Magnetic particle separation device

Abstract

Provided is a magnetic particle separation device in which an adsorption amount of magnetic powder in a liquid in an end part can be made substantially equal to that in a center part even when using a specimen container having the same size as a conventional one.　The magnetic particle separation device includes: a plurality of specimen containers in which a mixture composed of a liquid and magnetic particles is accommodated; a first magnet arranged for each of the plurality of specimen containers and capable of moving up and down independently of the specimen container; a magnetic body container that covers the first magnet; and a hole plate having holes into which the plurality of specimen containers can be placed. In the hole plate, at least side surface parts of the holes close to the specimen containers are made of a soft magnetic body.

Description

Magnetic particle separation device

[0001] The present invention relates to a magnetic particle separator that separates magnetic particles from a liquid in which the magnetic particles are suspended.

[0002] In recent years, information obtained through nucleic acid analysis, such as cancer genome testing using next-generation sequencing (NGS) systems, has been utilized in various fields, including medicine, clinical testing, the pharmaceutical industry, and the food industry. For this nucleic acid analysis, nucleic acid extraction from various biological samples, such as blood, tissue, and cultured cells, is an essential pretreatment step.

[0003] Nucleic acid extraction methods generally do not use harmful organic solvents such as phenol or chloroform, but rather rely on the property of nucleic acids binding to silica in the presence of a chaotropic agent or on the property of nucleic acids binding to silica in the presence of an organic solvent. Using these methods, nucleic acid extraction methods using a nucleic acid capture chip incorporating a silica-containing solid phase as a nucleic acid capture carrier, and methods using magnetic beads (nucleic acid capture carriers) with silica-coated surfaces have been reported. These methods include a step of binding nucleic acids to the nucleic acid capture carrier and an elution step of eluting the nucleic acids from the nucleic acid capture carrier using an eluent.

[0004] In the method using magnetic beads, after the elution step, the magnetic beads are recovered from the eluate using a magnet. Patent Document 1 describes a method for efficiently collecting magnetic beads, in which the magnetic beads are first collected on the wall of a container and then recovered using a magnetic rod. The method discloses that adjacent magnets are magnetically coupled by arranging the magnetization directions of the adjacent magnets in different directions, thereby minimizing the difference in magnetic flux density distribution between the edge and center of the reagent container.

[0005] Special Publication No. 2007-520331

[0006] In a DNA extraction and purification device in which multiple specimen containers containing a mixture of liquid containing DNA and magnetic particles that have been surface-treated to adsorb DNA are arranged vertically and horizontally, a combination of a magnetic rod and magnet and the magnetic container move up and down independently relative to the specimen container, causing a DNA adsorption reaction through stirring.After this, the magnetic particles are collected on the surface of the magnetic container near the magnet, and in this "magnetic collection" process, magnetic coupling occurs between the magnets in small containers where the distance between each container is short, and the magnetic flux density distribution in the liquid differs between the edges and center of the specimen container, resulting in different amounts of magnetic powder being adsorbed.

[0007] In Patent Document 1, the magnetization directions of adjacent magnets are set in different directions to magnetically couple them, thereby suppressing the difference in magnetic flux density distribution between the edge and center of a sample container, but the edge magnets have few adjacent magnets, resulting in a difference between the edge and center. The difference can be reduced by arranging more magnets than the number of sample containers, but this requires a larger space and a larger number of magnets, which is an issue.

[0008] An object of the present invention is to provide a magnetic particle separator that can make the amount of magnetic powder adsorbed in liquid at the ends and the center almost the same even when using a specimen container of the same size as conventional ones.

[0009] To achieve the above object, the present invention is configured as follows: A magnetic particle separation device comprising: a plurality of sample containers each containing a mixture of liquid and magnetic particles; a first magnet disposed for each of the plurality of sample containers and movable up and down independently of the sample container; a magnetic container covering the first magnet; and a hole plate having holes on which the plurality of sample containers can be placed, wherein at least the side portion of the hole plate that is closest to the sample container is made of a soft magnetic material.

[0010] It is possible to provide a particle separator that can make the amount of magnetic powder adsorbed in the liquid at the end and the center substantially the same even when the specimen container is the same size as a conventional one.

[0011] 10. A perspective view of a nucleic acid extraction device according to the present invention. A transparent cross-sectional view of a nucleic acid extraction device according to the present invention. A diagram showing an example of an 8 x 12 array magnetic container 4. A top view of a hole plate and holes. A transparent cross-sectional view of a hole plate. A diagram showing the positions of magnetic flux density evaluation points. A diagram showing the difference in magnetic flux density between the end and center of a hole plate. A diagram showing a state where the hole plate position has been changed. Calculation results of the magnetic flux density distribution difference in FIG. 8. A diagram showing the positions of the upper end face and lower end face of the hole plate. Calculation results of the magnetic flux density distribution difference in FIG. 10. A diagram showing a state where the position of the upper magnet has been changed. A diagram showing an embodiment using an upper magnet and a lower magnet. A diagram showing the magnetic collection flow of magnetic particles using an upper magnet and a lower magnet. A diagram showing the positions of the upper end face and lower end face of the hole plate. Calculation results of the magnetic flux density distribution difference in FIG. 15. An embodiment with tapered holes. An embodiment with grooves. A diagram showing the difference in driving force depending on whether tapering is performed or not.

