Method for refining biological substance, magnetic stand, and biological substance refining device

Abstract

Provided is a biological substance refining method, a magnetic stand, and a biological substance refining device that can effectively remove a cleaning solution and suppress the occurrence of adverse situations due to the entry of the cleaning solution into a subsequent process. The refining method has: an adsorption process in which a magnetic bead having a saturation magnetization of 50 [emu / g] or more, a biological substance, and a liquid are placed in a container, the biological substance is adsorbed to the magnetic bead, and then the liquid is removed; a cleaning process in which the magnetic bead is cleaned; and an elution process in which the biological substance adsorbed to the magnetic bead is eluted. The cleaning process includes: a magnetic bead fixation process in which a magnet is disposed so that an extension line of a direction of a magnetic pole does not overlap the container, and the magnetic bead is fixed; a cleaning solution discharge process in which the cleaning solution in the container is discharged; and a cleaning solution gasification process in which, after the cleaning solution discharge process, the cleaning solution is gasified while the magnet is disposed so that the extension line of the direction of the magnetic pole overlaps the container, and a magnetic field gradient in the container is 40 [T / m] or more.

Description

Technical Field

[0001] This invention relates to a method for refining biological substances, a magnetic scaffold, and a device for refining biological substances. Background Technology

[0002] Methods using magnetic beads are known to be used for extracting biological substances such as nucleic acids.

[0003] For example, Patent Document 1 discloses a method for extracting biological substances, comprising a dissolution, adsorption, washing, and leaching process. In the dissolution and adsorption process, after nucleic acids are adsorbed from a nucleic acid-containing sample onto magnetic beads, a magnetic separation technique is used to remove the liquid. In the washing process, after the magnetic beads are washed with a washing solution, a magnetic separation technique is used to remove the washing solution. In the leaching process, after the nucleic acids captured by the magnetic beads are dissolved using a leaching solution, a magnetic separation technique is used to recover the leaching solution.

[0004] Furthermore, Patent Document 1 discloses a process in which, after the cleaning solution is discharged, the cleaning solution adhering to the magnetic beads is dried and removed. During the drying process, spikes are generated on the magnetic beads, increasing the surface area. This improves drying efficiency.

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent document 1: Japanese Patent Application Publication No. 2024-140111.

[0008] However, when the magnetic beads are small in size and the magnetic field is weak, the increase in surface area caused by the spike phenomenon becomes insufficient, sometimes failing to adequately improve drying efficiency. As a result, cleaning solution remains on the magnetic beads, and its components are recovered along with the nucleic acid during the dissolution process. These recovered cleaning solution components are carried over into subsequent processes such as nucleic acid testing, leading to defects such as poor test results.

[0009] Therefore, effectively removing the cleaning fluid and preventing adverse conditions caused by the cleaning fluid being carried into subsequent processes has become a technical problem. Summary of the Invention

[0010] The biological substance purification method involved in the application examples of the present invention has the following characteristics: In the adsorption process, magnetic beads, biological material, and liquid with a saturation magnetization of 50 [emu / g] or higher are placed in a container, and the biological material is adsorbed onto the magnetic beads. After that, the liquid is removed. The cleaning process, following the adsorption process, involves washing the magnetic beads with the adsorbed biological material using a cleaning solution; and The dissolution process, following the washing process, dissolves the biological material adsorbed on the magnetic beads. The cleaning process includes: The magnetic bead fixing process involves cleaning the magnetic beads, which have adsorbed the biological material, with the cleaning solution, and then fixing the magnetic beads by arranging magnets in a manner in which the extension lines of the orientation of the magnetic poles do not overlap with the container. The cleaning fluid is discharged by draining the cleaning fluid from the container while the magnetic beads are fixed in place; and The cleaning fluid is vaporized after the cleaning fluid is discharged. The magnet is arranged such that the extension line of the orientation of the magnetic pole overlaps with the container and the magnetic field gradient in the container is 40 [T / m] or more.

[0011] The magnetic support involved in the application examples of the present invention, The magnetic support separates the magnetic beads from the liquid by applying a magnetic field generated by a magnet to a container containing magnetic beads, biological material, and liquid. A base having an insertion hole extending along a first axis for inserting the container; and A magnetic field applying part is disposed on the base to apply a magnetic field to the insertion hole. The magnetic field applying part has: The plurality of said magnets; and A movable part that allows the position of the magnet to change. The movable part is rod-shaped and extends along a second axis that intersects the first axis, supports a plurality of magnets arranged along the second axis, and changes the orientation of the magnetic field generated by the plurality of magnets by translating along the second axis or rotating around the second axis.

[0012] The biological substance purification apparatus according to the application example of the present invention comprises: The magnetic support involved in the application examples of the present invention; A support drive unit that drives the magnetic field application unit of the magnetic support; The cleaning fluid supply unit supplies cleaning fluid to the container inserted into the insertion hole of the magnetic support; The cleaning fluid discharge section discharges the cleaning fluid from the container; and The control unit controls the operation of the support drive unit, the cleaning fluid supply unit, and the cleaning fluid discharge unit. Attached Figure Description

[0013] Figure 1 This is a perspective view showing the magnetic support involved in the embodiment.

[0014] Figure 2 yes Figure 1 The top view of the magnetic bracket shown.

[0015] Figure 3 yes Figure 1 The top view of the magnetic bracket shown.

[0016] Figure 4 yes Figure 2 The magnetic support shown is a cross-sectional view of the container mounted on the magnetic support.

[0017] Figure 5 yes Figure 3 The magnetic support shown is a cross-sectional view of the container mounted on the magnetic support.

[0018] Figure 6 This is a process diagram illustrating the structure of the biological substance purification method involved in the embodiment.

[0019] Figure 7 It is used for explanation Figure 6 The diagram shows a method for refining biological substances.

[0020] Figure 8 It is used for explanation Figure 6 The diagram shows a method for refining biological substances.

[0021] Figure 9 It is used for explanation Figure 6 The diagram shows a method for refining biological substances.

[0022] Figure 10 It is used for explanation Figure 6 The diagram shows a method for refining biological substances.

[0023] Figure 11 This is a cross-sectional view showing the magnetic bead.

[0024] Figure 12 This is a cross-sectional view used to illustrate the configuration of the magnets relative to the container.

[0025] Figure 13 This is a top view of the magnetic support involved in a variation of the implementation method.

[0026] Figure 14 yes Figure 13 The magnetic support shown is a cross-sectional view of the container mounted on the magnetic support.

[0027] Figure 15 yes Figure 13 The magnetic support shown is a cross-sectional view of the container mounted on the magnetic support.

[0028] Figure 16 This is a schematic diagram showing the general structure of the biological substance purification apparatus involved in the embodiment.

[0029] Figure 17 Table 1 shows the conditions of the cleaning process for nucleic acid purification when using magnetic beads from the comparative example, as well as the evaluation results of nucleic acid purification.

[0030] Figure 18 Table 2 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0031] Figure 19 Table 3 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0032] Figure 20 Table 4 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0033] Symbol Explanation

[0034] 1. Magnetic support; 2. Magnetic bead; 3. Liquid; 6. Pipette; 9. Container; 11. Base; 12. Magnetic field application part; 13. Insertion hole; 22. Magnetic metal particles; 24. Coating layer; 71. Support drive part; 72. Cleaning fluid supply part; 73. Cleaning fluid discharge part; 74. Supply and discharge drive part; 75. Control part; 90. Internal space; 91. Opening; 100. Biological substance purification device; 112. Upper plate; 113. Through hole; 114. Lower plate; 115. Recess; 116. Side plate; 117. Side plate; 118. Back Plate; 119, Insertion hole for magnetic field application part; 122, Magnet; 122a, First magnet; 122b, Second magnet; 122c, Magnet; 122d, Magnet; 124, Movable part; 127A, Magnet pair; 127B, Magnet pair; 127C, Magnet pair; 127D, Magnet pair; AX, Rotation shaft; AX1, Shaft; CP, Midpoint; EL, Extension line; H9, Height; Lm, Magnetic flux line; MP, Magnetic pole; S102, Adsorption process; S104, Cleaning process; S106, Dissolution process; TL, Tangent; θ, Angle. Detailed Implementation

[0035] Hereinafter, preferred embodiments of the biological substance purification method, magnetic scaffold, and biological substance purification apparatus of the present invention will be described in detail based on the accompanying drawings.

[0036] 1. Magnetic support

[0037] First, the magnetic support involved in the implementation method will be described.

[0038] Figure 1 This is a perspective view showing the magnetic support 1 according to the embodiment. Figure 2 as well as Figure 3 They are Figure 1 The top view of the magnetic support 1 shown.

[0039] It should be noted that in the figures of this application, the X-axis, Y-axis, and Z-axis are defined as three mutually orthogonal axes. Each axis is indicated by an arrow, with the leading edge designated as "positive" and the base edge as "negative." In the following description, for example, "X-axis direction" includes both the positive and negative sides of the X-axis. The same applies to the Y-axis and Z-axis directions. Furthermore, in the following description, sometimes the positive side of the Z-axis is designated as "up" and the negative side as "down."

[0040] Figure 1 The magnetic support 1 shown is a magnetic field generating device that holds containers 9 such as microtubes and applies a magnetic field to the contents of containers 9.

[0041] Container 9 contains a mixture comprising magnetic beads, biological material, and a dispersion medium. In a magnetic support 1, by applying a magnetic field to the mixture contained in container 9, the magnetic beads, which are the solid phase, and the dispersion medium, which is the liquid phase, are separated. This process is called "magnetic separation."

[0042] Figure 1 The magnetic support 1 shown has a base 11 and a magnetic field application part 12.

[0043] The base 11 has an insertion hole 13. The insertion hole 13 extends along the Z-axis (first axis) and the container 9 is inserted therein.

[0044] The magnetic field applying part 12 includes a magnet 122 and a movable part 124. The magnet 122 applies a magnetic field to the insertion hole 13. This allows a magnetic field to be applied to the container 9 inserted into the insertion hole 13. The movable part 124 is a rod-shaped part extending along a Y-axis (second axis) intersecting the Z-axis, supporting a plurality of magnets 122 arranged along the Y-axis. Furthermore, the movable part 124 is capable of translation along the Y-axis. Translation of the movable part 124 changes the position of the plurality of magnets 122, thus changing the orientation of the magnetic field generated by the magnets 122.

