Magnetic bead separation method and magnetic bead separation device

Abstract

To provide a magnetic bead separation method, a magnetic bead separation device and a sample tube which suppress carry-over of liquid by selectively separating magnetic beads.SOLUTION: A magnetic bead separation method includes the steps of: storing mixed liquid containing magnetic beads which has Fe group metal soft magnetic particles and a film covering the Fe group metal soft magnetic particles and has saturation magnetization of 50 emu / g or more and 250 emu / g or less, and liquid containing an objective molecule in a container, and adsorbing the objective molecule to the magnetic beads; applying an external magnetic field to the container, and magnetically attracting at least a part of the magnetic beads by the external magnetic field; imparting acceleration to the container in the state where the magnetic beads are magnetically attracted by the external magnetic field, and desorbing the liquid attached to the magnetic beads.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a magnetic bead separation method. and Magnetic Bead Separator Place It is related to. [Background technology]

[0002] Magnetic bead separation is a known method for extracting target molecules such as proteins, antibodies, peptides, and nucleic acids. Magnetic bead separation uses magnetic force to separate and recover beads, allowing for rapid separation.

[0003] For example, Patent Document 1 discloses a method for separating phospholipid vesicles, which includes an adsorption step, an aggregation step, a separation step, and a redispersion step. In the adsorption step, cationic magnetic particles complexed with a substance having a cationic functional group by covalent bonding or physical adsorption are mixed with phospholipid vesicles such as viruses to form a conjugate. In the aggregation step, the conjugate is mixed with an aggregating agent to obtain a water-insoluble complex. In the separation step, a pellet of the complex is formed by magnetic separation, and the supernatant is removed. In the redispersion step, the pellet is dispersed in a liquid. This separation method allows viruses and the like to be easily separated and also reduces the effects of inhibitors of virus diagnosis. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-112904 Summary of the Invention [Problem to be solved by the invention]

[0005] In the separation method described in Patent Document 1, when the supernatant is removed in the separation step, a portion of the supernatant tends to remain inside or on the surface of the pellet of the complex, i.e., near the surface of the magnetic beads. The remaining supernatant is transferred into the liquid in the redispersion step. This is called carryover. If such carryover occurs, for example, impurities contained in the supernatant also transfer into the liquid in the redispersion step. This may cause the impurities to adversely affect virus diagnosis, etc. [Means for solving the problem]

[0006] A magnetic bead separation method according to an application example of the present invention includes: A mixed liquid containing magnetic beads having Fe-based soft magnetic metal particles and a coating covering the Fe-based soft magnetic metal particles, the magnetic beads having a saturation magnetization of 50 emu / g or more and 250 emu / g or less, and a liquid containing target molecules. The container has a cylindrical body with a bottom and a lid for closing the body. a step of placing the target molecule in a container and adsorbing the target molecule onto the magnetic beads; An external magnetic field is applied to the container, and at least a portion of the magnetic beads are induced by the external magnetic field. To the lid a step of magnetically attracting; The magnetic beads are To the lid When magnetically attracted, The container is rotated around the rotation axis while tilting the container so that the bottom is positioned farther from the rotation axis than the opening of the main body. In the container Centrifugation applying acceleration to remove the liquid adhering to the magnetic beads; The present invention is characterized by having the following.

[0007] A magnetic bead separation method according to an application example of the present invention includes: The magnetic beads have Fe-based soft magnetic metal particles and a coating that coats the Fe-based soft magnetic metal particles, and the saturation magnetization is 50 emu / g or more and 250 emu / g or less. The magnetic beads have a liquid containing target molecules. The magnetic beads are provided with a container mounting section on which a container containing a mixed liquid is mounted, and the container is adapted to apply centrifugal acceleration to the container. Around the axis of rotation A rotating body of revolution; an external magnetic field application unit that applies an external magnetic field to the container; Equipped with 、 the container placement portion has an insertion hole into which the container is inserted, the insertion hole is inclined so that the bottom is located farther from the rotation axis than the opening, the external magnetic field applying unit includes a head unit having a magnet, The head portion is attached to the lid portion of the container inserted into the insertion hole. It is characterized by: [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a cross-sectional view showing a magnetic bead separation device according to an embodiment. [Figure 2] FIG. 2 is an enlarged view of the angle rotor shown in FIG. [Figure 3] FIG. 3 is a cross-sectional view of the magnetic bead shown in FIG. [Figure 4] FIG. 2 is a cross-sectional view illustrating a sample tube according to an embodiment. [Figure 5] 1 is a flowchart illustrating a magnetic bead separation method according to an embodiment. [Figure 6] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. [Figure 7] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. [Figure 8] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. [Figure 9] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. [Figure 10] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. [Figure 11] 1 is a schematic diagram for explaining a magnetic bead separation method according to an embodiment. FIG. DETAILED DESCRIPTION OF THE INVENTION

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the magnetic bead separation method, magnetic bead separation device, and sample tube of the present invention will be described in detail below with reference to the accompanying drawings.

[0011] 1. Magnetic bead separator First, a magnetic bead separation device according to an embodiment will be described.

[0012] FIG. 1 is a cross-sectional view showing a magnetic bead separation device according to an embodiment. In FIG. 1, three mutually orthogonal axes are set: an X axis, a Y axis, and a Z axis. Each axis is represented by an arrow, with the tip end being "plus" and the base end being "minus." In the following description, for example, "X-axis direction" includes both the positive and negative directions of the X axis. In the following description, the positive side of the Z axis may be referred to as "up" and the negative side of the Z axis as "down."

