Magnetic bead reagent

Abstract

To provide a magnetic bead reagent that has excellent dispersibility of magnetic beads and suppresses the elution of Fe ions due to ultrasonic treatment.SOLUTION: A magnetic bead reagent contains: magnetic beads containing Fe-based metal soft magnetic particles and silica films having an average thickness of 3 nm or more and covering the Fe-based metal soft magnetic particles; a surfactant; and a dispersion medium in which the magnetic beads are dispersed. The surfactant preferably should be a nonionic surfactant.SELECTED DRAWING: Figure 2

Description

Technical Field

[0007] ,

[0001] The present invention relates to magnetic bead reagents.

Background Art

[0002] As a method for extracting target molecules such as proteins, antibodies, peptides, and nucleic acids, a magnetic bead separation method is known. Since the magnetic bead separation method is a method of separating and recovering beads by magnetic force, a rapid separation operation is possible.

[0003] For example, Patent Document 1 discloses extracting nucleic acids from a blood sample using highly magnetized magnetic beads having a core of metallic Fe and a silica coating, adsorbing a target biological substance on the bead surface, and separating and collecting the nucleic acids by combination with a permanent magnet, and enabling automation of nucleic acid extraction.

[0004] Also, it is disclosed that highly magnetized magnetic beads are dispersed in advance in a buffer solution or a polar organic solvent. And it is disclosed that by performing such an operation, the amount of recovered nucleic acids is improved.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the nucleic acid extraction method described in Patent Document 1, when highly magnetized magnetic beads are dispersed in advance, there is a risk of aggregation. When aggregation of highly magnetized magnetic beads occurs, the redispersibility decreases, leading to a decrease in efficiency in washing the highly magnetized magnetic beads adsorbed with nucleic acids and eluting the nucleic acids.

[0007] Furthermore, if aggregation of highly magnetized magnetic beads occurs, friction between the beads during ultrasonic agitation can damage the silica coating. When the silica coating is damaged, the core is exposed, causing Fe ions to leach out. These leached Fe ions reduce the amount of nucleic acid recovered. [Means for solving the problem]

[0008] The magnetic bead reagent according to the application example of the present invention is Fe-based metal soft magnetic particles, and a coating with an average thickness of 3 nm over the Fe-based metal soft magnetic particles. Above 40nm A silica film comprising Average particle size between 0.8 μm and 5.0 μm Magnetic beads and, Nonionic surfactants and, A dispersion medium for dispersing the magnetic beads, Contains Magnetic bead reagent, The content of the nonionic surfactant in the magnetic bead reagent is 0.05% by mass or more and 0.1% by mass or less. The magnetic beads have a coercivity Hc of 1500 A / m or less. It is characterized by the following: [Brief explanation of the drawing]

[0009] [Figure 1] This is a flowchart illustrating an example of a nucleic acid extraction method using magnetic bead reagents. [Figure 2] This is a schematic cross-sectional view showing the magnetic beads contained in the magnetic bead reagent according to the embodiment. [Modes for carrying out the invention]

[0010] Hereinafter, preferred embodiments of the magnetic bead reagent of the present invention will be described in detail with reference to the attached drawings.

[0011] 1. Method for extracting the target molecule First, we will describe an example of a method for extracting a target molecule using magnetic bead reagents. Examples of target molecules include proteins, antibodies, peptides, and nucleic acids. The following explanation will focus on the case where the target molecule is a nucleic acid, but the same principles apply to other target molecules. Note that nucleic acids may exist in biological samples such as cells and tissues, viruses, bacteria, etc. Furthermore, the nucleic acid may be either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).

[0012] Figure 1 is a flowchart illustrating an example of a nucleic acid extraction method using magnetic bead reagent. Figure 2 is a schematic cross-sectional view showing the magnetic beads contained in the magnetic bead reagent according to the embodiment. The nucleic acid extraction method shown in Figure 1 comprises a dispersion step S102, a mixing step S104, a separation step S106, a washing step S108, and an elution step S110.

[0013] 1.1.Dispersion process In the dispersion step S102, the magnetic beads 2 and surfactant shown in Figure 2 are dispersed in the dispersion medium. For this dispersion, for example, ultrasonic irradiation is used. As a result, the magnetic beads 2 are dispersed almost uniformly in the dispersion medium.