[0045] In this magnetic support 1, since the orientation of the magnetic field can be changed as described above, the fixation state of the magnetic beads contained in the container 9 can be altered. The fixation state, as explained later, refers to states such as the magnetic beads being concentrated into smaller clusters or expanding into needle-like shapes. By changing the fixation state in this way, the cleaning solution remaining after cleaning the magnetic beads can be effectively removed in the biological substance purification method described later. This helps to prevent adverse conditions caused by the cleaning solution being carried into subsequent processes.

[0046] 1.1.Abutment

[0047] Figure 1 The base 11 shown includes an upper plate 112 and a lower plate 114, and side plates 116 and 117 and a back plate 118 that connect them to each other. The upper plate 112 and the lower plate 114 are plate-shaped extending in the XY plane. The side plates 116 and 117 are plate-shaped extending in the XZ plane. Furthermore, the back plate 118 is plate-shaped extending in the YZ plane.

[0048] The upper plate 112 has a plurality of through holes 113. The through holes 113 extend through the upper plate 112 along the Z-axis. In addition, a plurality of through holes 113 are arranged at predetermined intervals along the Y-axis.

[0049] The lower plate 114 is disposed below and separate from the upper plate 112. The lower plate 114 has a plurality of recesses 115. The recesses 115 open upward. In addition, a plurality of recesses 115 are arranged at predetermined intervals along the Y-axis. Furthermore, in the XY plane, the positions of corresponding through holes 113 coincide with the positions of the recesses 115. Thus, a pair of through holes 113 and recesses 115 constitute an insertion hole 13 extending along the axis AX1. When the container 9 is inserted from above the through hole 113, the container 9 is held by the through hole 113 and the recesses 115. That is, the container 9 is held in the insertion hole 13 in an upright state.

[0050] The base 11 only needs to have one insertion hole 13, but preferably has multiple insertion holes 13. The insertion holes 13 are arranged at predetermined intervals along the Y-axis. As an example, Figures 1 to 3 The base 11 shown has four insertion holes 13.

[0051] It should be noted that, Figure 1 The inner wall of the through hole 113 shown is in a continuous annular shape surrounding the shaft AX1, but it is not limited to this and can also be partially interrupted. Similarly, Figure 1 The inner wall of the recess 115 shown is also in a continuous annular shape, but it is not limited to this and may also be in a partially interrupted shape. In addition, the recess 115 may also penetrate the lower plate 114. Furthermore, if the position of the container 9 can be maintained solely by the through hole 113, the recess 115 may be omitted.

[0052] Side plate 116 connects the negative Y-axis end of upper plate 112 and the negative Y-axis end of lower plate 114. Side plate 117 connects the positive Y-axis end of upper plate 112 and the positive Y-axis end of lower plate 114. The frame is composed of upper plate 112, lower plate 114, side plate 116, and side plate 117.

[0053] The back plate 118 is provided to block the opening on the positive X-axis side of the frame. Furthermore, a magnetic field application insertion hole 119 is formed on the back plate 118. This magnetic field application insertion hole 119 opens on the negative Y-axis side of the side plate 116 and extends into the back plate 118 towards the positive Y-axis side. A magnetic field application part 12, described later, is inserted into the magnetic field application insertion hole 119. By inserting the magnetic field application part 12 into the magnetic field application insertion hole 119, the magnet 122 of the magnetic field application part 12 can be positioned near the container 9 provided on the magnetic support 1. Furthermore, by translating the movable part 124 relative to the magnetic field application insertion hole 119, the orientation of the magnetic field generated by the magnet 122 can be changed.

[0054] The material of the base 11 is not particularly limited. For example, it can be a resin material such as ABS (acrylonitrile-butadiene-styrene), polypropylene, nylon, or a metal material such as aluminum alloy.

[0055] Furthermore, the shape of the base 11 is not limited to the shape shown in the figure; it can be any shape as long as it has the insertion hole 13.

[0056] 1.2. Magnetic field application section

[0057] The magnetic field application part 12 is located on the positive side of the X-axis of the insertion hole 13 and is disposed between the upper plate 112 and the lower plate 114. For example... Figure 2 as well as Figure 3 As shown, the magnetic field application part 12 has a magnet 122 and a movable part 124.

[0058] Magnet 122 can also be an electromagnet, but a permanent magnet is preferred. This eliminates the need for a power source for the magnetic support 1 and facilitates miniaturization and weight reduction. Furthermore, the increased mobility of the magnetic support 1 provides greater flexibility in its placement.

[0059] Examples of permanent magnets include neodymium iron boron magnets, samarium cobalt magnets, ferrite magnets, and alnico magnets.

[0060] The magnetic flux density (surface magnetic flux density) on the surface of magnet 122 is preferably 50 mT or more, more preferably 200 mT or more. This increases the moving speed of the magnetic beads during magnetic separation and also suppresses the shedding of fixed magnetic beads. The surface magnetic flux density of magnet 122 is measured, for example, using a gaussmeter with a Hall element.

[0061] The size of magnet 122 can be appropriately selected according to the size of container 9, etc.

[0062] Furthermore, the number of magnets 122 in the magnetic field application section 12 is preferably more than twice the number of insertion holes 13. For example, such as Figures 1-3As shown, when there are four insertion holes 13, the number of magnets 122 is preferably eight or more. This allows two magnets 122 with different pole orientations to be paired with one insertion hole 13. Consequently, by changing the relative position of the magnets 122 with respect to the insertion holes 13 using the movable part 124, the orientation of the magnetic field relative to the container 9 can be changed.

[0063] Furthermore, the period of the arrangement of the magnets 122 is preferably half the period of the arrangement of the insertion holes 13. This allows the two magnets 122 to be correctly aligned with one insertion hole 13.

[0064] Here, the operation of the magnetic field application unit 12 will be explained.

[0065] Figure 2 as well as Figure 3 The diagram illustrates states where the relative position of the magnetic field application unit 12 with respect to the base 11 is different. That is, by making the position of the magnetic field application unit 12 different from that of the base 11... Figure 2 Moving the magnetic field applying unit 12 at the position shown toward the negative Y-axis allows the magnetic field applying unit 12 to be positioned in... Figure 3 The location shown.

[0066] Figure 4 yes Figure 2 The magnetic support 1 and the container 9 mounted on the magnetic support 1 are shown in cross-sectional view. Figure 5 yes Figure 3 The magnetic support 1 and the container 9 mounted on the magnetic support 1 are shown in cross-sectional view.

[0067] exist Figure 4 as well as Figure 5 The diagram shows how to insert into Figure 2 as well as Figure 3 The container 9 in one of the insertion holes 13 shown and a portion of the magnetic field application part 12 adjacent thereto.

[0068] Among the multiple magnets 122 in the magnetic field application unit 12, the magnet whose extension line of the orientation of the magnetic pole MP does not overlap with the container 9 is designated as "first magnet 122a", and the magnet whose extension line of the orientation of the magnetic pole MP overlaps with the container 9 is designated as "second magnet 122b".

[0069] When container 9, which becomes the object, is closest to magnet 122, it is determined whether the extension line of the orientation of magnetic pole MP overlaps with container 9. Therefore, as Figure 4 as well as Figure 5As shown, the magnet 122 with its magnetic pole MP facing the negative Y-axis is designated as the first magnet 122a, and the magnet 122 with its magnetic pole MP facing the negative X-axis is designated as the second magnet 122b. Furthermore, in this specification, "the orientation of the magnetic pole MP" refers to the orientation from the S pole to the N pole inside the magnet 122 when viewed from the Z-axis direction. Additionally, when viewed from the Z-axis direction, the "extension line of the orientation of the magnetic pole MP" passes through the middle of the width of the magnet 122 (the width in the direction orthogonal to the orientation of the magnetic pole MP), becoming a straight line parallel to the magnetic pole MP. Figure 4 as well as Figure 5 In the middle, it is represented as the extension line EL.

[0070] like Figure 4 as well as Figure 5 As shown, in the magnetic field application section 12, the first magnet 122a and the second magnet 122b are arranged alternately adjacent to each other in the Y-axis direction.

[0071] The relative position of the magnetic field application part 12 is Figure 4 At the position shown, a magnetic field generated by the first magnet 122a is applied to container 9. Thus, as... Figure 4 As shown, the magnetic flux line Lm from the N pole to the S pole of the first magnet 122a contains a significant portion parallel to the Y-axis. The magnetic field represented by this flux line Lm acts on the magnetic bead 2 contained in the container 9.

[0072] On the other hand, the relative position of the magnetic field application part 12 is Figure 5 At the position shown, a magnetic field generated by the second magnet 122b is applied to container 9. Thus, as... Figure 5 As shown, the magnetic flux line Lm from the N pole to the S pole of the second magnet 122b contains a significant portion parallel to the X-axis. The magnetic field represented by this flux line Lm acts on the magnetic bead 2 contained in the container 9.

[0073] As described above, the movable part 124 is in the shape of a rod extending along the Y-axis (second axis) and supports a plurality of magnets 122 arranged along the Y-axis.

[0074] The movable part 124 can translate along the Y-axis. By translating the movable part 124 along the Y-axis, the positions of the multiple magnets 122 can be changed, and the orientation of the magnetic field generated by the magnets 122 can be changed.

[0075] The material of the movable part 124 is not particularly limited; for example, resin, ceramic, and non-magnetic metal materials can be listed. However, from the viewpoints of moldability and impact resistance, resin is preferred.

[0076] like Figure 2 as well as Figure 3As shown, when the magnetic field applying part 12 is inserted into the magnetic field applying part insertion hole 119 of the base 11, a portion of the movable part 124 extends out from the magnetic field applying part insertion hole 119. This extended portion becomes a gripping part held by the operator, enabling relative movement of the magnetic field applying part 12. It should be noted that the movable part 124 can also be connected to a drive unit (not shown) and moved by power generated from the drive unit.

[0077] Based on the structure described above, a magnetic support 1 can be realized in which the orientation of the magnetic field applied to the container 9 can be easily changed by translating the magnetic field application part 12 in the Y-axis direction. Furthermore, changing the orientation of the magnetic field also changes the distance between the N pole and the S pole relative to the container 9. Therefore, a magnetic support 1 can be realized that allows for easy modification of the magnetic flux density formed within the container 9 and easy modification of the fixation state of the magnetic beads 2. Such a magnetic support 1 is particularly effective in the biological material purification method described later.