[0013] The magnetic bead separator 1 shown in FIG. 1 includes an angle rotor 11 (rotating body), a motor 12, a drive shaft 13, a rotor chamber 14, an upper door 15, and an external magnetic field application unit 16.

[0014] The rotor chamber 14 is cylindrical and has an upper opening 142 and a bottom 144, and accommodates the angle rotor 11 therein. An upper door 15 is provided at the upper opening 142 of the rotor chamber 14. The upper door 15 can be opened and closed.

[0015] A motor 12 is provided below the rotor chamber 14. The motor 12 and the angle rotor 11 are connected via a drive shaft 13, which is a rotation axis AX extending parallel to the Z axis. The drive shaft 13 passes through a bottom 144 of the rotor chamber 14. The angle rotor 11 is rotated around the rotation axis AX by the motor 12 via this drive shaft 13. Note that the extension direction of the rotation axis AX of the angle rotor 11 is not limited to the Z axis.

[0016] The angle rotor 11 has a plurality of container placement portions 112 on which sample tubes 5 (containers) are placed. The angle rotor 11 is disk-shaped, and has a truncated cone-shaped recess 114 opening upward.

[0017] Fig. 2 is an enlarged view of the angle rotor 11 shown in Fig. 1. Fig. 1 and Fig. 2 are cross-sectional views of the magnetic bead separation device 1 cut along a plane including the Z axis.

[0018] The container mounting portion 112 is an insertion hole that opens into the inner surface of the recess 114 and into which the sample tube 5 is inserted, and has an opening 115 and a bottom 116. The axis of the container mounting portion 112 is defined as an axis A112.

[0019] The axis A112 of the container mounting portion 112 is disposed so as to be inclined with respect to the rotation axis AX. Specifically, the angle θ formed between the axis A112 and the rotation axis AX is set to be greater than 0° so that the bottom 116 is located farther from the rotation axis AX than the opening 115. The angle θ is preferably set to be greater than or equal to 10° and less than or equal to 90°, and more preferably greater than or equal to 30° and less than or equal to 80°.

[0020] When the angle rotor 11 is rotated around the rotation axis AX with the sample tube 5 inserted in the container mounting section 112, centrifugal acceleration toward the outside of the rotation axis AX is applied to the sample tube 5. This centrifugal acceleration allows the sample contained inside the sample tube 5 to undergo centrifugal sedimentation.

[0021] The shape of sample tube 5 is not particularly limited, but in FIG. 2, as an example, sample tube 5 is a cylindrical body with a bottom and a long axis along axis A112, and includes main body 55 having opening 52 and bottom 54, and openable / closable lid 56. Therefore, centrifugal acceleration is applied to sample tube 5 from opening 52 to bottom 54. Note that a container of any shape may be used instead of sample tube 5.

[0022] A mixed liquid 4 containing magnetic beads 2 and a liquid 3 containing target molecules is contained in a sample tube 5. As a result, the target molecules are adsorbed onto the magnetic beads 2. The target molecules that have been transferred to the magnetic beads 2 are then transferred to an elution liquid by an elution operation and recovered.

[0023] Examples of target molecules contained in the liquid 3 include proteins, antibodies, peptides, nucleic acids, etc. In the following explanation, a case where the target molecule is a nucleic acid will be described, but the following explanation also applies to other target molecules.

[0024] Nucleic acids may be present in, for example, biological samples such as cells and biological tissues, viruses, bacteria, etc. Nucleic acids may be DNA (deoxyribonucleic acid) or RNA (ribonucleic acid).

[0025] As will be described later, the magnetic beads 2 are magnetized and are therefore magnetically attracted by the external magnetic field applied from the external magnetic field application unit 16. Furthermore, when centrifugal acceleration is applied to the sample tube 5, the magnetically attracted magnetic beads 2 can be efficiently separated from the liquid 3 that is not magnetically attracted. Therefore, the magnetic bead separation device 1 can combine this magnetic attraction operation with a separation operation using centrifugal acceleration. Furthermore, by using new liquids, such as a washing liquid and an elution liquid, instead of the liquid 3 and performing these magnetic attraction and separation operations, nucleic acids can be efficiently washed and eluted.

[0026] FIG. 3 is a cross-sectional view of the magnetic bead 2 shown in FIG. 3, the magnetic beads 2 include Fe-based metal soft magnetic particles 21 and coatings 22. The Fe-based metal soft magnetic particles 21 are made of an Fe-based metal and are particles having soft magnetism.

[0027] An Fe-based metal is a metal whose main component is Fe. "Main component" means that the Fe content in the Fe-based metal is 50% or more in terms of atomic ratio. Such Fe-based metals have higher saturation magnetization, toughness, and hardness than ferrites. Therefore, they have excellent magnetic separation properties and good durability. Furthermore, "soft magnetism" refers to the properties of low coercive force and high magnetic permeability.

[0028] The Fe-based metal may contain, in addition to Fe, an element that exhibits ferromagnetism by itself, such as Ni or Co, or may contain at least one element selected from the group consisting of Cr, Nb, Cu, Al, Mn, Mo, Si, Sn, B, C, P, Ti, and Zr, depending on the target properties. Furthermore, the soft magnetic material may contain unavoidable impurities to the extent that the effects of the embodiment are not impaired.