[0012] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0013] 1 and 2 are a perspective view and a cross-sectional perspective view, respectively, of a nucleic acid extraction apparatus 100 according to the present invention. The nucleic acid extraction apparatus 100 is an apparatus for extracting nucleic acids from a sample containing nucleic acids and magnetic particles.

[0014] The nucleic acid extraction apparatus 100 includes a linear motion mechanism A1 that moves the magnetic rod 3 vertically, a linear motion mechanism A2 that moves the magnetic container 4 vertically, a linear motion mechanism B that moves the specimen container 1 horizontally together with a hole plate (container holding member) 2 that holds the specimen container 1, and a motor 101 that drives these linear motion mechanisms. The specimen container 1 is held by the hole plate (container holding member) 2, which will be described later.

[0015] The linear motion mechanism B moves the magnetic container 4 below the magnetic bar 3, and the linear motion mechanism A1 moves the magnetic bar 3 up and down, thereby attaching and detaching the magnetic container 4 to and from the magnetic bar 3. Furthermore, the linear motion mechanism B moves the sample container 1 (with the magnetic container 4 attached) below the magnetic bar 3, and by moving the magnetic container 4 (and the magnetic bar 3) up and down, the liquid in the sample container 1 can be stirred by the magnetic container 4. Note that in this figure, the magnetic containers 4 have different thicknesses, but they may be of the same thickness. The nucleic acid extraction device 100 shown in FIG. 2 is equipped with a lower magnet 10, but the lower magnet 10 is used in Example 2 and not in Example 1. The lower magnet 10 will be described again in Example 2.

[0016] In this embodiment, six magnetic bars 3 are arranged in a horizontal row, but the device may also have multiple rows of magnetic bars 3. For example, it is possible to have eight rows of magnetic bars 3, with 6 x 8 = 48 magnetic bars 3. With this configuration, magnetic bars 3 can be inserted simultaneously into all sample containers placed on the hole plate 2, allowing magnetic particle collection to be performed simultaneously in all sample containers. In this case, the magnetic containers 4 are also configured in a matrix, just like the magnetic bars 3. Figure 3 shows an example of magnetic containers 4 with 8 x 12 rows.

[0017] Next, the details of the hole plate are described. Figure 4 shows a hole plate 2 with holes 9 that can accommodate an array of eight vertical and twelve horizontal sample containers 1. Figure 5 is a cross-sectional perspective view of the hole plate 2. By inserting the sample containers 1 into the holes 9 of the hole plate 2, the sample containers 1 can be arranged side by side. To collect only the magnetic particles 7 from the sample 6 in which magnetic particles 7 are suspended in the sample container 1, a magnetic rod 3, an upper magnet 5 connected to the magnetic rod 3, and a magnetic container 4 (also called a magnetic cover) that covers them are inserted into the sample container 1. The magnetic container 4 is then moved up and down to stir and cause a DNA adsorption reaction. This results in "magnetic collection," in which the magnetic particles 7 are collected on the surface of the magnetic container 4 near the upper magnet 5. In Figure 5, the upper magnets 5 are all magnetized in the same direction (the north pole is positioned downward in the drawing).

[0018] Magnetic field analysis was used to calculate the magnetic flux density at magnetic flux density evaluation points 8 (points marked with "x" in Figure 6) in a liquid containing magnetic particles 7. Figure 7 shows the results of comparing the difference in magnetic flux density between the edge and center of the hole plate in Figure 4. A 14% difference was observed when the upper magnets 5 were magnetized in the same direction and there was no hole plate 2 made of soft magnetic material ("NN without hole plate" - NN means that the magnets are arranged with a north pole, north pole, etc.). This difference could be reduced to 5% by using the "NS without hole plate" (meaning that the magnets are arranged with a north pole, south pole, north pole, etc.) in which the magnetization directions are mutually aligned, as described in Patent Document 1. It can be seen that a further reduction of 1% can be achieved by using the "NN with hole plate" or "NS with hole plate" of the present invention.