[0078] It should be noted that the magnetic beads 2 mentioned above are a group of particles used for magnetic separation to adsorb biological substances. Magnetic separation is a technique that separates the solid and liquid phases by applying a magnetic field to a container containing a solid phase containing magnetic beads 2 and a liquid phase containing a solvent, etc., thereby attracting the solid phase magnetically.

[0079] Biological substances include, for example, nucleic acids such as DNA and RNA or mixtures thereof. Biological substances can also be other biologically derived components besides nucleic acids. It should be noted that nucleic acids and biologically derived components can exist in biological samples such as cells and biological tissues, viruses, and bacteria. The purification method for biological substances described later involves refining such biological substances through processes such as adsorption, separation, washing, and dissolution. It should be noted that the following description uses nucleic acids as an example of biological substances, but the following description is equally applicable to biologically derived components other than nucleic acids.

[0080] 2. Methods for refining biological substances

[0081] Next, the method for purifying biological substances involved in the embodiments will be described.

[0082] Figure 6 This is a process diagram illustrating the structure of the biological substance purification method according to the embodiment. Figures 7 to 10 These are for explanation Figure 6 The diagram shows a method for refining biological substances.

[0083] 2.1. Summary

[0084] Figure 6The method for refining biological substances shown is a method for refining biological substances, which includes an adsorption step S102, a washing step S104, and a dissolution step S106.

[0085] In the adsorption process S102, Figure 7 In container 9, a mixture is prepared by combining magnetic beads 2 saturated with magnetization of 50 emu / g or higher, nucleic acid (biological material) not shown, and a dissolving and adsorption solution (liquid 3). Then, the nucleic acid is adsorbed onto the magnetic beads 2 in the mixture. Next, the magnetic beads 2 with adsorbed nucleic acid are magnetically separated from the mixture within container 9.

[0086] In the cleaning process S104, the magnetic beads 2 adsorbed with nucleic acid are cleaned with cleaning solution in the container 9.

[0087] In the dissolution process S106, nucleic acids are dissolved from magnetic beads 2 that have been adsorbed with nucleic acids in container 9.

[0088] In this embodiment, the cleaning process S104 includes magnetic bead fixing, cleaning fluid discharge, and cleaning fluid vaporization.

[0089] In the magnetic bead immobilization process, after washing the magnetic beads 2 adsorbed with nucleic acid with a washing solution, as follows: Figure 4 As shown, magnet 122 is arranged such that the extension line EL of the orientation of magnetic pole MP does not overlap with container 9. Thus, as... Figure 8 As shown, the magnetic bead 2 can be fixed to the inner wall of the container 9.

[0090] During the cleaning fluid discharge process, with magnetic bead 2 fixed in place, as follows: Figure 9 As shown, use pipette 6 to drain the cleaning solution from container 9.

[0091] In the vaporization treatment of cleaning fluid, after the cleaning fluid is discharged and treated, such as Figure 5 As shown, magnet 122 is configured such that the extension line of the orientation of the magnetic pole MP of magnet 122 overlaps with container 9, and the magnetic field gradient within container 9 is 40 [T / m] or more. That is, during the cleaning liquid vaporization process, the magnetic support 1 is operated to change the fixed state of magnetic bead 2 from the fixed state during the cleaning liquid discharge process. Then, the cleaning liquid is vaporized in the changed fixed state.

[0092] Based on this structure, the immobilization state of the magnetic beads 2 can be optimized during the vaporization of the cleaning solution, thus ensuring thorough drying of the cleaning solution. This helps to prevent adverse effects caused by the cleaning solution being carried into subsequent processes. Examples of such subsequent processes include nucleic acid PCR-based testing.

[0093] Furthermore, based on the structure described above, the fixation state of the magnetic bead 2 can be optimized during the cleaning fluid discharge process, thereby suppressing interference between the fixed magnetic bead 2 and the pipette 6. This improves the workability of the cleaning fluid discharge process and effectively removes the cleaning fluid.

[0094] The following is a description of each process step in turn.

[0095] 2.2. Adsorption process

[0096] In the adsorption process S102, a dissolution and adsorption solution containing nucleic acid of the sample is added. Figure 7 The liquid 3 shown and the magnetic bead 2 are placed in Figure 7 In container 9 as shown. Then, the contents of container 9 are mixed. Thus, as Figure 7 As shown, magnetic beads 2 are dispersed in a dissolving and adsorption solution within container 9. Nucleic acids are typically enclosed within a cell membrane and nucleus. The nucleic acids are extracted from the sample by dissolving and removing the so-called outer shell of the cell membrane and nucleus through the dissolving and adsorption solution. Then, the nucleic acids are captured by the magnetic beads 2 through adsorption by the dissolving and adsorption solution.

[0097] As a dissolving and adsorbing liquid, a liquid containing proteinase K, a dissociating agent, etc., can be used. Proteinase K breaks down proteins, making nucleic acids soluble. The dissociating agent generates dissociating ions in the aqueous solution, reducing the interaction of water molecules and thus destabilizing the structure. This facilitates the adsorption of nucleic acids.

[0098] The dissolving and adsorbing solution can be water, such as sterile water. Alternatively, any additives can be added to the dissolving and adsorbing solution. Examples of additives include salts, buffers, surfactants, organic solvents, acids, and bases.

[0099] Examples of salts include sodium chloride, lithium chloride, potassium carbonate, and trisodium citrate.

[0100] Examples of buffers include acetic acid buffers, phosphate buffers, formic acid buffers, citric acid buffers, and tartaric acid buffers.

[0101] Examples of surfactants include tritium-based surfactants like Triton X-100, Tween-based surfactants like Tween 20 (nonionic surfactants), and anionic surfactants like sodium N-lauroyl sarcosinate (SDS). Examples of organic solvents include alcohols like ethanol.

[0102] In the adsorption process S102, the contents of container 9 can also be stirred. Therefore, as... Figure 7As shown, since the magnetic beads 2 are dispersed in the dissolving and adsorption solution (liquid 3), the probability of nucleic acids being captured by the magnetic beads 2 is increased. Stirring can be used, for example, with a vortex mixer, hand shaker, pipette, etc.

[0103] Figure 11 This is a cross-sectional view showing magnetic bead 2. Figure 11 The magnetic bead 2 shown has magnetic metal particles 22 and a coating layer 24. The magnetic metal particles 22 are made of magnetic metal powder. The coating layer 24 contains inorganic oxide. The magnetic bead 2 will be described in detail later.

[0104] Next, a magnetic field is applied to the mixture containing magnetic beads 2 with adsorbed nucleic acids, magnetically attracting them to the inner wall of container 9. Thus, as... Figure 8 As shown, the magnetic beads 2 are moved toward and fixed to the inner wall of the container 9. As a result, the magnetic beads 2, which are the solid phase, and the dissolved adsorption liquid (liquid 3), which is the liquid phase, can be separated.

[0105] Next, with the magnetic beads 2 fixed, the dissolved and adsorbed liquid in container 9 is drained. Specifically, as follows... Figure 9 As shown, the dissolved adsorbent solution (liquid 3) accumulated at the bottom of container 9 is aspirated and drained using pipette 6. Thus, magnetic beads 2 containing captured nucleic acids remain inside container 9.

[0106] It should be noted that after dissolving and adsorbing liquid is discharged, acceleration can be applied to container 9 as needed. This can shake off the dissolved and adsorbed liquid adhering to magnetic beads 2, thus reducing the amount of unseparated dissolved and adsorbed liquid. The acceleration can also be centrifugal acceleration. A centrifuge can be used to apply the centrifugal acceleration.

[0107] 2.3. Cleaning Process

[0108] In the cleaning process S104, the magnetic beads 2 adsorbed with nucleic acids are cleaned. Cleaning refers to the process of removing impurities trapped by the magnetic beads 2 by contacting the adsorbed nucleic acid magnetic beads 2 with the cleaning solution in the container 9, followed by a separation process to transfer the impurities into the cleaning solution. In this case, Figures 7 to 9 Liquid 3 in the solution becomes the cleaning solution.

[0109] In the cleaning process S104, firstly, the contents of container 9 are stirred. Thus, as... Figure 7 As shown, since the magnetic beads 2 are dispersed in the cleaning solution (liquid 3), the cleaning efficiency of the magnetic beads 2 can be improved. Stirring can be performed using, for example, a vortex mixer, a hand-cranked vibrator, or a pipette. It should be noted that this stirring is preferably performed without applying a magnetic field.

[0110] Next, the magnetic beads are fixed, the cleaning fluid is discharged, and the cleaning fluid is vaporized in sequence.

[0111] 2.3.1. Magnetic bead fixing treatment

[0112] In the magnetic bead immobilization process, after cleaning the magnetic beads 2 adsorbed with nucleic acid with a cleaning solution, the container 9 is placed on the magnetic support 1. Additionally, as... Figure 4 As shown, magnet 122 is arranged such that the extension line EL of the orientation of magnetic pole MP does not overlap with container 9. That is, magnet 122 is arranged in such a way that the extension line EL of the orientation of magnetic pole MP does not overlap with container 9. Figure 8 The first magnet 122a shown is positioned close to the container 9. Thus, as... Figure 8 As shown, the magnetic bead 2 can be fixed to the inner wall of container 9. The extension line EL does not overlap with container 9, as shown... Figure 4 As shown, this means that the extension line EL does not pass through the internal space 90 of container 9.

[0113] 2.3.2. Cleaning fluid discharge treatment

[0114] During the cleaning fluid discharge process, with magnetic bead 2 fixed in place, as follows: Figure 9 As shown, the cleaning fluid (liquid 3) accumulated at the bottom of container 9 is drained using pipette 6. As a result, the cleaned magnetic beads 2 remain in container 9.

[0115] In the magnetic bead fixing process described above, such as Figure 4 As shown, since the magnet 122 is arranged so that its extension line EL does not overlap with the container 9, the magnetic flux line Lm generated by the magnet 122 is as follows: Figure 4 As shown, it contains a significant amount of Y-axis component. The magnetic flux density of such flux lines Lm, extending from the inner wall of container 9 towards the center of container 9, is low. Therefore, as... Figure 9 As shown, the magnetic beads 2 are fixed to the inner wall of the container 9 in a concentrated, small configuration. This suppresses the occurrence of a needle-like arrangement of the magnetic beads 2 (spike phenomenon). Consequently, sufficient space is ensured within the internal space 90 of the container 9 for inserting and removing the pipette 6. As a result, the discharge of cleaning fluid via the pipette 6 can be performed efficiently.

