic beads.
The magnetic beads were washed with ethanol and acetone, respectively, and then dried at 65° C. for 30 minutes and baked at 200° C. for 90 minutes.

The magnetic beads obtained by the above method were evaluated for Ct value, redispersibility, and the presence or absence of sedimentation (sedimentation). In addition, the magnetic properties (coercivity and saturation magnetization) of the obtained magnetic beads were measured using a vibrating sample magnetometer (VSM).

The Ct value is the cycle number in PCR when a certain amount of amplified product is produced and the fluorescence brightness reaches or exceeds a certain value. In other words, the smaller the Ct value (the smaller the cycle number), the higher the extraction efficiency of the substance being tested, indicating that the testing time can be shortened.
In this example, the magnetic beads were first used to carry out a conventional nucleic acid extraction reaction (pretreatment), and the resulting extract was added to a PCR reaction solution to carry out a real-time PCR reaction to determine the Ct value. PCR was carried out under the same conditions for each magnetic bead using Escherichia coli DNA as the target nucleic acid.

Regarding redispersibility, the redispersibility of the magnetic beads after the application of a magnetic field was repeated seven times as a nucleic acid extraction step was judged visually.
As for sedimentation properties, the presence or absence of sedimentation 30 minutes after the stirring treatment in the nucleic acid extraction process was also visually evaluated.

The above evaluation results are shown in Table 2.
As shown in Table 2, it was confirmed that the magnetic properties were excellent in both the magnetic beads of Examples 1 and 2. In addition, the magnetic beads of Examples 1 and 2 both had smaller Ct values than the comparative example, and were excellent in both redispersibility and sedimentation properties, and it was confirmed that the extraction speed and extraction efficiency of nucleic acid were significantly improved.

Claims (12)

A magnetic bead having a magnetic metal powder and a coating layer that coats the surface of the magnetic metal powder,
The magnetic metal powder has a 50% particle diameter D50 of 0.1 to 1.5 μm in particle size distribution;
The ratio D90/D50 of the 90% particle diameter D90 to the 50% particle diameter D50 of the magnetic metal powder in the particle size distribution is 2.00 or less,
The magnetic metal powder has a coercive force Hc of 1500 A/m or less and a saturation magnetization of 1.1 T or more .
A magnetic bead characterized by: 2. The magnetic beads according to claim 1 , wherein the ratio of particles having a circularity of 0.60 or less in the magnetic metal powder is 2.0% or less. 3. The magnetic beads according to claim 1, wherein the magnetic metal powder is an Fe-based alloy powder produced by an atomization method. The magnetic bead according to any one of claims 1 to 3, characterized in that the coating layer is made of 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 and Zr . 5. The magnetic bead according to claim 1, wherein the coating layer has a thickness of 1 nm to 100 nm. a magnetic metal powder producing step for obtaining magnetic metal powder;
a liquid classification process for classifying the magnetic metal powder in a liquid to extract magnetic metal powder having a 50% particle diameter D50 of 0.1 to 1.5 μm and a ratio D90/D50 of the 90% particle diameter D90 of the magnetic metal powder to the 50% particle diameter D50 in a particle size distribution of 2.00 or less;
A coating step of forming a coating layer on the magnetic metal powder;
having
A method for producing magnetic beads , wherein the magnetic metal powder has a coercive force Hc of 1500 A/m or less and a saturation magnetization of 1.1 T or more . 7. The method for producing magnetic beads according to claim 6 , wherein the submerged classification step classifies the magnetic beads by centrifugal force or gravity. 8. The method for producing magnetic beads according to claim 7 , wherein in the magnetic metal powder producing step, the magnetic metal powder is produced by an atomization method. The method for producing magnetic beads described in claim 7 or 8, characterized in that the coating layer is made of 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 and Zr. 10. The method for producing magnetic beads according to claim 7, wherein in the coating step, the coating layer is formed to a thickness of 1 nm to 100 nm. The method for producing magnetic beads according to any one of claims 7 to 10 , characterized in that a dispersant is used in the submerged classification step. 12. The method for producing magnetic beads according to claim 7, further comprising a classification step between the magnetic metal powder production step and the submerged classification step.