[0029] Inevitable impurities are impurities that are unintentionally mixed into raw materials or during manufacturing. Examples of inevitable impurities include O, N, S, Na, Mg, and K.

[0030] Such Fe-based metals are not particularly limited, but examples thereof include pure iron, carbonyl iron, Fe-Si-Al-based alloys such as sendust, and Fe-based alloys such as Fe-Ni-based, Fe-Co-based, Fe-Ni-Co-based, Fe-Si-B-based, Fe-Si-BC-based, Fe-Si-B-Cr-C-based, Fe-Si-Cr-based, Fe-B-based, Fe-PC-based, Fe-Co-Si-B-based, Fe-Si-B-Nb-based, Fe-Si-B-Nb-Cu-based, Fe-Zr-B-based, Fe-Cr-based, and Fe-Cr-Al-based alloys.

[0031] The Fe-based metal may be either an amorphous metal or a crystalline metal, but amorphous metals are preferred because amorphous metals have high toughness and hardness, which can suppress wear, chipping, and the resulting elution of metal ions.

[0032] The saturation magnetization of the magnetic beads 2 is set to 50 emu / g or more and 250 emu / g or less, preferably 100 emu / g or more, and more preferably 100 emu / g or more and 200 emu / g or less. If the saturation magnetization of the magnetic beads 2 is within this range, it is possible to prevent the magnetic beads 2 fixed by the external magnetic field from falling off when the liquid 3 adhering to the magnetic beads 2 is released by a separation operation using centrifugal acceleration. This makes it possible to further increase the accuracy of separation between the magnetic beads 2 and the liquid 3.

[0033] The saturation magnetization of the magnetic beads 2 is measured using, for example, a vibrating sample magnetometer (VSM). The saturation magnetization of the Fe-based metallic soft magnetic particles 21 may be regarded as the saturation magnetization of the magnetic beads 2.

[0034] 1 is a device that applies such centrifugal acceleration, but the direction of acceleration is not limited to the centrifugal direction and may be a linear direction. For example, the magnetic bead separation device may be a device that applies linear acceleration by repeatedly shaking the sample tube 5 vigorously down and then slowly pulling it up.

[0035] The external magnetic field applying unit 16 shown in FIG. 2 includes a head portion 162 attached to the lid portion 56 of each sample tube 5, and a permanent magnet 164 built into the head portion 162.

[0036] By attaching the head portion 162 to the lid portion 56, the permanent magnet 164 is brought into close proximity to the lid portion 56. When the lid portion 56 is closed in this state, the magnetic beads 2 contained in the sample tube 5 can be magnetically attracted to the lid portion 56. Furthermore, when the lid portion 56 is opened with the magnetic beads 2 magnetically attracted to the lid portion 56, the liquid 3 contained in the sample tube 5 can be discharged or supplied.

[0037] The external magnetic field application unit 16 may be independent of the angle rotor 11, or may be connected to it via a flexible connecting member 166 as shown in Fig. 2. Providing the connecting member 166 makes it possible to open and close the lid 56 while the head 162 is attached to the lid 56. This also improves the ease of attaching the head 162 to another sample tube 5.

[0038] The permanent magnet 164 may be replaced by an electromagnet. Examples of the permanent magnet 164 include a neodymium magnet, a ferrite magnet, a samarium-cobalt magnet, and an alnico magnet.

[0039] As described above, the magnetic bead separation device 1 according to the embodiment includes an angle rotor 11 (rotating body) that accommodates a mixed liquid 4, and an external magnetic field application unit 16. The mixed liquid 4 contains magnetic beads 2 that have Fe-based metal soft magnetic particles 21 and coatings 22 that coat the Fe-based metal soft magnetic particles 21 and have a saturation magnetization of 50 emu / g or more and 250 emu / g or less, and a liquid 3 that contains nucleic acids. The angle rotor 11 includes a container mounting unit 112 on which a sample tube 5 (container) is mounted, and rotates so as to apply centrifugal acceleration to the sample tube 5. The external magnetic field application unit 16 applies an external magnetic field to the sample tube 5.

[0040] With this configuration, it is possible to combine the magnetic attraction operation with the separation operation using centrifugal acceleration, so that the liquid 3 adhering to the magnetic beads 2 can be detached while the magnetic beads 2 are fixed by an external magnetic field. In other words, since the magnetically attracted magnetic beads 2 are fixed, the liquid 3 can be selectively moved by applying centrifugal acceleration to the sample tube 5. This allows the magnetic beads 2 and the liquid 3 to be separated with high precision.

[0041] It is also possible to suppress carryover of Liquid 3. Carryover refers to the transfer of Liquid 3 into a new liquid, such as a washing solution or elution solution described below, when Liquid 3 attached to the magnetic beads 2 is immersed in the new liquid. Carryover of Liquid 3 also involves the transfer of impurities contained in Liquid 3, and various adverse effects caused by these impurities are of concern.

[0042] The magnetic bead separation device 1 can suppress such carryover of the liquid 3. This makes it possible to minimize the adverse effects of contaminants when finally analyzing the recovered nucleic acids.

[0043] 2. Sample tube modifications Next, a modified example of a sample tube having a structure different from that described above will be described.