[0116] 2.3.3. Vaporization treatment of cleaning fluid

[0117] In the vaporization treatment of the cleaning liquid, the magnetic field application part 12 of the magnetic support 1 is operated to adjust the relative position of the magnetic field application part 12 with respect to the base 11 from... Figure 2 The location shown has been changed to Figure 3 The location shown. That is, as indicated. Figure 5 As shown, magnet 122 is arranged such that the extension line EL of the orientation of the magnetic pole MP of magnet 122 overlaps with container 9. The term "extension line EL overlaps with container 9" means that... Figure 5 As shown, this refers to the extension line EL passing through the internal space 90 of container 9. Therefore, as... Figure 5As shown, the magnetic flux lines Lm generated by magnet 122 contain a greater proportion of the X-axis component. The component of such flux lines Lm extending from the inner wall of container 9 towards the center of container 9 has a high magnetic flux density. Therefore, on the magnetic bead 2, as... Figure 10 As shown, the magnetic beads 2 exhibit a needle-like arrangement (spike phenomenon). When the spike phenomenon occurs, the overall surface area of ​​the fixed magnetic beads 2 increases. This allows the cleaning fluid adhering to the magnetic beads 2 to be effectively vaporized. Consequently, the cleaning fluid can be effectively removed, and the carryover of cleaning fluid components into subsequent processes can be prevented.

[0118] exist Figure 1 In the magnetic support 1 shown, the state of the first magnet 122a being positioned close to the container 9 and the state of the second magnet 122b being positioned close to the container 9 can be easily switched by simply translating the movable part 124 along the Y-axis (second axis).

[0119] In addition, during the cleaning fluid vaporization process, magnetic beads 2 with a saturation magnetization of 50 [emu / g] or higher are used, and magnets 122 are arranged such that the magnetic field gradient within container 9 is 40 [T / m] or higher. Then, the cleaning fluid is vaporized in this state.

[0120] This structure allows the cleaning fluid to be fully vaporized, improving drying efficiency. As a result, the amount of cleaning fluid components carried into subsequent processes can be minimized.

[0121] When the saturation magnetization falls below the lower limit, the magnetic attraction force generated on the magnetic bead 2 decreases, and the occurrence of the spike phenomenon becomes insufficient. As a result, the drying efficiency decreases, and cleaning solution may remain.

[0122] Furthermore, the saturation magnetization of magnetic beads 2 is preferably 100 [emu / g] or more, more preferably 150 [emu / g] or more. Saturation magnetization refers to the value of magnetization exhibited by a magnetic material when a sufficiently large magnetic field is applied from the outside, and the magnetization remains constant regardless of the magnetic field. If the saturation magnetization is within this range, the drying efficiency of magnetic beads 2 can be significantly improved. In addition, since the movement speed of magnetic beads 2 in the magnetic field can be increased, the time required for magnetic separation can be shortened. Furthermore, the saturation magnetization of magnetic beads 2 is influenced by the adsorption force when fixed by the magnetic field. If the saturation magnetization is within this range, a sufficiently high adsorption force can be obtained, thus preventing the magnetic beads 2 from being discharged along with the liquid 3 when the liquid 3 is discharged while the magnetic beads 2 are fixed. As a result, the decrease in nucleic acid yield that accompanies the reduction of magnetic beads 2 can be suppressed.

[0123] It should be noted that there is no particular limit to the upper limit of saturation magnetization, but from the viewpoint of ease of material selection that is suitable for balancing performance and cost, it is preferably below 300 [emu / g].

[0124] The saturation magnetization of magnetic bead 2 can be measured using a vibrating sample magnetometer (VSM). Examples of vibrating sample magnetometers include the TM-VSM1230-MHHL manufactured by Tamagawa Corporation. The maximum applied magnetic field for measuring saturation magnetization is, for example, 0.5 T or more.

[0125] On the other hand, when the magnetic field gradient is below the lower limit, the magnetic attraction force generated on the magnetic bead 2 decreases, and the occurrence of the spike phenomenon becomes insufficient. As a result, the drying efficiency decreases, and cleaning solution may remain.

[0126] Furthermore, the magnetic field gradient is preferably 100 [T / m] or more, more preferably 150 [T / m] or more. If the magnetic field gradient is within this range, the drying efficiency of the magnetic beads 2 can be significantly improved. Additionally, since the movement speed of the magnetic beads 2 in the magnetic field can be increased, the time required for magnetic separation can be shortened. Furthermore, the magnetic field gradient within the container 9 is influenced by the adsorption force when the magnetic field is fixed. If the magnetic field gradient is within this range, a sufficiently high adsorption force can be obtained, thus preventing the magnetic beads 2 from being discharged along with the liquid 3 when the liquid 3 is discharged while the magnetic beads 2 are fixed. This suppresses the decrease in nucleic acid yield that accompanies the reduction of the magnetic beads 2.

[0127] It should be noted that there is no particular limit to the upper limit of the magnetic field gradient, but from the perspective of ease of selection of magnet 122 and constraints on the distance between container 9 and magnet 122, it is preferably below 400 [T / m].

[0128] The point on the surface of magnet 122 closest to container 9 is taken as measurement point a, and the point inside the space 90 of container 9 closest to magnet 122 is taken as measurement point b. The magnetic field gradient G can be considered as the difference in magnetic fields between measurement point a and measurement point b. Therefore, the distance [m] between measurement point a and measurement point b is set as Δs. In addition, the magnetic field [T] at measurement point a is set as Ba, and the magnetic field [T] at measurement point b is set as Bb. Thus, the magnetic field gradient G is obtained by the following equation (1).

[0129]

[0130] Figure 12 This is a cross-sectional view used to illustrate the configuration of magnet 122 relative to container 9.

[0131] In the vaporization treatment of the cleaning liquid, it is sufficient to arrange the magnet 122 so that its extension line EL overlaps with the container 9. However, it is preferable to arrange the magnet 122 so that the angle θ formed by the extension line EL and the tangent TL of the inner wall of the container 9 is 45° or more and 90° or less. With this structure, the magnetic beads 2 can be extended over a wider range throughout the internal space 90 of the container 9. That is, the surface area of ​​the magnetic beads 2, where the peak phenomenon occurs, can be further increased. As a result, the drying efficiency of the magnetic beads 2 can be particularly improved.

[0132] It should be noted that when the angle θ is lower than the lower limit, the peak phenomenon may not be generated sufficiently, and the surface area of ​​the magnetic bead 2 may not be fully expanded.

[0133] Furthermore, the tangent TL of the inner wall of container 9 refers to the tangent line of the inner wall at the location closest to the magnetic field application part 12 in a cross-sectional view orthogonal to the axis of container 9. For example, in Figure 12 In the example shown, the tangent of the inner wall extending parallel to the movable part 124 of the magnetic field application part 12 can be set as tangent TL.

[0134] In addition, angle θ is the smallest angle among the angles formed between the extension line EL and the tangent line TL.

[0135] In the cleaning fluid vaporization process, it is preferable to place the container 9 with the magnet 122 positioned so that its extension line EL overlaps with the container 9 for a certain period of time. Specifically, it is preferable to allow the cleaning fluid vaporization process to last for 3 minutes or more, more preferably 10 minutes or more. This allows the cleaning fluid to vaporize sufficiently, thus further reducing the probability that components of the cleaning fluid will be carried into subsequent processes.

[0136] It should be noted that during the vaporization treatment of the cleaning fluid, it can be left to dry naturally as described above. However, forced drying, accompanied by heating and blowing, can also be performed as needed. Forced drying can further reduce the probability of cleaning fluid residue and shorten the drying time.

[0137] Furthermore, in the vaporization treatment of the cleaning fluid, the magnet 122 can be positioned arbitrarily along the height direction (Z-axis direction) of container 9, but if... Figure 10 As shown, the magnetic bead 122 is preferably positioned above the midpoint CP of the height H9 of the container 9 (near the opening 91 of the container 9). That is, the center of the magnet 122 in the Z-axis direction only needs to be above the midpoint CP. By positioning it in this way, the distance between the fixed magnetic bead 2 and the opening 91 of the container 9 is shortened, improving the gas exchange efficiency around the magnetic bead 2. As a result, the drying efficiency of the magnetic bead 2 can be further improved.

[0138] It should be noted that in the cleaning process S104, the magnetic bead fixing treatment, cleaning fluid discharge treatment, and cleaning fluid vaporization treatment can also be grouped into one group, and then performed as two or more groups. In this case, the composition of the cleaning fluid can also be different in each group.

[0139] The cleaning solution can be any liquid that does not promote the dissolution of nucleic acids and does not promote the binding of impurities to magnetic beads 2. There are no particular limitations. For example, in addition to water, organic solvents such as ethanol, isopropanol, and acetone, or their aqueous solutions or low-salt concentration aqueous solutions can also be listed.

[0140] In addition, various additives can be added to the cleaning solution. Examples of additives include buffers such as acetic acid buffer and phosphate buffer, salts such as sodium chloride, and surfactants.

[0141] 2.4. Dissolution process

[0142] In the dissolution step S106, the nucleic acids captured by the magnetic beads 2 are dissolved into the dissolution solution. Dissolution refers to the process of bringing the magnetic beads 2, with adsorbed nucleic acids, into contact with the dissolution solution within the container 9, transferring the nucleic acids to the dissolution solution, followed by separation, thereby recovering the dissolution solution. In this case, Figures 7 to 9 Liquid 3 in the solution becomes the dissolution solution.

[0143] Specifically, firstly, the magnetic beads 2 and the dissolution solution are stirred in the container 9. The dissolution solution comes into contact with the magnetic beads 2, and the nucleic acid dissolves into the dissolution solution. This is preferably performed without applying a magnetic field. As a result, the dissolution efficiency is improved because the magnetic beads 2 are redispersed in the dissolution solution.

[0144] Next, a magnetic field is applied to the mixture containing magnetic beads 2, magnetically attracting it to the inner wall of container 9. Thus, as... Figure 8 As shown, the magnetic bead 2 is moved toward and fixed to the inner wall of the container 9. As a result, the magnetic bead 2, which is the solid phase, and the dissolution liquid (liquid 3), which is the liquid phase, can be separated.

[0145] Next, with the magnetic bead 2 fixed in place, the dissolved liquid in container 9 is recovered. Specifically, as follows... Figure 9 As shown, pipette 6 is used to aspirate and recover the dissolution liquid (liquid 3) accumulated at the bottom of container 9.