[0014] The surfactant reduces the surface tension of the magnetic bead reagent, suppressing the aggregation of magnetic beads 2. This improves the dispersibility of the dispersion medium.

[0015] 1.1.1. Magnetic Beads The magnetic beads 2 shown in Figure 2 comprise Fe-based metal soft magnetic particles 21 and a silica film 22 coating them. The Fe-based metal soft magnetic particles 21 are particles composed of an Fe-based metal and possessing soft magnetism. The silica film 22 is a coating with a hydrophilic surface capable of adsorbing and retaining nucleic acids. Adsorption refers to reversible physical bonding. By providing the silica film 22 with a predetermined thickness, damage to the silica film 22 is suppressed even if friction occurs between the magnetic beads 2 due to ultrasonic irradiation or the like. As a result, the Fe-based metal soft magnetic particles 21 are less likely to be exposed, and the elution of Fe ions and the like can be suppressed.

[0016] Since the magnetic beads 2 have magnetization, they are magnetically attracted by applying an external magnetic field. Therefore, by using the magnetic beads 2, it is possible to selectively separate the magnetic beads 2 adsorbed with nucleic acids, that is, the solid phase, and the liquid phase containing impurities.

[0017] The Fe-based metal is a metal mainly composed of Fe. The main component means that the content ratio of Fe in the Fe-based metal is 50% or more in terms of atomic ratio. Such an Fe-based metal has a higher saturation magnetization than ferrite or the like, and also has higher toughness and hardness. Therefore, it has excellent magnetic separability and good durability. Also, soft magnetism refers to the property of having a low coercive force and a high magnetic permeability.

[0018] In addition to Fe, the Fe-based metal may contain an element that exhibits ferromagnetism alone, such as Ni or Co, and may contain at least one selected from the group consisting of Cr, Nb, Cu, Al, Mn, Mo, Si, Sn, B, C, P, Ti, and Zr according to the target characteristics. In particular, at least one selected from the group consisting of Cr, Nb, Cu, Si, B, C, and P can be preferably contained to form an amorphous metal or a nanocrystalline metal described later. Also, the soft magnetic material may contain unavoidable impurities as long as the effects of the embodiments are not impaired. Unavoidable impurities are impurities that are unintentionally mixed during raw material or manufacturing. Examples of unavoidable impurities include O, N, S, Na, Mg, K, etc.

[0019] Such Fe-based metals are not particularly limited, and examples include pure iron, carbonyl iron, and Fe-Si-Al alloys such as Sendust, Fe-Ni-based, Fe-Co-based, Fe-Ni-Co-based, Fe-Si-B-based, Fe-Si-B-C-based, Fe-Si-B-Cr-C-based, Fe-Si-Cr-based, Fe-B-based, Fe-P-C-based, Fe-Co-Si-B-based, Fe-Si-B-Nb-based, Fe-Si-B-Nb-Cu-based, Fe-Zr-B-based, Fe-Cr-based, Fe-Cr-Al-based Fe-based alloys, etc.

[0020] Furthermore, the Fe-based metal may be an Fe-based amorphous metal or a nanocrystalline metal, or an Fe-based crystalline metal, but Fe-based amorphous metals or Fe-based nanocrystalline metals are preferred. Here, amorphous metals are non-crystalline metals that do not contain crystals, and nanocrystalline metals are metals that contain fine crystals with a grain size of approximately 100 nm. Amorphous metals or nanocrystalline metals have higher toughness and hardness compared to metal oxides, for example, so wear and chipping, and the resulting leaching of metal ions, especially Fe ions, can be suppressed. Also, by using amorphous metals or nanocrystalline metals, the coercivity becomes lower, and as described above, the dispersibility of magnetic beads 2 is improved.