[0044] In the following, a sample tube 5A having a different structure from the above sample tube 5 will be described as a sample tube according to the embodiment.

[0045] Fig. 4 is a cross-sectional view illustrating a sample tube according to an embodiment of the present invention, in which the same components as those in Fig. 2 are denoted by the same reference numerals.

[0046] The magnetic bead separation device 1 described above is provided with an external magnetic field application unit 16. Therefore, even if the sample tube 5 does not have a magnet or the like, an external magnetic field can be applied.

[0047] 4 includes a permanent magnet 164 (magnet) provided on the lid portion 56. That is, the sample tube 5A includes a main body portion 55, a lid portion 56, and a permanent magnet 164. As described above, the main body portion 55 is cylindrical with a bottom and has an opening 52. The lid portion 56 opens and closes the opening 52 of the main body portion 55.

[0048] With this sample tube 5A, the permanent magnet 164 is provided on the lid 56, so that the permanent magnet 164 can be operated integrally when the lid 56 is opened or closed. Therefore, by opening the lid 56 with the magnetic beads 2 fixed by magnetic attraction to the underside of the lid 56, it is possible to easily discharge the liquid 3 contained in the main body 55 or supply new liquid. Therefore, with the sample tube 5A, even in a centrifuge that does not have an external magnetic field application unit 16, it is possible to perform an operation that combines the magnetic attraction operation by the magnetic bead separation device 1 and the separation operation by centrifugal acceleration. This makes it possible to suppress carryover of the liquid 3 even in a centrifuge that does not have an external magnetic field application unit 16.

[0049] The permanent magnet 164 may be replaced by an electromagnet. 4 is built into the head portion 162. The head portion 162 is detachable from the lid portion 56. This allows the permanent magnet 164 to be reused with multiple lid portions 56. As a result, the permanent magnet 164 can be used effectively, and the cost of the sample tube 5A can be reduced.

[0050] 3. Magnetic bead separation method Next, a magnetic bead separation method according to an embodiment will be described. In the following description, a method using the magnetic bead separation device 1 described above will be described, but the device used in this method is not limited to this.

[0051] Fig. 5 is a flowchart illustrating the magnetic bead separation method according to the embodiment. Fig. 6 to Fig. 11 are schematic diagrams illustrating the magnetic bead separation method according to the embodiment.

[0052] 5 includes an adsorption step S102, a magnetic attraction step S104, a separation step S106, a washing step S108, and an elution step S110. Each step will be explained below in order.

[0053] 3.1.Adsorption process 6, in the adsorption step S102, a mixture 4 containing magnetic beads 2 and a liquid 3 containing nucleic acid is placed in a sample tube 5. The sample tube 5 is a cylindrical container with a bottom, and has an opening 52 at one end. When the magnetic beads 2 come into contact with the liquid 3 in the sample tube 5, the nucleic acid is adsorbed to the magnetic beads 2.

[0054] 3, the magnetic beads 2 include Fe-based metal soft magnetic particles 21 and coatings 22. The Fe-based metal soft magnetic particles 21 are made of an Fe-based metal and are particles having soft magnetism.

[0055] The coercive force of the magnetic beads 2 is preferably 100 Oe or less, more preferably 30 Oe or less, and even more preferably 10 Oe or less. Such magnetic beads 2 have a sufficiently low coercive force that 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 the magnetic attraction step S104 described below, when the operation of transitioning to a magnetically attracted state by an external magnetic field is performed and when the operation of releasing the magnetically attracted state thereafter is performed, can be improved.

[0056] The coercive force of the magnetic beads 2 is measured using, for example, a vibrating sample magnetometer (VSM). The coercive force of the Fe-based metallic soft magnetic particles 21 may be regarded as the coercive force of the magnetic beads 2.

[0057] The saturation magnetization of the magnetic beads 2 is set to 50 emu / g or more and 250 emu / g or less, and preferably 100 emu / g or more and 200 emu / g or less. If the saturation magnetization of the magnetic beads 2 is within this range, when the liquid 3 adhering to the magnetic beads 2 is released in the separation step S106, the magnetic beads 2 fixed by the external magnetic field are less likely to fall off even when acceleration is applied. Therefore, the accuracy of separation of the magnetic beads 2 and the liquid 3 in the separation step S106 can be further improved.

[0058] If the saturation magnetization of the magnetic beads 2 falls below the lower limit, the magnetic beads 2 fixed by the external magnetic field may fall off due to inertial force when acceleration is applied. On the other hand, if the saturation magnetization of the magnetic beads 2 exceeds the upper limit, the magnetic beads 2 may continue to be fixed even if an attempt is made to release the fixed magnetic beads 2 by intentionally reducing the magnetic flux density of the external magnetic field applied to the sample tube 5. In other words, the operability of magnetic attraction may be reduced.

[0059] Coating 22 is a coating having a hydrophilic surface that can adsorb and retain nucleic acids contained in liquid 3. Adsorption refers to reversible physical bonding. The constituent material of coating 22 is not particularly limited as long as it is a material that can form the aforementioned hydrophilic surface, but it is, for example, a material containing silicon dioxide. Specific examples include silica, silicon-containing glass, and diatomaceous earth. It may also be a composite material in which the surface of any material is modified with such a silicon oxide-containing material.