[0146] The dissolution solution can be any liquid that promotes the dissolution of nucleic acids captured by magnetic beads 2, without any particular limitation. For example, sterile water and pure water can be listed.

[0147] 3. Magnetic beads

[0148] Next, an example of magnetic bead 2 will be described. It should be noted that the structure of magnetic bead 2 used in the biological substance purification method is not limited to the structure described below.

[0149] Figure 11 The magnetic bead 2 shown has magnetic metal particles 22 and a coating layer 24. The coating layer 24 preferably covers the entire surface of the magnetic metal particles 22, but it may also have interrupted portions. Furthermore, the coating layer 24 can be provided as needed or omitted.

[0150] 3.1. Magnetic metal particles

[0151] Magnetic metal particles 22 are particles containing magnetic metal. Examples of constituent materials for magnetic metal particles 22 include Fe-based alloys, Co-based alloys, and Ni-based alloys. In particular, from the viewpoint of obtaining high saturation magnetization, Fe-based alloys (alloys with Fe as the main component) are preferred as constituent materials for magnetic metal particles 22.

[0152] Fe-based alloys are any alloys in which the Fe content is 50% or more in terms of atomic percentage, and the Fe content is preferably 70% or more.

[0153] Fe-based alloys are mainly composed of Fe, and depending on the target properties, they may also contain one or more of the following groups: Co, Ni, Cr, Nb, Cu, Al, Mn, Mo, Si, Sn, B, C, P, Ti, and Zr.

[0154] As an example of Fe-based alloys, alloys in which the Si content is preferably 1.0 atomic% or more and 30.0 atomic% or less, more preferably 1.5 atomic% or more and 13.0 atomic% or less, and even more preferably 2.0 atomic% or more and 7.0 atomic% or less. Such alloys, due to their high magnetic permeability, tend to have high saturation magnetization.

[0155] In addition, the Fe-based alloy may also contain at least one of B (boron) at a concentration of 5.0 atomic% or more and 16.0 atomic% or less, and C (carbon) at a concentration of 0.5 atomic% or more and 5.0 atomic% or less. These elements are amorphous-promoting elements, which help to form a stable amorphous or nanocrystalline structure on the magnetic metal particles 22.

[0156] Furthermore, the Fe-based alloy may contain Cr (chromium) at a concentration of 1.0 atomic% or more and 8.0 atomic% or less. This improves the corrosion resistance of the magnetic metal particles 22.

[0157] It should be noted that the total impurity content is preferably 1.0 atomic% or less. If this level is achieved, even with impurities, the aforementioned effects of the magnetic metal particles 22 are unlikely to be impaired. In this specification, impurities refer to elements that are unintentionally introduced into the raw materials of the magnetic metal particles 22 or during manufacturing.

[0158] The main metallic structure constituting the magnetic metal particles 22 can take various forms, such as crystalline structure, amorphous structure, and nanocrystalline structure. Amorphous structure refers to an amorphous structure without crystals, while nanocrystalline structure refers to a structure dominated by fine crystals with a grain size of less than 100 nm. Both amorphous and nanocrystalline structures impart high hardness to the magnetic metal particles 22. Furthermore, by forming an amorphous or nanocrystalline structure, the coercivity of the magnetic beads 2 becomes particularly low, which helps to improve the redispersibility of the magnetic beads 2. It should be noted that the volume fraction of the amorphous or nanocrystalline structure in the magnetic metal particles 22 is preferably 40% or more, more preferably 60% or more. This volume fraction is determined by the results of crystal structure analysis using X-ray diffraction.

[0159] The metallic structure of the magnetic metal particles 22 can be identified by analyzing their crystal structure using X-ray diffraction. Furthermore, it can be determined by observing the microstructure or diffraction pattern of the cut sample using transmission electron microscopy (TEM). For example, in the case of an amorphous structure, diffraction peaks originating from α-Fe phase metal crystals are not visible in X-ray diffraction peak analysis. Additionally, in the case of an amorphous structure, a so-called halo pattern is formed in the TEM electron beam diffraction pattern, and the formation of light spots caused by crystallization is not visible. Nanocrystalline structures, consisting of crystalline structures with a particle size of, for example, less than 100 nm, can be confirmed from TEM images.

[0160] The magnetic metal contained in the magnetic metal particles 22 is particularly preferably an Fe-based amorphous alloy containing Fe, Cr, Si, and B. An Fe-based amorphous alloy is an Fe-based alloy containing an amorphous structure. Such magnetic metal particles 22 have high magnetic permeability and low coercivity. Therefore, by using such magnetic metal particles 22, magnetic beads 2 with good separation and excellent redispersibility in magnetic separation can be achieved.

[0161] 3.2. Coating layer

[0162] The coating layer 24 coats the magnetic metal particles 22 and contains gold or inorganic oxide. Based on this structure, the coating layer 24 can be given the function of protecting the magnetic metal particles 22. This suppresses the leaching of metal ions and the like from the magnetic metal particles 22. As a result, the occurrence of refining defects and inspection defects associated with metal ions and the like can be suppressed. It should be noted that the coating layer 24 can be provided as needed or omitted.

[0163] Examples of inorganic oxides include silicon oxide, magnesium oxide, calcium oxide, aluminum oxide, titanium oxide, zirconium oxide, boron oxide, yttrium oxide, and molybdenum oxide. One or a mixture of two or more of these can be used.

[0164] The inorganic oxide is preferably silicon oxide. Since silicon oxide is chemically stable, it can effectively suppress the oxidation and corrosion of the magnetic metal particles 22, thereby significantly improving the corrosion resistance of the magnetic beads 2. Furthermore, it can impart good adsorption properties to the coating layer 24 for nucleic acids.

[0165] Silicon oxide is represented by the formula SiO. x (0 < x ≤ 2), preferably SiO2. In addition, silicon oxide can also form a composite oxide or complex with one or more of the following materials: Al, Ti, V, Nb, Cr, Mn, Sn and Zr.

[0166] The coating layer 24 may contain substances other than the inorganic oxide (impurities) in a proportion of less than 50% by mass of the aforementioned inorganic oxide, without impairing its effectiveness. For example, when silicon oxide is used as the inorganic oxide, C, N, P, etc., can be listed as impurities. The composition of the inorganic oxide can be confirmed by, for example, EDX analysis, Auger electron spectroscopy, etc.

[0167] The thickness of the coating layer 24 is preferably 10 nm or more and 200 nm or less, more preferably 15 nm or more and 100 nm or less, and even more preferably 20 nm or more and 100 nm or less. Therefore, even if the magnetic beads 2 collide with each other or with the inner wall of the container 9, damage or peeling of the coating layer 24 can be suppressed. As a result, the dissolution of iron ions and the like accompanying the exposure of the magnetic metal particles 22 can be suppressed. Furthermore, the reduction in magnetization per unit volume of the magnetic beads 2 can be suppressed, and the reduction in the moving speed of the magnetic beads 2 can be suppressed.

[0168] Gold has high corrosion resistance, thus effectively suppressing the oxidation and corrosion of magnetic metal particles 22. By providing a gold-containing coating layer 24, the reduction in magnetization of magnetic metal particles 22 can be suppressed, and the decrease in the adsorption efficiency of nucleic acids accompanying the dissolution of iron ions can also be suppressed.

[0169] Gold can exist in the form of monomers or alloys. In addition, if the coating layer 24 contains gold, it may also contain impurities other than gold.

[0170] The thickness of the gold-containing coating layer 24 is preferably 0.5 nm or more and 50 nm or less, more preferably 1 nm or more and 30 nm or less, and even more preferably 2 nm or more and 20 nm or less. This ensures particularly good corrosion resistance of the magnetic bead 2. Furthermore, it suppresses the decrease in magnetization per unit volume of the magnetic bead 2 and suppresses the decrease in the movement speed of the magnetic bead 2.

[0171] It should be noted that the coating layer 24 can coat the surface of one magnetic metal particle 22, or it can coat multiple magnetic metal particles 22 together.

[0172] The thickness of the coating layer 24 can be determined, for example, by observing cross-sectional images of the magnetic bead 2 obtained using a transmission electron microscope or a scanning electron microscope. Specifically, it can be calculated by obtaining multiple cross-sectional images of the coating layer 24 and averaging the measurements from image processing, etc. For example, for one magnetic bead 2, the thickness of the coating layer 24 can be measured at five or more locations, the average value can be calculated, and then the average value can be further averaged using ten or more magnetic beads 2 to obtain the thickness.

[0173] 3.3. Characteristics of Magnetic Beads

[0174] The average particle size D50 of the magnetic beads 2 is preferably 0.5 μm or more and 30 μm or less, more preferably 1 μm or more and 20 μm or less, even more preferably 2 μm or more and 15 μm or less, and particularly preferably 3 μm or more and 10 μm or less. When the average particle size D50 of the magnetic beads 2 is within this range, the specific surface area of ​​the magnetic beads 2 can be sufficiently increased, and the magnetic beads 2 can generate attractive and adsorption forces suitable for magnetic separation. Furthermore, the aggregation of the magnetic beads 2 can be suppressed, and dispersibility can be improved.

[0175] It should be noted that when the average particle size D50 of magnetic beads 2 is below the lower limit, the magnetization value of magnetic beads 2 decreases, and they become more prone to aggregation. As a result, the adsorption efficiency of nucleic acids may decrease, or impurities may easily be incorporated. Additionally, the movement speed of magnetic beads 2 decreases, potentially increasing the time required for magnetic separation. On the other hand, when the average particle size D50 of magnetic beads 2 is above the upper limit, the specific surface area of ​​magnetic beads 2 decreases, thus failing to adsorb a sufficient amount of nucleic acid, potentially reducing the nucleic acid yield. Furthermore, magnetic beads 2 tend to settle, reducing the number of magnetic beads 2 that aid in nucleic acid extraction, potentially further decreasing the nucleic acid yield.

[0176] It should be noted that the average particle size D50 of magnetic bead 2 can be determined by measuring the particle size distribution on a volume basis using laser diffraction / dispersion method, and then calculated from the cumulative distribution curve obtained from this particle size distribution. Specifically, in the cumulative distribution curve, the particle size (median particle size) with a cumulative value of 50% from the small diameter side is the average particle size D50 of magnetic bead 2. Examples of devices for determining particle size distribution using laser diffraction / dispersion method include the MT3300 series manufactured by Microtrac BEL.