[0019] During magnetic collection, the upper magnet 5 and magnetic vessel 4 move synchronously in the vertical direction to collect magnetic particles 7. The hole plate 2 (also called the specimen vessel holder) has a hole that accommodates the specimen vessel. Ideally, the hole 9 would completely cover the upper magnet 5, specimen 6, and magnetic bar 3. However, specimen vessels 1 typically have a hemispherical bottom and a cylindrical top, making it difficult to design a hole plate that covers the top of the specimen vessel 1. Figure 9 shows the difference in magnetic flux density between the edge and center of a 5 mm-thick hole plate positioned at the bottom, middle, and top of the specimen vessel 1, as shown in Figure 8. Figure 9 shows that the edge of the hole does not need to completely cover the upper magnet 5 or magnetic bar 3; it is most effective to cover a distance of approximately 5 mm below the tip of the magnet, as shown in Figure 8. It was also found that placing the hole plate at the top of the vessel did not necessarily affect the difference in magnetic flux density.

[0020] Furthermore, Figure 11 shows the difference in magnetic flux density between the end and center when the thickness H from the magnet tip to the upper end face of the hole plate is changed in 1 mm increments, as shown in Figure 10. From this, it was found that it is desirable for the upper end face of the hole plate to be about 2 mm higher than the magnet tip face, and for the thickness to the lower end of the hole plate to be 7 mm or more.

[0021] Next, the relationship between the sample liquid level and the magnet position is shown in Figure 12. If we consider that the magnetic container 4 and upper magnet 5 can move independently of the sample container 1, and that the attraction of the main magnetic powder occurs when the tip of the magnetic container 4 descends and touches the sample liquid level, in the "magnet position: up" state, then in the case of the dimensional design shown in this figure, it is clear that it is desirable for the upper end surface of the hole plate to be at least 2 mm higher than the tip surface of the magnet when the sample liquid level and magnetic container 4 come into contact, and at least 5 mm above the tip surface of the magnet when the magnetic container 4 is in the "magnet position: down" state when it touches the bottom of the sample container 1.

[0022] It goes without saying that the above ideal dimensional values ​​will differ depending on the device, since the inner and outer diameters of the sample container 1, the size of the upper magnet 5, and the amount of sample 6 differ depending on the device, and the magnetic coupling state between the upper magnet 5 and the hole plate 2 also differs.

[0023] The hole plate 2 may be formed by drilling holes in a soft magnetic material, or may have a lattice structure made by combining processed metal plates. When a metallic soft magnetic material is used, it may also have a temperature control function due to its excellent thermal conductivity. A structure in which holes are drilled in a non-magnetic material such as resin and cylindrical or container-shaped soft magnetic material is placed inside may also be used. The hole plate 2 may be structured to hold only one sample container, as it strengthens the magnetic force and has a magnetic shielding effect. Soft magnetic materials include permalloy, pure iron, iron-based alloys, nickel alloys, cobalt alloys, ferrite, and resin materials containing magnetic powder.

[0024] This embodiment is an embodiment further provided with a lower magnet 10 (also referred to as a bottom magnet). The description will be made again using FIG. 2 . The nucleic acid extraction device 100 is equipped with the lower magnet 10. The lower magnet 10 is arranged so as to be positioned below the sample container 1 when the sample container 1 moves below the magnetic bar 3. The lower magnet 10 can be moved up and down, for example, by a linear motion mechanism A2. When the lower magnet 10 approaches the bottom surface of the sample container 1, it can magnetically attract magnetic beads to the bottom surface of the sample container 1. Multiple types of lower magnets 10 may be arranged to allow selection, such as one for use with large sample containers 1 and one for use with small sample containers 1, or only one lower magnet 10 may be used.

[0025] Figure 13 shows a perspective cross-sectional view of the hole plate when a lower magnet 10 is used. The lower magnet 10 can be driven up and down independently of the upper magnet 5. The tip of the magnetic container 4 has a hemispherical surface to promote stirring of the specimen 6 and magnetic particles 7, and in consideration of ease of molding, it is thick, which has the disadvantage of increasing the distance between the upper magnet 5 and the surface of the magnetic container 4 and reducing the attractive force. On the other hand, the lower magnet 10 is placed at the bottom of the thin magnetic container 4, which has the advantage of having a high attractive force and being able to shorten the magnetic collection time.

[0026] The magnetic collection flow of magnetic particles 7 using the upper magnet 5 and lower magnet 10 is described using Figure 14. First, the magnetic container 4 alone is immersed in the liquid containing suspended magnetic particles 7 in the specimen container 1, and the liquid is mixed by moving the magnetic container 4 up and down (1). Next, the magnetic container 4 is raised above the liquid surface, and the lower magnet 10 is moved upward until it approaches the bottom of the specimen container 1. This allows the magnetic particles 7 suspended in the liquid to be collected in one location near the lower magnet 10 (2). Next, the upper magnet 5 is immersed in the liquid simultaneously with the magnetic container 4, and the magnetic particles 7 collected in one location are collected by the lower magnet 10 (3). During magnetic collection, the magnetic container 4 may be moved up and down in the liquid. The magnetic container 4 is immersed together with the upper magnet 5 in the liquid in a separate container, and then the upper magnet 5 is moved upward. By moving only the magnetic vessel 4 up and down in the liquid in the separate vessel, the magnetic particles 7 collected on the surface of the magnetic vessel 4 are suspended in the liquid in the separate vessel (4).