[0060] The average particle size of the magnetic beads 2 is preferably 0.05 μm or more and 20.0 μm or less, more preferably 0.5 μm or more and 10.0 μm or less, and even more preferably 1.0 μm or more and 5.0 μm or less. If the average particle size of the magnetic beads 2 is within this range, the magnetic attraction state of the magnetic beads 2 due to the external magnetic field is unlikely to be released when acceleration is applied to the sample tube 5 in the separation step S106. If the average particle size of the magnetic beads 2 is below the lower limit, the magnetic beads 2 are likely to aggregate, which may reduce the efficiency of nucleic acid adsorption. On the other hand, if the average particle size of the magnetic beads 2 exceeds the upper limit, the magnetic attraction state may be released when centrifugal force is applied to the magnetic beads 2.

[0061] The average particle size of the magnetic beads 2 is determined as the particle size D50 at which the cumulative size reaches 50% from the smallest diameter side in the volume-based particle size distribution obtained by laser diffraction.

[0062] The content of the Fe-based metal in the magnetic beads 2 is preferably 50% by volume or more, more preferably 70% by volume or more, and even more preferably 90% by volume or more. Such magnetic beads 2 have a sufficiently high content of the Fe-based metal, so they can obtain a large magnetic attractive force even with a small diameter. On the other hand, if the content of the Fe-based metal falls below the lower limit, the magnetic attractive force decreases, and there is a risk that the separability between the magnetic beads 2 and the liquid 3 will decrease.

[0063] The content of the Fe-based metal in the magnetic beads 2 is calculated based on the area ratio occupied by the Fe-based metal by observing the cross section of the magnetic beads 2 with an electron microscope. If necessary, element mapping may be performed to calculate the area ratio occupied by the Fe-based metal.

[0064] The liquid 3 is a dispersion medium for dispersing nucleic acids, and examples thereof include water, saline, alcohols, etc. The liquid 3 may also contain impurities other than nucleic acids.

[0065] When the magnetic beads 2 and the liquid 3 are placed in a sample tube 5 , the nucleic acid is adsorbed to the magnetic beads 2 .

[0066] In addition to the magnetic beads 2 and liquid 3, a dissolution solution may be added to the mixed solution 4. For example, a liquid containing a chaotropic substance is used as the dissolution solution. Chaotropic substances generate chaotropic ions in aqueous solution, which have the effect of increasing the water solubility of hydrophobic molecules and contribute to the adsorption of nucleic acids to the 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.

[0067] The concentration of the chaotropic substance in the lysis solution varies depending on the chaotropic substance, but is preferably, for example, 1.0 M to 8.0 M. In particular, when guanidine thiocyanate is used, the concentration is preferably 3.0 M to 5.5 M. Furthermore, in particular, when guanidine hydrochloride is used, the concentration is preferably 4.0 M to 7.5 M.

[0068] The lysis solution may contain a surfactant. The surfactant is used for the purpose of disrupting cell membranes or denaturing proteins contained in cells. The surfactant is not particularly limited, but examples thereof include nonionic surfactants such as Triton (registered trademark)-X and Tween (registered trademark) 20 surfactants, and anionic surfactants such as sodium N-lauroylsarcosinate (SDS). Of these, nonionic surfactants are preferred.

[0069] The concentration of the surfactant in the solution is not particularly limited, but is preferably 0.1% by mass or more and 2.0% by mass or less.

[0070] The dissolution solution 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).

[0071] The concentration of the reducing agent in the dissolution solution is not particularly limited, but is preferably 0.2 M or less. The concentration of the chelating agent in the dissolution solution is not particularly limited, but is preferably 0.2 mM or less. The pH of the dissolution solution is not particularly limited, but is preferably neutral, between 6 and 8.

[0072] In the adsorption step S102, the mixture 4 is stirred as needed using an ultrasonic homogenizer, a vortex mixer, manual shaking, etc. The stirring time is not particularly limited, but is preferably from 5 seconds to 30 minutes.

[0073] 3.2.Magnetic attraction process In the magnetic attraction step S104, an external magnetic field generated by the external magnetic field application unit 16 is applied to the sample tube 5. This causes the external magnetic field to act on at least some of the magnetic beads 2 to which nucleic acid is adsorbed and contained in the sample tube 5, causing them to be magnetically attracted. As a result, the magnetic beads 2 to which nucleic acid is adsorbed are fixed to the lid 56 of the sample tube 5, as shown in FIG. 7. At this time, most of the liquid 3 falls to the bottom 54 of the sample tube 5, but some of the liquid 3 continues to remain around the magnetic beads 2, as shown in FIG. 7.

[0074] The magnetic flux density of the external magnetic field is preferably 0.5 T or more, more preferably 0.5 T to 1.5 T, and even more preferably 0.7 T to 1.3 T. By setting the magnetic flux density of the external magnetic field within the above range, the magnetic beads 2 can be more reliably fixed to the inner wall surface of the sample tube 5.

[0075] If the magnetic flux density of the external magnetic field falls below the lower limit, the magnetic attraction force may become insufficient, and the fixed magnetic beads 2 may fall off in the separation step S106, depending on factors such as the particle size of the magnetic beads 2 and the magnitude of the acceleration applied in the separation step S106. On the other hand, if the magnetic flux density of the external magnetic field exceeds the upper limit, smooth operation may become difficult when wishing to release the magnetic attraction state, depending on factors such as the particle size of the magnetic beads 2 and the magnitude of the acceleration applied in the separation step S106.