[0177] The coercivity Hc of the magnetic metal particles 22 is preferably 100 A / m or less, more preferably 80 A / m or less, even more preferably 60 A / m or less, and particularly preferably 50 A / m or less. Coercivity 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. That is, coercivity Hc refers to the resistance to an external magnetic field. The smaller the coercivity Hc of the magnetic metal particles 22, the less likely the magnetic beads 2 are to agglomerate when switching from a state with an applied magnetic field to a state without an applied magnetic field, allowing the magnetic beads 2 to be uniformly dispersed in the liquid 3. Furthermore, even when switching between applied magnetic fields repeatedly, the redispersibility of the magnetic beads 2 can be improved. It should be noted that the lower limit of the coercivity Hc of the magnetic metal particles 22 is not particularly limited, but from the viewpoint of ease of material selection that balances performance and cost, it is preferably 5 A / m or more.

[0178] The coercivity Hc of the magnetic metal particle 22 is the same as that of the saturation magnetization described above, and can be measured using a vibrating sample type magnetometer or the like. The maximum applied magnetic field for measuring the coercivity Hc is, for example, 15 kOe.

[0179] 3.4. Manufacturing method of magnetic beads

[0180] Next, an example of the manufacturing method of magnetic bead 2 will be described.

[0181] First, magnetic metal particles 22 are prepared. The magnetic metal particles 22 are manufactured using a standard method for manufacturing general metal powders. Examples of manufacturing methods include melting processes that melt, solidify, and pulverize metal; chemical processes that produce powders through reduction and carbonyl processes; and mechanical processes that mechanically crush materials of larger shapes, such as ingots, to obtain powder. Among these, melting processes such as atomization are suitable for manufacturing magnetic metal particles 22.

[0182] Next, a coating layer 24 is formed on the surface of the magnetic metal particles 22. Methods for forming the coating layer 24 include, for example, wet formation methods such as sol-gel methods and plating methods, vacuum evaporation methods, sputtering methods, and dry formation methods such as ALD (Atomic Layer Deposition). Among these, the Stuber method and the ALD method, both of which are types of sol-gel methods, are preferred.

[0183] As described above, a magnetic bead 2 with magnetic metal particles 22 and a coating layer 24 can be obtained.

[0184] 4. Variations

[0185] Next, the magnetic support involved in the modified embodiment will be described.

[0186] Figure 13 This is a top view of the magnetic support 1 according to a variation of the embodiment.

[0187] The following description describes variations, but focuses on the differences from the embodiments described above; identical details are omitted. It should be noted that... Figure 13 In this document, structures identical to those described in the preceding embodiments are marked with the same symbols.

[0188] Except for the different structure of the magnetic field application part 12, the modified example is the same as the embodiment described above.

[0189] Figure 13 The magnetic field applying unit 12 shown has magnet pairs 127A (first magnet pair), 127B (second magnet pair), 127C, and 127D, which serve as magnets 122. Additionally, Figure 13 The movable part 124 shown is a rod-shaped part extending along the Y-axis (second axis), supporting magnet pairs 127A, 127B, 127C, and 127D arranged along the Y-axis. Magnet pairs 127A, 127B, 127C, and 127D each have a magnet 122c (third magnet) and a magnet 122d (fourth magnet).

[0190] Magnet 122c is the same as the first magnet 122a in the previously described embodiment, and the extension line EL of the orientation of the magnetic pole MP does not overlap with the container 9.

[0191] On the other hand, magnet 122d is the same as the second magnet 122b in the previously described embodiment, and the extension line EL of the orientation of the magnetic pole MP overlaps with the container 9.

[0192] in addition, Figure 13 The movable part 124 shown can rotate around the Y-axis. By rotating the movable part 124, the positions of the multiple magnets 122 (magnets 122c, 122d) change, thereby changing the orientation of the magnetic field generated by the magnets 122.

[0193] Based on this magnetic support 1, similar to the embodiment described above, the orientation of the magnetic field can be changed, thus changing the fixed state of the magnetic beads 2 contained in the container 9. Therefore, in the biological substance purification method, the cleaning fluid remaining after cleaning the magnetic beads 2 can be effectively removed. As a result, adverse conditions caused by the cleaning fluid being carried into subsequent processes can be suppressed.

[0194] Next, regarding Figure 13 The operation of the magnetic support 1 shown will be explained.

[0195] Figure 14 as well as Figure 15 They are Figure 13The magnetic support 1 and the container 9 mounted on the magnetic support 1 are shown in cross-sectional view.

[0196] In the magnetic bead immobilization process of cleaning step S104, after cleaning the magnetic beads 2 adsorbed with nucleic acid with cleaning solution, the container 9 is placed on the magnetic support 1. Then, Figure 14 The magnet 122c shown is positioned close to container 9. Magnet 122c is identical to the first magnet 122a in the previously described embodiment, as... Figure 14 As shown, the extension line EL of the orientation of the magnetic pole MP extends in the Y-axis direction and does not overlap with container 9. Therefore, as Figure 14 As shown, the magnetic bead 2 can be fixed to the inner wall of the container 9 in a concentrated, smaller state.

[0197] During the cleaning fluid discharge process, with magnetic bead 2 fixed in place, as follows: Figure 9 As shown, the cleaning fluid (liquid 3) accumulated at the bottom of container 9 is drained using pipette 6. As a result, the cleaned magnetic beads 2 remain in container 9.

[0198] In the vaporization treatment of cleaning fluid, the operation Figure 13 The magnetic field applying part 12 of the magnetic support 1 shown is used to adjust the relative posture of the magnetic field applying part 12 with respect to the base 11 from... Figure 14 The posture shown has changed to Figure 15 The posture shown. Then, make Figure 15 The magnet 122d shown is positioned close to the container 9. Magnet 122d is identical to the second magnet 122b in the previously described embodiment, as... Figure 15 As shown, the extension line EL of the orientation of the magnetic pole MP extends in the X-axis direction and overlaps with the container 9. Therefore, on the magnetic bead 2, as... Figure 15 As shown, the magnetic beads 2 exhibit a needle-like arrangement (spike phenomenon). This allows the cleaning liquid adhering to the magnetic beads 2 to be effectively vaporized. Consequently, the cleaning liquid can be effectively removed, and the carryover of cleaning liquid components into subsequent processes can be prevented.

[0199] exist Figure 13 In the magnetic support 1 shown, the states of magnet 122c being positioned close to container 9 and magnet 122d being positioned close to container 9 can be easily switched simply by rotating the movable part 124 around the rotation axis AX. Specifically, when the rotation angle of the movable part 124 is at the first rotation angle, as... Figure 14 As shown, magnet 122c (the third magnet) approaches container 9. On the other hand, when the rotation angle of movable part 124 is at a second rotation angle that has rotated 180° from the first rotation angle, as... Figure 15As shown, magnet 122d (the fourth magnet) is close to container 9. With this structure, the magnetic flux density formed inside container 9 can be easily changed, and the fixed state of magnetic bead 2 can be easily changed.

[0200] In the variations described above, the same effects as in the previously described embodiments can be obtained.

[0201] 5. Biological substance purification device

[0202] The apparatus for purifying biological substances involved in the embodiments will be described.

[0203] Figure 16 This is a schematic diagram showing the general structure of the biological substance purification apparatus 100 according to the embodiment.

[0204] The following description describes the biological substance purification apparatus 100 according to the embodiments. However, the description will focus on the differences from the biological substance purification method according to the embodiments described above, and descriptions of identical structures will be omitted. It should be noted that in... Figure 16 In this document, structures identical to those described in the preceding embodiments are marked with the same symbols.

[0205] Figure 16 The biological substance purification device 100 shown includes a magnetic support 1, a support drive unit 71, a cleaning liquid supply unit 72, a cleaning liquid discharge unit 73, a supply and discharge drive unit 74, and a control unit 75.

[0206] The support drive unit 71 causes the magnetic field application unit 12 of the magnetic support 1 to translate or rotate. As a result, the orientation of the magnetic field applied to the container 9 provided on the magnetic support 1 changes.

[0207] The cleaning fluid supply unit 72 supplies cleaning fluid to the container 9 mounted on the magnetic support 1. The cleaning fluid discharge unit 73 discharges cleaning fluid from the container 9 mounted on the magnetic support 1. An electric pipette can be cited as an example of the cleaning fluid supply unit 72 and the cleaning fluid discharge unit 73.

[0208] The supply and discharge drive unit 74 changes the relative positions of the cleaning fluid supply unit 72 and the cleaning fluid discharge unit 73 relative to the magnetic support 1. This allows cleaning fluid to be supplied to or discharged from a predetermined container 9 from among multiple containers 9.

[0209] The control unit 75 controls the operation of the support drive unit 71, the cleaning fluid supply unit 72, the cleaning fluid discharge unit 73, and the supply and discharge drive unit 74. This enables the automatic execution of the biological substance purification method described in the preceding embodiments.

[0210] Specifically, in the cleaning process S104, firstly, the cleaning liquid supply unit 72 supplies cleaning liquid to the container 9 for cleaning. Next, a magnetic bead fixing process is performed. Specifically, the support drive unit 71 drives the magnetic field application unit 12 of the magnetic support 1, bringing the first magnet 122a close to the container 9. Next, a cleaning liquid discharge process is performed. Specifically, the cleaning liquid discharge unit 73 discharges the cleaning liquid from the container 9. At this time, since the fixing state of the magnetic bead 2 is optimized, interference between the fixed magnetic bead 2 and the cleaning liquid discharge unit 73 can be suppressed. This improves the reliability of the cleaning liquid discharge process and effectively removes the cleaning liquid. Next, a cleaning liquid vaporization process is performed. Specifically, the support drive unit 71 drives the magnetic field application unit 12, bringing the second magnet 122b close to the container 9. This state is then maintained for a certain period of time. At this time, since the fixing state of the magnetic bead 2 is optimized, the cleaning liquid can be thoroughly dried. This suppresses the occurrence of adverse conditions caused by the cleaning liquid being carried into subsequent processes.

[0211] It should be noted that, in addition to the above-described structure, the biological substance purification device 100 may also include a heating mechanism and a ventilation mechanism. The heating mechanism heats the cleaning liquid or the magnetic beads 2, promoting the vaporization of the cleaning liquid. The ventilation mechanism improves the gas exchange efficiency around the magnetic beads 2, further promoting the vaporization of the cleaning liquid.