[0021] The metallic structure of Fe-based metal soft magnetic particles 21 can be identified by X-ray diffraction or transmission electron microscopy (TEM) on the Fe-based metal soft magnetic particles 21 before the formation of magnetic beads 2 or silica film 22. More specifically, amorphous metals can be identified by the absence of diffraction peaks originating from metal crystals such as the αFe phase in peak analysis using X-ray diffraction, and by the formation of a so-called halo pattern in the electron diffraction pattern using TEM, where no spots formed by crystals are observed. Nanocrystalline metals consist of a crystalline structure with a particle size of approximately 100 nm or less, which can be confirmed from TEM observation images. More precisely, the average particle size can be calculated from multiple TEM microstructure observation images containing multiple crystals using image processing. Furthermore, the crystal grain size can be estimated from the diffraction peak of the target crystalline phase using the Scherrer method. For larger crystalline structures, the crystal grain size can be observed and measured by methods such as observing the cross-section using an optical microscope or SEM.

[0022] The Fe-based metal soft magnetic particles 21 may be particles manufactured by any method. Examples of manufacturing methods include various atomization methods such as water atomization, gas atomization, and rotary water flow atomization, as well as pulverization methods. Of these, atomization methods yield Fe-based metal soft magnetic particles 21 with a particle shape closer to a perfect sphere. Such Fe-based metal soft magnetic particles 21 contribute to the realization of magnetic beads 2 with a high packing density and a large amount of nucleic acid recovered per unit volume. Furthermore, water atomization and rotary water flow atomization are suitable for obtaining amorphous metals or nanocrystalline metals because they have high rapid cooling rates. Alternatively, the metal powder produced by the above manufacturing method may be classified using various classifiers to adjust the particle size, and the resulting metal powder may be used as Fe-based metal soft magnetic particles 21.

[0023] The lower limit of the saturation magnetization of the magnetic beads 2 is preferably 50 emu / g or higher, and more preferably 100 emu / g or higher. If the lower limit of the saturation magnetization of the magnetic beads 2 is within the above range, a sufficient magnetic attraction force acts on the magnetic beads 2, allowing the magnetic beads 2 to be fixed more securely, and the movement speed of the magnetic beads 2 due to the magnetic field can be improved, thereby shortening the inspection time. On the other hand, the upper limit of the saturation magnetization of the magnetic beads 2 is not particularly limited, but it may be set to 220 emu / g or lower, which is the saturation magnetization of pure iron. This allows for more accurate separation of the liquid phase and the solid phase.

[0024] The saturation magnetization of the magnetic beads 2 is measured, for example, using a vibrating sample magnetometer (VSM). Alternatively, the saturation magnetization of the Fe-based metal soft magnetic particles 21 may be considered as the saturation magnetization of the magnetic beads 2.

[0025] The coercivity of the magnetic beads 2 is preferably 1500 A / m or less, and more preferably 800 A / m or less. Because such magnetic beads 2 have sufficiently low coercivity, they are magnetized only when an external magnetic field is applied, and return to their original state when the application of the external magnetic field is stopped. Therefore, by using such magnetic beads 2, the operability of operations such as magnetic attraction using an external magnetic field and subsequent operations to release magnetic attraction can be improved. Furthermore, because the coercivity is sufficiently low, even if magnetic attraction and release are repeated, aggregation of the magnetic beads 2 is suppressed after the magnetic attraction is released, and the magnetic beads 2 can be uniformly dispersed in the dispersion medium.

[0026] The average particle size of the magnetic beads 2 is preferably 0.05 μm or more and 10.0 μm or less, more preferably 0.10 μm or more and 5.0 μm or less, and even more preferably 0.3 μm or more and 2.0 μm or less. If the average particle size of the magnetic beads 2 is within the above range, the specific surface area of ​​the magnetic beads 2 will be sufficiently large, and the amount of nucleic acid recovered can be increased. If the average particle size of the magnetic beads 2 falls below the lower limit, the magnetic beads 2 will be more prone to aggregation, which may reduce the nucleic acid adsorption efficiency and decrease the amount recovered. On the other hand, if the average particle size of the magnetic beads 2 exceeds the upper limit, the specific surface area of ​​the magnetic beads 2 will be small, which may reduce the amount of nucleic acid recovered. In addition, depending on the magnitude of the magnetic attraction force, the operability of the operation to fix the magnetic beads 2 by magnetic attraction may decrease.