[0076] The magnetic flux density of the external magnetic field is a value measured on the outer surface of the sample tube 5. For example, a Tesla meter is used to measure the magnetic flux density. When the external magnetic field application unit 16 has a permanent magnet 164, the residual magnetic flux density of the permanent magnet 164 may be regarded as the magnetic flux density of the external magnetic field.

[0077] In the magnetic attraction step S104, while the external magnetic field is applied, the contents of the sample tube 5 are stirred as needed using an ultrasonic homogenizer, a vortex mixer, manual shaking, etc. This increases the probability that the magnetic beads 2 in the mixture 4 will be magnetically attracted by the external magnetic field.

[0078] 3.3.Separation process In the separation step S106, the sample tube 5 is inserted into the container mounting portion 112 of the angle rotor 11. At this time, an external magnetic field application unit 16 is attached to the sample tube 5, so that an external magnetic field is applied to the magnetic beads 2 to which nucleic acids are adsorbed. In this state, the sample tube 5 is rotated around the rotation axis AX. As a result, centrifugal acceleration is applied to the sample tube 5.

[0079] The external magnetic field application unit 16 applies an external magnetic field to the lid 56, so that the magnetic beads 2 with the nucleic acid adsorbed thereon are magnetically attracted to the lid 56. Meanwhile, the centrifugal acceleration is applied from the opening 52 of the sample tube 5 toward the bottom 54, so that the liquid 3 moves in accordance with the direction of the centrifugal acceleration.

[0080] As a result, the magnetic beads 2 with adsorbed nucleic acid remain on the lid 56 due to magnetic attraction, while the liquid 3 moves due to centrifugal force toward the bottom 54 located below the lid 56, as shown by the white arrow in Figure 8. As a result, the magnetic beads 2 with adsorbed nucleic acid and the liquid 3 can be separated from each other, as shown in Figure 9.

[0081] In this embodiment, as described above, the acceleration applied to the sample tube 5 is centrifugal acceleration, and its magnitude is preferably 10 G or more and 1000 G or less, and more preferably 50 G or more and 500 G or less. If the magnitude of the centrifugal acceleration is within the above range, the magnetic beads 2 and the liquid 3 can be separated more efficiently.

[0082] If the magnitude of the centrifugal acceleration is below the lower limit, the centrifugal force generated in the liquid 3 will be insufficient, and the liquid 3 may tend to remain around the nucleic acid-adsorbed magnetic beads 2. On the other hand, if the magnitude of the centrifugal acceleration is above the upper limit, the centrifugal force generated in the magnetic beads 2 will exceed the magnetic attractive force, causing the magnetic beads 2 to fall off, and making it difficult to separate the nucleic acid-adsorbed magnetic beads 2 from the liquid 3.

[0083] After separating the magnetic beads 2 with adsorbed nucleic acid from the liquid 3 in the above manner, the lid 56 is opened. At this time, by opening the lid 56 while the magnetic beads 2 with adsorbed nucleic acid remain fixed to the lid 56, the magnetic beads 2 can be temporarily moved from inside the sample tube 5 to the outside. Then, the liquid 3 inside the sample tube 5 is discharged using a pipette or the like.

[0084] The magnetic beads 2 may be fixed to a location other than the lid portion 56, for example, to the wall surface of the main body portion 55. The same applies to the following steps.

[0085] 3.4.Cleaning process In the washing step S108, the magnetic beads 2 to which nucleic acids are adsorbed are washed. Washing is an operation to remove impurities adsorbed to the magnetic beads 2 by bringing the magnetic beads 2 to which nucleic acids are adsorbed into contact with a washing solution 6 and then separating them again to remove the impurities.

[0086] Specifically, first, as shown in FIG. 10, a washing solution 6 is supplied into the sample tube 5 using a pipette or the like. Then, the lid 56 is closed and the washing solution 6 is stirred. This brings the washing solution 6 into contact with the magnetic beads 2, and the magnetic beads 2 to which nucleic acids are adsorbed are washed. At this time, the application of the external magnetic field may be stopped temporarily, for example by removing the external magnetic field application unit 16. This allows the magnetic beads 2 to disperse in the washing solution 6, thereby further improving washing efficiency. In this case, the external magnetic field can be applied again after washing.

[0087] Next, the lid 56 is opened and the washing solution 6 is discharged. By repeating the supply and discharge of the washing solution 6 as described above once or twice or more times, the magnetic beads 2 can be washed.

[0088] The washing solution 6 is not particularly limited as long as it does not promote the elution of nucleic acids and does not promote the binding of contaminants 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, Pipette, and phosphate are preferably used.

[0089] The washing solution 6 may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), SDS, etc. The pH of the washing solution 6 is not particularly limited.

[0090] In the washing step S108, the contents of the sample tube 5 are agitated as needed using an ultrasonic homogenizer, a vortex mixer, or manual shaking while the washing solution 6 is in contact with the magnetic beads 2. This can improve the washing efficiency.

[0091] The cleaning step S108 may be performed as needed, and may be omitted if cleaning is not required.

[0092] 3.5. Elution process In the elution step S110, the nucleic acids are eluted from the magnetic beads 2 to which the nucleic acids are adsorbed. Elution is a procedure in which the magnetic beads 2 to which the nucleic acids are adsorbed are brought into contact with an elution solution 7, and then separated again, thereby transferring the nucleic acids to the elution solution 7.