[0027] In Example 1, which is an embodiment that does not use the lower magnet 10, magnetic collection is performed using only the upper magnet. In contrast, in Example 2, the magnetic particles 7 are once collected in one location using the lower magnet 10, and the collected magnetic particles 7 can then be collected by the upper magnet 5, so the time required for magnetic collection is shorter than in Example 1, i.e., throughput is higher. On the other hand, Example 2 uses the lower magnet 10 and a mechanism for moving the lower magnet 10 up and down, so the cost is higher than the device in Example 1 and the device may also be slightly larger. It is desirable to appropriately select whether to use only the upper magnet 5 or also the lower magnet 10 depending on the magnetic collection performance required for the device.

[0028] Next, the optimal thickness of the hole plate when using the lower magnet 10 will be explained using Figure 15. As shown in Figure 15, assuming that the main attraction force occurs when the tip of the magnetic vessel 4 containing the upper magnet 5 touches the liquid surface of the sample 6 or when the lower magnet 10 approaches 10 mm from the bottom of the sample 6, it is desirable that the upper end face of the hole plate 2 be at least 2 mm below the tip face of the upper magnet 5 when the tip of the magnetic vessel 4 touches the liquid surface of the sample 6, and that the lower end face of the hole plate 2 be at least 2 mm below the position when the lower magnet 10 is 10 mm from the bottom of the sample 6. Figure 16 shows the analysis results of the difference in magnetic flux density between the edge and center when the distance between the end faces of the upper and lower magnets is 5 mm and the center of the plate thickness is located at the center of the upper and lower magnet ends. It can be seen that the difference in magnetic flux density is minimized when the plate thickness is 9 mm or more, and an overlap of at least 2 mm between the magnets is sufficient.

[0029] When using a hole plate 2 made of a soft magnetic material as in Examples 1 and 2, there is a problem that when a magnet is moved closer to or further away from the hole plate 2, the magnetic force increases suddenly when a certain distance is reached between the hole plate 2 and the magnet, increasing the load on the drive source (motor, etc.) for driving the magnetic rod 3, etc. To address this problem, tapered holes or grooves of different depths may be provided at the (bottom) end of the hole plate 2, as shown in Figures 17 and 18, so that the magnetic force between the soft magnetic material and the magnet gradually decreases. The taper angle and shape may be different for each hole, shifting the position where the load peaks (shifting the phase) and reducing the total load.

[0030] The area between the upper tapered boundary 11 and the lower tapered boundary 12 in Fig. 17 is narrower relative to the specimen container 1, and corresponds to the upper end face and the lower end face of the hole plate shown in Examples 1 and 2, and is a range that has the effect of minimizing the difference in magnetic flux density between the end and the center. Similarly, the area between the upper groove end 13 and the lower groove end 14 in Fig. 18 corresponds to the upper end face and the lower end face of the hole plate in Examples 1 and 2, and is a range that has the effect of minimizing the difference in magnetic flux density between the end and the center.

[0031] Figure 19 shows the magnetic force generated between the magnet and the hole plate 2 at each position. It can be seen that tapering makes the peak position of the magnetic force smaller than without tapering, and also reduces the total force (energy) required for driving, as shown by the integrated value of the graph in Figure 19.

[0032] 1 sample container, 2 hole plate, 3 magnetic bar, 4 magnetic body container, 5 upper magnet, 6 sample, 7 magnetic particles, 9 hole, 10 lower magnet.

Claims

1. A magnetic particle separation device comprising: a plurality of specimen containers containing a mixture of a liquid and magnetic particles; a first magnet disposed for each of the plurality of specimen containers and vertically movable independently of the specimen containers; a magnetic container covering the first magnet; and a hole plate having holes on which the plurality of specimen containers can be placed, wherein at least a side surface portion of the hole plate approaching the specimen containers is made of a soft magnetic material.

2. The magnetic particle separation device according to claim 1, wherein the soft magnetic material forms a magnetic path between the plurality of specimen containers.

3. The magnetic particle separation device according to claim 1, wherein the plurality of first magnets are arranged such that all have magnetic poles in the same direction.

4. The magnetic particle separation device according to claim 3, further comprising a second magnet that can be disposed at the bottom of the holes on which the plurality of specimen containers can be placed, and the magnetic poles of the second magnet are arranged in the same direction as the magnetic poles of the first magnet.

5. The magnetic particle separation device according to any one of claims 1 to 4, wherein the holes are provided with a taper in which the hole diameter expands toward the opening, or grooves having different depths.

Images