[0027] The average particle size of magnetic beads 2 is determined as the particle size D50, which is the point at which the cumulative size from the smallest diameter side reaches 50% in the volume-based particle size distribution obtained by laser diffraction.

[0028] The constituent material of the silica film 22 is not particularly limited as long as it is a material capable of forming the hydrophilic surface described above, but for example, it is a material containing silicon dioxide. Specifically, examples include silica, silicon-containing glass, diatomaceous earth, etc. Alternatively, it may be a composite material in which the surface of any material is modified with one of these silicon dioxide-containing materials.

[0029] The average thickness of the silica film 22 is set to 3 nm or more, preferably 30 nm or more, and more preferably 40 nm or more. If the average thickness of the silica film 22 is within the above range, the silica film 22 is formed uniformly, so the elution of Fe ions and the like due to ultrasonic irradiation can be suppressed more reliably.

[0030] The upper limit of the average thickness of the silica film 22 is not particularly limited, but considering the ratio of metal in the entire magnetic beads 2, the adhesion of the silica film 22, the saturation of nucleic acid extraction performance, and the increase in the film formation time of the silica film 22, it is preferably 1000 nm or less, and more preferably 100 nm or less.

[0031] The average thickness of the silica film 22 is the average of the film thickness measured at 10 or more locations by observing the cross-section of the magnetic beads 2 with an electron microscope. Preferably, the measurement locations span across different magnetic beads 2.

[0032] The method for forming the silica film 22 is not particularly limited, but examples include wet deposition methods such as the sol-gel method and the Stöber method, which is a type of sol-gel method; dry deposition methods such as chemical vapor deposition (CVD), atomic layer deposition (ALD), and ion plating. Of these, the Stöber method and the ALD method are preferably used as methods for forming the silica film 22.

[0033] The Fe-based metal content in the magnetic beads 2 is preferably 50 volume% or more, more preferably 70 volume% or more, and even more preferably 90 volume% or more. Because such magnetic beads 2 have a sufficiently high Fe-based metal content, a large magnetic attraction force can be obtained even with a small diameter. On the other hand, if the Fe-based metal content falls below the lower limit, the magnetic attraction force will decrease, and the separation between the magnetic beads 2 and the liquid phase may decrease.

[0034] The Fe-based metal content in magnetic beads 2 is calculated based on the area percentage occupied by the Fe-based metal, obtained by observing the cross-section of magnetic beads 2 with an electron microscope. If necessary, elemental mapping may be performed to calculate the area percentage occupied by the Fe-based metal.

[0035] The content of magnetic beads 2 in the magnetic bead reagent is not particularly limited, but is preferably 10% by mass or more and 80% by mass or less, more preferably 20% by mass or more and 50% by mass or less, and even more preferably 30% by mass or more and 45% by mass or less. By setting the content of magnetic beads 2 within the above range, nucleic acids can be recovered efficiently. In addition, it is possible to suppress the significantly increased collision frequency between magnetic beads 2 due to ultrasonic irradiation, etc., and to suppress wear of the silica film 22.

[0036] 1.1.2. Surfactants Examples of surfactants include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants, but nonionic surfactants are preferred. This suppresses the influence of ionic surfactants when analyzing nucleic acids after extraction. As a result, electrophoretic analysis becomes possible, expanding the range of analytical methods available.

[0037] 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 lauryl sulfate (also known as sodium dodecyl sulfate: SDS), sodium N-lauroyl sarcosinate, sodium cholate, and sarcosine. Examples of amphoteric surfactants include phosphatidylethanolamine. These surfactants can be used individually or in combination of two or more.

[0038] The surfactant content in the magnetic bead reagent is preferably above 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. When the surfactant content is above the critical micelle concentration, the surfactant is more likely to form a layer around the magnetic beads 2. This further enhances the effect of suppressing the aggregation of the magnetic beads 2.

[0039] 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 magnetic bead reagent is preferably 0.05% by mass or more and 3.0% by mass or less, regardless of the critical micelle concentration.

[0040] For ultrasonic irradiation, an ultrasonic disperser such as an ultrasonic homogenizer is used. Ultrasonic irradiation generates tiny bubbles (cavitation), which allows for more uniform dispersion of the magnetic beads 2 and the surfactant.