[0093] Specifically, first, as shown in FIG. 11, elution solution 7 is supplied into sample tube 5 using a pipette or the like. Next, lid 56 is closed and elution solution 7 is stirred. This brings elution solution 7 into contact with magnetic beads 2, allowing nucleic acid to be eluted. At this time, application of the external magnetic field may be stopped temporarily by, for example, removing external magnetic field application unit 16. This allows magnetic beads 2 to disperse in elution solution 7, thereby further improving elution efficiency. In this case, the external magnetic field can be applied again after the nucleic acid has been eluted.

[0094] Next, the cover 56 is opened and the eluate 7 containing the eluted nucleic acid is discharged, thereby recovering the nucleic acid.

[0095] The elution solution 7 is not particularly limited as long as it is a liquid that promotes the elution of nucleic acids from the magnetic beads 2 to which the nucleic acids are adsorbed. For example, in addition to water such as sterilized water or pure water, a TE buffer solution, i.e., an aqueous solution containing 10 mM Tris-HCl buffer and 1 mM EDTA and having a pH of 8, is preferably used.

[0096] The elution solution 7 may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), or SDS.

[0097] In the elution step S110, the contents of the sample tube 5 are agitated, if necessary, using an ultrasonic homogenizer, a vortex mixer, or manual shaking, while the elution solution 7 is in contact with the magnetic beads 2 to which nucleic acids are adsorbed. This can improve the elution efficiency.

[0098] Furthermore, in the elution step S110, the elution solution 7 may be heated, thereby facilitating the elution of nucleic acids. The heating temperature of the elution solution 7 is not particularly limited, but is preferably 70°C or higher and 200°C or lower, more preferably 80°C or higher and 150°C or lower, and even more preferably 95°C or higher and 125°C or lower.

[0099] Examples of the heating method include a method of supplying preheated eluate 7, and a method of supplying unheated eluate 7 to the sample tube 5 and then heating it. The heating time is not particularly limited, but is preferably 30 seconds or more and 10 minutes or less.

[0100] The elution step S110 may be performed as needed, and may be omitted, for example, when the only purpose is to separate the magnetic beads 2 from the liquid 3 in the separation step S106.

[0101] 1 includes an adsorption step S102, a magnetic attraction step S104, and a separation step S106. In the adsorption step S102, a mixture 4 containing magnetic beads 2, each of which has a saturation magnetization of 50 emu / g or more and 250 emu / g or less and a coating 22 coating the Fe-based metal soft magnetic particles 21, and a liquid 3 containing nucleic acid, is placed in a sample tube 5 (container), and the nucleic acid is adsorbed to the magnetic beads 2. In the magnetic attraction step S104, an external magnetic field is applied to the sample tube 5, and at least a portion of the magnetic beads 2 is magnetically attracted by the external magnetic field. In the separation step S106, while the magnetic beads 2 are magnetically attracted by the external magnetic field, acceleration is applied to the sample tube 5, causing the liquid 3 adhering to the magnetic beads 2 to detach.

[0102] With this configuration, by applying acceleration while the magnetic beads 2 are fixed by an external magnetic field, the liquid 3 can be efficiently separated from the magnetic beads 2. Meanwhile, since the magnetic beads 2 are fixed by the external magnetic field, their movement is suppressed. This allows the magnetic beads 2 and the liquid 3 to be separated with high precision.

[0103] The magnetic bead separation method shown in FIG. 1 further includes a washing step S108 and an elution step S110.

[0104] In the washing step S108, a washing solution 6 is placed in the sample tube 5 and stirred while the magnetic beads 2 are magnetically attracted by an external magnetic field. The sample tube 5 is then placed on the container mounting section 112, and centrifugal acceleration is applied by the rotation of the angle rotor 11, causing the washing solution 6 adhering to the magnetic beads 2 to selectively detach. This makes it possible to suppress carryover of the washing solution 6. As a result, it is possible to suppress the migration of substances contained in the washing solution 6 into the elution solution 7.

[0105] In the elution step S110, the eluate 7 is placed in the sample tube 5 and stirred while the magnetic beads 2 are magnetically attracted by an external magnetic field. The sample tube 5 is then placed on the container mounting section 112, and centrifugal acceleration is applied by the rotation of the angle rotor 11, causing the eluate 7 adhering to the magnetic beads 2 to selectively detach. This can increase the yield of nucleic acids.

[0106] The magnetic bead separation method, magnetic bead separation device, and sample tube of the present invention have been described above based on the illustrated embodiments, but the present invention is not limited to these. For example, the magnetic bead separation method of the present invention may be obtained by adding any desired process to the above-described embodiments. Furthermore, the magnetic bead separation device and sample tube of the present invention may be obtained by replacing each part of the above-described embodiments with any configuration having the same function, or by adding any component to the above-described embodiments. [Example]

[0107] Next, specific examples of the present invention will be described. 4. Magnetic Bead Separation Example 1 First, magnetic beads and pure water were mixed, and the resulting mixture was placed in a sample tube. The magnetic beads used were soft magnetic particles comprising Fe-based metal soft magnetic particles and a coating, as shown in Table 1. In this example, pure water was used as the liquid containing nucleic acids.

[0108] Next, the lid of the sample tube was closed, and a permanent magnet was attached to the lid, and the mixture in the sample tube was stirred by manual shaking.