[0212] 6. Effects of the implementation method

[0213] As described above, the biological substance purification method according to the embodiment includes an adsorption step S102, a washing step S104, and a dissolution step S106. In the adsorption step S102, magnetic beads 2 saturated with a magnetization of 50 emu / g or higher, biological substance, and liquid 3 are placed in a container 9, and after the biological substance is adsorbed onto the magnetic beads 2, the liquid 3 is removed. In the washing step S104, after the adsorption step S102, the magnetic beads 2 with adsorbed biological substance are washed with a washing solution. In the dissolution step S106, after the washing step S104, the biological substance adsorbed on the magnetic beads 2 is dissolved.

[0214] In addition, the cleaning process S104 includes magnetic bead fixing, cleaning liquid discharge, and cleaning liquid vaporization. In the magnetic bead fixing process, after cleaning the magnetic beads 2 that have adsorbed biological material with the cleaning liquid, the magnetic beads 2 are fixed by arranging magnets 122 such that the extension line EL of the orientation of the magnetic pole MP does not overlap with the container 9. In the cleaning liquid discharge process, the cleaning liquid is discharged from the container 9 while the magnetic beads 2 are fixed. In the cleaning liquid vaporization process, after the cleaning liquid discharge process, the cleaning liquid is vaporized while the magnets 122 are arranged such that the extension line EL of the orientation of the magnetic pole MP overlaps with the container 9 and the magnetic field gradient within the container 9 is 40 [T / m] or higher.

[0215] With this structure, the fixation state of the magnetic beads 2 can be optimized during the cleaning fluid vaporization process, thus ensuring thorough drying of the cleaning fluid. This prevents undesirable situations caused by the cleaning fluid being carried into subsequent processes. Furthermore, with the same structure, the fixation state of the magnetic beads 2 can be optimized during the cleaning fluid discharge process, thus suppressing interference between the fixed magnetic beads 2 and the pipette 6. This improves the workability of the cleaning fluid discharge process and effectively removes the cleaning fluid.

[0216] In the biological substance purification method involved in the embodiments, it is preferable that the saturation magnetization of the magnetic beads 2 is 100 [emu / g] or more, and the magnetic field gradient in the container 9 for cleaning liquid vaporization treatment is 100 [T / m] or more.

[0217] Based on this structure, the drying efficiency of magnetic bead 2 can be significantly improved. Furthermore, since the moving speed of magnetic bead 2 in the magnetic field can be increased, the time required for magnetic separation can be shortened.

[0218] In the biological material purification method of the embodiment, the cleaning liquid vaporization treatment is preferably carried out when the angle θ between the extension line EL of the orientation of the magnetic pole MP of the magnet 122 and the tangent TL of the inner wall of the container 9 is 45° or more and 90° or less.

[0219] With this structure, the magnetic beads 2 can be extended over a wider range within the internal space 90 of the container 9. This, in turn, significantly improves the drying efficiency of the magnetic beads 2.

[0220] In the biological material purification method described in the embodiments, the duration of the cleaning liquid vaporization treatment is preferably 3 minutes or more.

[0221] With this structure, the cleaning fluid can be fully vaporized, thus further reducing the probability that components of the cleaning fluid will be carried into subsequent processes.

[0222] In the biological material purification method of the embodiment, the position of the magnet 122 in the cleaning liquid vaporization treatment is preferably closer to the opening than the midpoint CP of the height H9 of the container 9.

[0223] With this structure, the distance between the fixed magnetic bead 2 and the opening 91 of the container 9 is shortened, and the gas exchange efficiency around the magnetic bead 2 is improved. As a result, the drying efficiency of the magnetic bead 2 can be further improved.

[0224] The magnetic support 1 described in this embodiment is a magnetic support that separates the magnetic beads 2 and the liquid 3 by applying a magnetic field generated by a magnet 122 to a container 9 containing magnetic beads 2, biological material, and liquid 3. It includes a base 11 and a magnetic field applying part 12. The base 11 extends along the Z-axis (first axis) and has an insertion hole 13 for inserting the container 9. The magnetic field applying part 12 is provided on the base 11 and applies a magnetic field to the insertion hole 13.

[0225] In addition, the magnetic field applying part 12 has a plurality of magnets 122 and a movable part 124 for changing the position of the magnets 122. Furthermore, the movable part 124 is rod-shaped and extends along the Y-axis (second axis) that intersects the Z-axis (first axis), supports the plurality of magnets 122 arranged along the Y-axis, and changes the orientation of the magnetic field generated by the plurality of magnets 122 by translating along the Y-axis or rotating around the Y-axis.

[0226] Based on this structure, the orientation of the magnetic field can be changed, thus altering the fixed state of the magnetic beads 2 contained within the container 9. Therefore, in the biological substance refining method, the cleaning solution remaining after cleaning the magnetic beads 2 can be effectively removed. As a result, adverse conditions caused by the cleaning solution being carried into subsequent processes can be suppressed.

[0227] In the magnetic support 1 according to the embodiment, the magnetic field applying part 12 may also have a first magnet 122a and a second magnet 122b as magnets 122. In this case, the movable part 124 translates along the Y-axis (second axis). In addition, the first magnet 122a is supported by the movable part 124 in such a way that the extension line EL of the orientation of the magnetic pole MP does not overlap with the container 9. In addition, the second magnet 122b is provided along the Y-axis at a position adjacent to the first magnet 122a, and is supported by the movable part 124 in such a way that the extension line EL of the orientation of the magnetic pole MP overlaps with the container 9.

[0228] With this structure, a magnetic support 1 can be realized in which the orientation of the magnetic field applied to the container 9 can be easily changed by translating the magnetic field application part 12 in the Y-axis direction. Therefore, a magnetic support 1 can be realized in which the magnetic flux density formed in the container 9 can be easily changed and the fixed state of the magnetic bead 2 can be easily changed.

[0229] In the magnetic support 1 according to the embodiment, the magnetic field applying part 12 may also have a magnet pair 127A (first magnet pair) and a magnet pair 127B (second magnet pair) as magnets 122. In this case, the movable part 124 rotates about the Y-axis (second axis) between a first rotation angle and a second rotation angle that has rotated 180° from the first rotation angle. In addition, the first magnet pair 127A and the second magnet pair 127B each include a magnet 122c (third magnet) and a fourth magnet, respectively. The magnet 122c (third magnet) is configured such that when the movable part 124 is in the posture of the first rotation angle, the extension line EL of the orientation of the magnetic pole MP does not overlap with the container 9, and the fourth magnet is configured such that when the movable part 124 is in the posture of the second rotation angle, the extension line EL of the orientation of the magnetic pole MP overlaps with the container 9.

[0230] With this structure, a magnetic support 1 can be realized in which the orientation of the magnetic field applied to the container 9 can be easily changed by rotating the magnetic field application part 12 around the Y-axis. Therefore, a magnetic support 1 can be realized in which the magnetic flux density formed in the container 9 can be easily changed and the fixed state of the magnetic bead 2 can be easily changed.

[0231] The biological substance purification apparatus 100 according to the embodiment includes a magnetic support 1, a support drive unit 71, a cleaning fluid supply unit 72, a cleaning fluid discharge unit 73, and a control unit 75, as described in the embodiment. The support drive unit 71 drives the magnetic field application unit 12 of the magnetic support 1. The cleaning fluid supply unit 72 supplies cleaning fluid to a container 9 inserted into an insertion hole 13 of the magnetic support 1. The cleaning fluid discharge unit 73 discharges cleaning fluid from the container 9. The control unit 75 controls the operation of the support drive unit 71, the cleaning fluid supply unit 72, and the cleaning fluid discharge unit 73.

[0232] Based on this structure, a method for refining biological materials, including magnetic bead fixation, cleaning fluid discharge, and cleaning fluid vaporization, can be performed automatically. Furthermore, in the cleaning fluid vaporization process, the fixation state of the magnetic beads 2 can be optimized, thus ensuring thorough drying of the cleaning fluid. This prevents undesirable situations caused by the cleaning fluid being carried into subsequent processes. Additionally, in the cleaning fluid discharge process, the fixation state of the magnetic beads 2 can be optimized, thus suppressing interference between the fixed magnetic beads 2 and the cleaning fluid discharge section 73. This improves the reliability of the cleaning fluid discharge process and effectively removes the cleaning fluid.

[0233] In the biological substance purification apparatus 100 according to the embodiment, the control unit 75 may operate the cleaning liquid discharge unit 73 while the support drive unit 71 is operating and the magnet 122 is arranged in such a way that the extension line EL of the orientation of the magnetic pole MP does not overlap with the container 9. After the cleaning liquid is discharged from the container 9, the support drive unit 71 is operated and the magnet 122 is arranged in such a way that the extension line EL of the orientation of the magnetic pole MP overlaps with the container 9.

[0234] Based on this structure, the fixation state of the magnetic beads 2 can be optimized during the cleaning fluid vaporization process, thus enabling the cleaning fluid to dry thoroughly. Furthermore, during the cleaning fluid discharge process, since the fixation state of the magnetic beads 2 can be optimized, interference between the fixed magnetic beads 2 and the cleaning fluid discharge section 73 can be suppressed.

[0235] The above description, based on the illustrated embodiments, illustrates the method for refining biological substances, the magnetic scaffold, and the apparatus for refining biological substances according to the present invention; however, the present invention is not limited thereto. For example, the method for refining biological substances according to the present invention may also be a method with additional steps for any purpose added to the described embodiments. Furthermore, the magnetic scaffold and the apparatus for refining biological substances according to the present invention may each have arbitrary structures added to the described embodiments, or may be a structure in which a portion of the structures in the described embodiments is replaced by other structures with equivalent functions.

[0236] Example

[0237] Next, specific embodiments of the present invention will be described.

[0238] 7. Fabrication of magnetic beads and purification of nucleic acids

[0239] After the magnetic beads for each embodiment and comparative example were prepared as described below, nucleic acid purification was performed using the prepared magnetic beads.

[0240] Figure 17 Table 1 shows the conditions of the cleaning process for nucleic acid purification when using magnetic beads from the comparative example, as well as the evaluation results of nucleic acid purification.

[0241] Figure 18 Table 2 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0242] Figure 19 Table 3 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0243] Figure 20Table 4 shows the conditions of the cleaning process for nucleic acid purification and the evaluation results of nucleic acid purification when using the magnetic beads of the examples and comparative examples.

[0244] 7.1. Example 1

[0245] First, the fabrication of the magnetic beads and the purification of nucleic acids in Example 1 will be explained.