[0041] On the other hand, if the magnetic beads 2 are subjected to impact by ultrasonic irradiation, the silica film 22 may be damaged. In addition to collisions between the magnetic beads 2, cavitation erosion is also a possible cause of damage to the silica film 22. If the silica film 22 is damaged, the Fe-based metal soft magnetic particles 21 may be exposed, and Fe ions and other substances may leach out.

[0042] In contrast, if a surfactant is added to the magnetic bead reagent and the average thickness of the silica film 22 is within the aforementioned range, damage to the silica film 22 due to ultrasonic irradiation is sufficiently suppressed. As a result, the elution of Fe ions and the like is suppressed. Furthermore, aggregation of the magnetic beads 2 is suppressed, and the magnetic beads 2 can be well dispersed in the magnetic bead reagent, thus easily increasing the adsorption efficiency of nucleic acids.

[0043] Furthermore, it is believed that the surfactant suppresses material wear due to cavitation erosion by lowering the surface tension of the magnetic bead reagent. When a surfactant is added, the surface tension of the magnetic bead reagent decreases. A positive correlation is known between surface tension and material wear due to cavitation erosion. Therefore, by lowering the surface tension of the magnetic bead reagent through the action of the surfactant, damage to the silica film 22 due to cavitation erosion can be suppressed.

[0044] The duration of ultrasonic irradiation is not particularly limited, but is preferably between 1 minute and 60 minutes, and more preferably between 5 minutes and 40 minutes.

[0045] 1.1.3.Dispersion medium Examples of dispersion media 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.

[0046] Furthermore, it is preferable to add a preservative to the dispersion medium to improve its long-term storage and preservative effect. Examples of preservatives include sodium azide. The concentration of the added preservative is preferably 0.02% by weight or more and less than 0.1%. Below 0.02% by weight, sufficient effects on long-term storage and preservation cannot be obtained, while above 0.1%, problems such as a decrease in the extraction efficiency of biomolecules arise.

[0047] A buffer solution may also be added for pH adjustment. Examples of buffer solutions include Tris buffer.

[0048] Furthermore, the magnetic bead reagent may contain components other than those mentioned above.

[0049] As described above, the magnetic bead reagent according to the embodiment contains magnetic beads 2, a surfactant, and a dispersion medium for dispersing the magnetic beads 2. The magnetic beads 2 comprises Fe-based metal soft magnetic particles 21 and a silica film 22 with an average thickness of 3 nm or more that coats the Fe-based metal soft magnetic particles 21.

[0050] With this configuration, the magnetic beads 2 are equipped with Fe-based metal soft magnetic particles 21 that have higher magnetization than ferrite, etc., and a silica film 22 of sufficient thickness. Furthermore, the magnetic bead reagent contains a surfactant, which suppresses damage to the silica film 22 caused by ultrasonic irradiation, etc., and suppresses the elution of Fe ions. In addition, the surfactant reduces the surface tension of the magnetic bead reagent, allowing the magnetic beads 2 to be dispersed well. This makes it possible to improve the adsorption efficiency of nucleic acids.

[0051] 1.2.Mixing process In mixing step S104, the sample containing nucleic acids is placed in a container, and the magnetic bead reagent and the dissolution adsorption solution are mixed into this container. As a result, the nucleic acids are adsorbed onto the magnetic beads 2.

[0052] For example, a liquid containing a chaotropic substance is used as the dissolution and adsorption solution. Chaotropic substances generate chaotropic ions in aqueous solutions, increasing the water solubility of hydrophobic molecules and contributing to the adsorption of nucleic acids onto magnetic beads 2. Chaotropic ions are monovalent anions with a large ionic radius. Examples of chaotropic substances include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, and sodium perchlorate. Of these, guanidine thiocyanate or guanidine hydrochloride, which have a strong protein denaturing effect, are preferably used.

[0053] The concentration of the chaotropic substance in the dissolved adsorbent solution varies depending on the chaotropic substance, but is preferably between 1.0 M and 8.0 M. In particular, when guanidine thiocyanate is used, it is preferably between 3.0 M and 5.5 M. Furthermore, when guanidine hydrochloride is used, it is preferably between 4.0 M and 7.5 M.