[0109] Next, the sample tube was inserted into the container mounting section of the angle rotor of the magnetic bead separator shown in Figure 1. The angle rotor was then rotated to apply centrifugal acceleration to the sample tube, which separated the magnetic beads and water from each other within the sample tube.

[0110] 4.2. Examples 2 to 11 The magnetic beads and water were separated from each other in the same manner as in Example 1, except that the centrifugal acceleration and other conditions were changed as shown in Table 1.

[0111] 4.3. Comparative Example 1 The magnetic beads and water were separated from each other in the same manner as in Example 1, except that the application of centrifugal acceleration was stopped.

[0112] 4.4. Comparative Example 2 The magnetic beads and water were separated from each other in the same manner as in Comparative Example 1, except that magnetic beads in which ferrite-based soft magnetic particles were coated with a silica film were used.

[0113] 4.5. Comparative Examples 3 to 8 The magnetic beads and water were separated from each other in the same manner as in Examples 1 to 6, except that magnetic beads in which ferrite-based soft magnetic particles were coated with a silica film were used.

[0114] 5. Evaluation of Magnetic Bead Separation 5.1. Mass of carried-over water In each example and comparative example, the separated water was discharged from the sample tube and the mass of the water was measured. The mass of the carried-over water was calculated based on the measured mass of the water and the mass of the water placed in the sample tube. The calculation results are shown in Table 1.

[0115] 5.2. Presence or absence of fallen magnetic beads In each example and comparative example, centrifugal acceleration was applied to the sample tube, and then it was visually confirmed whether or not magnetic beads had fallen to the bottom of the sample tube. The results are shown in Table 1.

[0116] [Table 1]

[0117] As shown in Table 1, in each example, the mass of carried-over water was able to be kept significantly lower than in each comparative example. It was also found that setting the centrifugal acceleration within an appropriate range can prevent magnetic beads from falling off.

[0118] In Comparative Examples 3 to 8, the magnetic beads fell off due to centrifugal force, so the mass of the carried-over water could not be measured. [Explanation of symbols]

[0119] 1...magnetic bead separation device, 2...magnetic beads, 3...liquid, 4...mixture, 5...sample tube, 5A...sample tube, 6...washing solution, 7...elution solution, 11...angle rotor, 12...motor, 13...drive shaft, 14...rotor chamber, 15...upper door, 16...external magnetic field application unit, 21...Fe-based metal soft magnetic particles, 22...coating, 52...opening, 54...bottom, 55...main body, 56...lid, 112...container mounting unit, 114...recess, 115...opening, 116...bottom, 142...upper opening, 144...bottom, 162...head, 164...permanent magnet, 166...connecting member, A112...axis, AX...rotating axis, S102...adsorption process, S104...magnetic attraction process, S106...separation process, S108...washing process, S110...elution process, θ...angle

Claims

1. a step of placing a mixed liquid containing magnetic beads, which are provided with Fe-based soft magnetic metal particles and a coating covering the Fe-based soft magnetic metal particles and have a saturation magnetization of 50 emu / g or more and 250 emu / g or less, and a liquid containing target molecules, in a container having a cylindrical main body with a bottom and a lid that closes the main body, and allowing the target molecules to adsorb onto the magnetic beads; applying an external magnetic field to the container to magnetically attract at least a portion of the magnetic beads to the lid portion by the external magnetic field; a step of rotating the container around the rotation axis to apply centrifugal acceleration to the container while the magnetic beads are magnetically attracted to the lid portion by the external magnetic field and tilting the container so that the bottom is located farther from the rotation axis than the opening of the main body portion, thereby removing the liquid adhering to the magnetic beads; A magnetic bead separation method comprising:

2. 2. The magnetic bead separation method according to claim 1, wherein the saturation magnetization of the magnetic beads is 100 emu / g or more and 200 emu / g or less.

3. 3. The magnetic bead separation method according to claim 1, wherein the magnetic flux density of the external magnetic field is 0.5 T or more and 1.5 T or less.

4. 4. The magnetic bead separation method according to claim 1, wherein the magnitude of the centrifugal acceleration is 10 G or more and 1000 G or less.

5. 5. The magnetic bead separation method according to claim 1, wherein the container is tilted so that the axis of the container is at an angle of 10° to 80° relative to the rotation axis.

6. a rotor having a container mounting section on which a container containing a mixed liquid including magnetic beads having Fe-based soft magnetic metal particles and a coating covering the Fe-based soft magnetic metal particles, the magnetic beads having a saturation magnetization of 50 emu / g or more and 250 emu / g or less, and a liquid containing target molecules, the rotor rotating around a rotation axis so as to apply centrifugal acceleration to the container; An external magnetic field application unit that applies an external magnetic field to the container; Equipped with the container placement portion has an insertion hole into which the container is inserted, the insertion hole is inclined so that the bottom is located farther from the rotation axis than the opening, the external magnetic field applying unit includes a head unit having a magnet, The magnetic bead separation device is characterized in that the head portion is attached to the lid portion of the container inserted into the insertion hole.

7. 7. The magnetic bead separator according to claim 6, wherein the axis of the insertion hole is inclined at an angle of 10° to 80° relative to the rotation axis.

8. the external magnetic field applying unit has a connecting member that connects the rotating body and the head unit, The magnetic bead separation device according to claim 6 or 7, wherein the connecting member is flexible.

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