[0246] 7.1.1. Fabrication of Magnetic Beads

[0247] First, Fe-based amorphous alloy powder, manufactured by water atomization, was prepared as magnetic metal particles.

[0248] Next, using the Stuber process, TEOS (tetraethoxysilane) was brought into contact with the surface of the magnetic metal particles, followed by sintering to form a coating layer composed of silicon oxide. This yielded magnetic beads. The saturation magnetization of the fabricated magnetic beads is shown in Table 2. Figure 18 )middle.

[0249] In addition, other properties of the manufactured magnetic beads are described below.

[0250] Average particle size D50: 10 μm

[0251] Coercivity: 50A / m

[0252] Coating thickness: 30nm

[0253] 7.1.2. Nucleic Acid Purification

[0254] First, the magnetic bead dispersion containing 20 mg of the magnetic beads from Example 1 was aliquoted into a 1.5 mL tube and placed at room temperature.

[0255] Next, prepare another 1.5mL tube (container), take out the sample solution containing nucleic acid, and place it at room temperature.

[0256] Next, an adsorption process was performed. Specifically, 750 μL of magnetic bead dispersion and dissolving adsorption solution were added to the tube containing the sample solution. Then, the contents of the tube were stirred using a vortex mixer for 9 minutes. The stirred tube was then placed in… Figure 1 The magnetic support shown has undergone magnetic separation and liquid discharge treatment.

[0257] Next, a cleaning process was performed. Specifically, 500 μL of cleaning solution was added to the tube after the adsorption process. The cleaning solution used was an 80% (v / v) aqueous ethanol solution. Then, the contents of the tube were stirred using a vortex mixer for 3 minutes. Next, a magnetic bead fixation process was performed. In this process, the tube was placed on a magnetic support and left for 20 seconds to fix the magnetic beads to the inner wall of the tube. At this time, the orientation of the magnetic poles was set as shown in Table 2. Next, a cleaning solution discharge process was performed. In this process, the supernatant cleaning solution was removed using a pipette. Next, a cleaning solution vaporization process was performed. In this process, the tube was placed on the magnetic support for the duration shown in Table 2. This vaporized the cleaning solution adhering to the magnetic beads. At this time, the magnetic field application part of the magnetic support was operated, and the orientation of the magnetic poles and the angle of intersection between the magnetic poles and the inner wall of the container were set as shown in Table 2. Furthermore, the surface magnetic flux density of the magnet and the distance between the magnet and the container were set such that the magnetic field gradient inside the tube was the value shown in Table 2.

[0258] Next, a dissolution process was performed. Specifically, 50 μL of room temperature dissolution solution was added to the tubes after the washing process. Ultrapure water was used as the dissolution solution. Then, the contents of the tubes were stirred using a vortex mixer for 3 minutes. This dissolved the nucleic acids captured by the magnetic beads. Then, magnetic separation was performed. In the magnetic separation process, the tubes were placed on a magnetic support and left for 30 seconds. Next, liquid discharge was performed, and the supernatant dissolution solution was recovered.

[0259] 7.2. Examples 2-8 and Comparative Examples 1-20

[0260] In addition to specifying the structure of the magnetic beads and the conditions for nucleic acid purification according to Table 1 ( Figure 17 Table 2 Figure 18 Table 3 Figure 19 ) and Table 4 ( Figure 20 Except for the changes shown, magnetic beads were made in the same manner as in Example 1, and nucleic acid purification was performed.

[0261] 8. Evaluation of nucleic acid purification

[0262] Next, the nucleic acid purification of each embodiment and each comparative example was evaluated.

[0263] 8.1. Presence or absence of PCR interference

[0264] The presence or absence of PCR interference in nucleic acid purification was evaluated as described below.

[0265] First, 2 μL of the recovered dissolution solution was added to a 10 μL real-time PCR reaction system, and the number of cycles until the PCR amplification curve rose was determined. In cases where washing solution or other contaminants are incorporated into the magnetic beads, leaving residual solution, this residual solution can sometimes transfer to the dissolution solution, causing PCR interference. This leads to an increase in the number of cycles. Therefore, the PCR interference caused by the transfer of washing solution was evaluated by comparing the number of cycles until the PCR amplification curve rose with the following evaluation criteria. The evaluation results are shown in Tables 1 to 4.

[0266] A: The PCR amplification curve rises at a sufficiently early moment.

[0267] B: Although the PCR amplification curve rises later than A, it does not hinder the detection.

[0268] C: The PCR amplification curve did not rise.

[0269] 8.2. Workability of cleaning fluid discharge treatment

[0270] The workability of the cleaning fluid discharge treatment in the cleaning process was evaluated as follows.

[0271] During the cleaning fluid discharge process, a pipette was used to remove the supernatant cleaning fluid. The workability of this process was evaluated according to the following evaluation criteria. The evaluation results are shown in Tables 1 to 4.

[0272] A: The distance between the pipette and the magnetic bead is sufficient, making it very easy to work with.

[0273] B: The distance between the pipette and the magnetic bead is narrow, making the operation slightly more time-consuming.

[0274] C: Interference occurs between the pipette and the magnetic bead, resulting in poor operability.

[0275] 8.3. Investigation

[0276] Based on the evaluation results shown in Tables 1 to 4, the following situations can be confirmed.

[0277] In the nucleic acid purification processes of each embodiment, PCR interference caused by the transfer of washing solution can be suppressed. This result confirms that, from the viewpoint of thoroughly and effectively removing washing solution and suppressing nucleic acid testing defects, washing solution vaporization is effective.

[0278] In the nucleic acid purification of each comparative example, sufficient results were not obtained from the perspective of inhibiting PCR interference. Reasons for this include insufficient saturation magnetization of the magnetic beads, the orientation of the magnetic poles during the vaporization treatment of the washing solution, and the magnetic field gradient.

[0279] In the nucleic acid purification process of each embodiment, it was confirmed that the workability of the cleaning solution discharge treatment in the cleaning step is high. Based on this result, it was confirmed that optimizing the orientation of the magnetic field during the cleaning solution discharge treatment is effective from the viewpoint of efficiently removing the cleaning solution.

Claims

1. A method for purifying biological substances, characterized in that, have: The adsorption process involves placing magnetic beads, biological material, and liquid that have been saturated with magnetization to 50 emu / g or higher into a container, allowing the biological material to adsorb onto the magnetic beads, and then removing the liquid. The cleaning process involves washing the magnetic beads, which have adsorbed the biological material, with a cleaning solution after the adsorption process. as well as The dissolution process, following the washing process, dissolves the biological material adsorbed on the magnetic beads. The cleaning process includes: The magnetic bead fixing process involves cleaning the magnetic beads, which have adsorbed the biological material, with the cleaning solution, and then fixing the magnetic beads by arranging magnets in a manner in which the extension lines of the orientation of the magnetic poles do not overlap with the container. The cleaning fluid is discharged by draining the cleaning fluid from the container while the magnetic beads are fixed in place; and The cleaning fluid is vaporized after the cleaning fluid is discharged. The magnet is arranged such that the extension line of the orientation of the magnetic pole overlaps with the container and the magnetic field gradient in the container is 40 T / m or more.

2. The method for purifying biological substances according to claim 1, wherein, The saturation magnetization of the magnetic beads is above 100 emu / g. The magnetic field gradient inside the container treated by the vaporization of the cleaning fluid is above 100 T / m.

3. The method for purifying biological substances according to claim 1 or 2, wherein, The cleaning fluid vaporization process is performed with the magnets arranged such that the angle between the extension of the magnetic poles and the tangent to the inner wall of the container is 45° or more and 90° or less.

4. The method for purifying biological substances according to claim 1 or 2, wherein, The duration of the cleaning fluid vaporization treatment is more than 3 minutes.

5. The method for purifying biological substances according to claim 1 or 2, wherein, The magnet in the vaporization process of the cleaning fluid is positioned closer to the opening than the midpoint of the container's height.

6. A magnetic support, characterized in that, The magnetic support separates the magnetic beads from the liquid by applying a magnetic field generated by a magnet to a container containing magnetic beads, biological material, and liquid. A base having an insertion hole extending along a first axis for inserting the container; and A magnetic field applying part is disposed on the base to apply a magnetic field to the insertion hole. The magnetic field applying part has: Multiple magnets; as well as A movable part that allows the position of the magnet to change. The movable part is rod-shaped and extends along a second axis that intersects the first axis, supports a plurality of magnets arranged along the second axis, and changes the orientation of the magnetic field generated by the plurality of magnets by translating along the second axis or rotating around the second axis.

7. The magnetic support according to claim 6, wherein, The magnetic field applying part has a first magnet and a second magnet, which serve as the magnet. The movable part translates along the second axis. The first magnet is supported on the movable part in such a way that the extension of the orientation of its magnetic poles does not overlap with the container. The second magnet is positioned adjacent to the first magnet along the second axis and is supported on the movable part in such a way that the extension of the orientation of the magnetic poles overlaps with the container.

8. The magnetic support according to claim 6, wherein, The magnetic field applying part has a first magnet pair and a second magnet pair, which serve as the magnets. The movable part rotates about the second axis between a first rotation angle and a second rotation angle that has rotated 180° from the first rotation angle. The first magnet pair and the second magnet pair each include a third magnet and a fourth magnet. The third magnet is configured such that when the movable part is in the posture of the first rotation angle, the extension line of the orientation of the magnetic pole does not overlap with the container. The fourth magnet is configured such that when the movable part is in the posture of the second rotation angle, the extension line of the orientation of the magnetic pole overlaps with the container.

9. A biological substance purification device, characterized in that, have: The magnetic support according to any one of claims 6 to 8; A support drive unit that drives the magnetic field application unit of the magnetic support; The cleaning fluid supply unit supplies cleaning fluid to the container inserted into the insertion hole of the magnetic support; A cleaning fluid discharge section discharges the cleaning fluid from the container; as well as The control unit controls the operation of the support drive unit, the cleaning fluid supply unit, and the cleaning fluid discharge unit.

10. The biological substance purification apparatus according to claim 9, wherein, The control unit activates the support drive unit, and in a state where the magnet is arranged such that the extension line of the magnetic pole orientation does not overlap with the container, the cleaning fluid discharge unit activates the support drive unit, and after the cleaning fluid is discharged from the container, the support drive unit activates the support drive unit, and the magnet is arranged such that the extension line of the magnetic pole orientation overlaps with the container, and the support drive unit is maintained in this state.

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