[0054] The solubilizing adsorbent may contain a surfactant. The surfactant is used to disrupt the cell membrane or denature proteins contained within the cell. While not particularly limited, examples of surfactants include nonionic surfactants such as Triton-type surfactants and Tween-type surfactants, and anionic surfactants such as sodium N-lauroyl sarcosinate. Of these, nonionic surfactants are preferred. This suppresses the influence of ionic surfactants when analyzing nucleic acids after extraction. As a result, electrophoretic analysis becomes possible, expanding the range of analytical methods available.

[0055] The concentration of the surfactant in the dissolved adsorbent is not particularly limited, but is preferably 0.1% by mass or more and 2.0% by mass or less.

[0056] The soluble adsorbent may contain at least one of a reducing agent and a chelating agent. Examples of reducing agents include 2-mercaptoethanol and dithiothreitol. Examples of chelating agents include EDTA (disodium salt dihydrate).

[0057] The concentration of the reducing agent in the dissolved adsorbent is not particularly limited, but is preferably 0.2 M or less. The concentration of the chelating agent in the dissolved adsorbent is not particularly limited, but is preferably 0.2 mM or less. The pH of the dissolved adsorbent is not particularly limited, but it is preferably neutral, between 6 and 8.

[0058] In mixing step S104, the contents of the container are stirred as needed using an ultrasonic homogenizer, vortex mixer, manual shaking, etc. The stirring time is not particularly limited, but is preferably between 5 seconds and 30 minutes.

[0059] 1.3.Separation process In separation step S106, an external magnetic field is applied to the magnetic beads 2 on which nucleic acids are adsorbed, causing magnetic attraction. This moves the magnetic beads 2 to the wall of the container and fixes them in place. As a result, the solid phase magnetic beads 2 and the liquid phase can be separated.

[0060] In separation step S106, while an external magnetic field is applied, the contents of the container are stirred as needed using an ultrasonic homogenizer, vortex mixer, or manual shaking. This increases the probability that the magnetic beads 2 will be magnetically attracted to the external magnetic field.

[0061] Furthermore, after fixing the magnetic beads 2, the container may be accelerated as needed. This allows any liquid adhering to the magnetic beads 2 to be shaken off, thus enabling more precise separation of the solid and liquid phases. The acceleration may be centrifugal acceleration. A centrifuge can be used to apply centrifugal acceleration.

[0062] After separating the magnetic beads 2 from the liquid phase as described above, the liquid phase inside the container is discharged using a pipette or the like while the magnetic beads 2 are fixed to the container wall.

[0063] 1.4. Washing Process In the washing step S108, the magnetic beads 2 on which nucleic acids are adsorbed are washed. Washing is an operation to remove impurities adsorbed on the magnetic beads 2 by bringing the magnetic beads 2 on which nucleic acids are adsorbed into contact with a washing solution and then separating them again.

[0064] Specifically, first, the washing solution is supplied into the container using a pipette or similar device. Then, the magnetic beads 2 and the washing solution are agitated. This causes the washing solution to come into contact with the magnetic beads 2, and the magnetic beads 2 on which nucleic acids are adsorbed are washed. At this time, the external magnetic field may be temporarily removed. This disperses the magnetic beads 2 in the washing solution, thereby further improving the washing efficiency.

[0065] Next, the magnetic beads 2 are fixed in place again, and the cleaning solution is drained. By repeating the supply and draining of the cleaning solution as described above one or more times, the magnetic beads 2 can be cleaned.

[0066] The washing solution is not particularly limited as long as it is a liquid that does not promote the elution of nucleic acids and does not promote the binding of impurities to the magnetic beads 2. Examples include organic solvents such as ethanol, isopropyl alcohol, and acetone, or aqueous solutions thereof, and low-salt aqueous solutions. Examples of low-salt aqueous solutions include buffer solutions. The salt concentration of the low-salt aqueous solution is preferably 0.1 mM to 100 mM, and more preferably 1 mM to 50 mM. The salt used to make the buffer solution is not particularly limited, but salts such as tris, hepes, pipes, and phosphoric acid are preferably used.

[0067] The cleaning solution may contain surfactants such as Triton®, Tween®, or sodium dodecyl sulfate. Furthermore, the pH of the cleaning solution is not particularly limited.

[0068] In the cleaning process S108, the contents of the container are stirred as needed using an ultrasonic homogenizer, vortex mixer, or manual shaking while the cleaning solution is in contact with the magnetic beads 2. This improves cleaning efficiency. The cleaning step S108 may be performed as needed, and may be omitted if cleaning is not required.

[0069] 1.5. Elution process In the elution step S110, nucleic acids are eluted from the magnetic beads 2 on which they are adsorbed. Elution is the process of transferring nucleic acids to the elution solution by bringing the magnetic beads 2 on which the nucleic acids are adsorbed into contact with the elution solution and then separating them again.

[0070] Specifically, first, the eluent is supplied into the container using a pipette or similar device. Then, the magnetic beads 2 and the eluent are stirred. This causes the eluent to come into contact with the magnetic beads 2, allowing the nucleic acids to be eluted. At this time, the external magnetic field may be temporarily removed. This disperses the magnetic beads 2 in the eluent, thereby further increasing the elution efficiency.

[0071] Next, the magnetic beads 2 are fixed again, and the eluate containing the eluted nucleic acid is drained. This allows the nucleic acid to be recovered.

[0072] The eluent is not particularly limited as long as it is a liquid that promotes the elution of nucleic acids from the magnetic beads 2 on which the nucleic acids are adsorbed. For example, in addition to water such as sterile water or pure water, an aqueous solution containing TE buffer, i.e., 10 mM Tris-HCl buffer and 1 mM EDTA, with a pH of 8, is preferably used.

[0073] The eluate may contain surfactants such as Triton®, Tween®, or sodium dodecyl sulfate.

[0074] In the elution step S110, the eluate is brought into contact with the magnetic beads 2 on which nucleic acids are adsorbed, and the contents of the container are stirred as needed using an ultrasonic homogenizer, vortex mixer, or manual shaking. This can improve the elution efficiency.

[0075] Furthermore, in the elution step S110, the eluate may be heated. This can promote the elution of nucleic acids. The heating temperature of the eluate is not particularly limited, but is preferably 70°C to 200°C, more preferably 80°C to 150°C, and even more preferably 95°C to 125°C.

[0076] Examples of heating methods include supplying a preheated eluate or supplying an unheated eluate to a container and then heating it. The heating time is not particularly limited, but it is preferably between 30 seconds and 10 minutes.

[0077] The elution step S110 may be performed as needed, and may be omitted if, for example, the only purpose is the separation of the magnetic beads 2 from the liquid phase in the separation step S106.

[0078] Although the magnetic bead reagent of the present invention has been described above based on the illustrated embodiment, the present invention is not limited thereto. For example, the magnetic bead reagent of the present invention may be in which each part of the above embodiment is replaced with any configuration having a similar function, or any configuration may be added to the above embodiment. [Examples]

[0079] Next, specific embodiments of the present invention will be described. 2. Recovery of nucleic acids 2.1. Example 1 First, the magnetic bead reagent in a container was subjected to ultrasonic irradiation for 30 minutes to disperse the magnetic beads. The mass of the magnetic beads used was 23 mg. Next, the magnetic bead reagent was mixed with the E. coli DNA and the lysis adsorbent. The contents of the container were then stirred for 10 minutes using a vortex mixer. The E. coli DNA content relative to the volume of the contents of the container was 20 ng / μL.

[0080] Next, with the magnetic beads magnetically separated, the liquid phase was discharged and replaced with a cleaning solution. The contents of the container were then agitated and washed. The cleaning solutions used were an 8M guanidine hydrochloride aqueous solution and a 70% ethanol aqueous solution. The former cleaning solution was used for two washes, followed by the latter cleaning solution for two washes.

[0081] Next, with the magnetic beads magnetically separated, the liquid phase was drained and replaced with an eluent. The contents of the container were then stirred to elute the nucleic acids. Pure water was used as the eluent. Next, the eluate was drained while the magnetic beads were magnetically separated. This allowed for the recovery of nucleic acids from the sample.

[0082] 2.2. Examples 2-9 Nucleic acids were recovered from the sample in the same manner as in Example 1, except that the composition of the magnetic bead reagent was changed as shown in Table 1.

[0083] 2.3. Comparative Example 1 Nucleic acids were recovered from the sample in the same manner as in Example 1, except that the addition of surfactants was omitted.

[0084] 2.4. Comparative Example 2 Nucleic acids in the sample were recovered in the same manner as in Example 2, except that the average thickness of the silica film was set to 1 nm.

[0085] 2.5. Comparative Example 3 Nucleic acids were recovered from the sample in the same manner as in Example 3, except that the addition of surfactants was omitted.

[0086] 3. Evaluation of recovered nucleic acids 3.1. Concentration of recovered nucleic acids For each example and comparative example, the amount of nucleic acid recovered using the magnetic bead reagent was measured using a real-time PCR instrument manufactured by Thermo Fisher Scientific Inc., and the concentration of nucleic acid in the eluate was calculated. The calculated concentration was then evaluated according to the following evaluation criteria.

[0087] AA: The concentration of the recovered nucleic acid is 12 ng / μL or higher. A: The concentration of the recovered nucleic acid is between 8 ng / μL and 12 ng / μL. B: The concentration of the recovered nucleic acid is 4 ng / μL or higher and less than 8 ng / μL. C: The concentration of the recovered nucleic acid is less than 4 ng / μL. The evaluation results are shown in Table 1.

[0088] 3.2. Suitability of analysis by electrophoresis The suitability of the nucleic acids recovered using the magnetic bead reagents in each example and comparative example for analysis by electrophoresis was evaluated according to the following evaluation criteria. Suitable: Electrophoretic analysis is appropriate. Unsuitable: Analysis by electrophoresis is unsuitable. The evaluation results are shown in Table 1.

[0089] [Table 1]

[0090] As shown in Table 1, it was confirmed that nucleic acids could be recovered at high concentrations by using the magnetic bead reagents of each example. In particular, it was found that the inclusion of a nonionic surfactant in the magnetic bead reagents enabled analysis by electrophoresis. Furthermore, no increase in Fe ions was observed in the magnetic bead reagents of each example even after ultrasonic irradiation.

[0091] In contrast, the magnetic bead reagents in Comparative Examples 1 and 3 did not contain surfactants, resulting in a low concentration of nucleic acids recovered using them. Furthermore, the magnetic bead reagent in Comparative Example 2 had a thin average silica film thickness in the magnetic beads, leading to a low concentration of nucleic acids recovered using it.

[0092] Furthermore, an increase in Fe ions was observed in the magnetic bead reagents of each comparative example after ultrasonic irradiation. Therefore, the low concentration of recovered nucleic acids is thought to be due to the influence of eluted Fe ions. In addition, the nucleic acids recovered using the magnetic bead reagents of each comparative example could not be analyzed by electrophoresis due to the effect of Fe ion elution. [Explanation of Symbols]

[0093] 2…Magnetic beads, 21…Fe-based metal soft magnetic particles, 22…Silica film, S102…Dispersion step, S104…Mixing step, S106…Separation step, S108…Washing step, S110…Elution step

Claims

1. Magnetic beads with an average particle size of 0.8 μm to 5.0 μm, comprising Fe-based metal soft magnetic particles and a silica film with an average thickness of 3 nm to 40 nm coating the Fe-based metal soft magnetic particles, Nonionic surfactants, A dispersion medium for dispersing the magnetic beads, A magnetic bead reagent containing, The content of the nonionic surfactant in the magnetic bead reagent is 0.05% by mass or more and 0.1% by mass or less. The magnetic beads have a coercivity Hc of 1500 A / m or less. A magnetic bead reagent characterized by the following features.

2. The magnetic bead reagent according to claim 1, wherein the content of the nonionic surfactant is equal to or greater than the critical micelle concentration of the nonionic surfactant.

3. The magnetic bead reagent according to claim 1 or 2, wherein the saturation magnetization of the magnetic beads is 50 emu / g or more and 220 emu / g or less.

4. The magnetic bead reagent according to any one of claims 1 to 3, wherein the Fe-based metal soft magnetic particles include an Fe-based amorphous metal or an Fe-based nanocrystalline metal.

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