Patent Application
20150247185
1. Technical Field
The present invention relates to a device and a method for purifying a target substance.
2. Related Art
Boom et al. reports a method for conveniently extracting nucleic acids from biological materials with the use of a nucleic acid-binding solid phase support such as silica particles, and a chaotropic agent (J. Clin. Microbiol., vol. 28 No. 3, p. 495-503 (1990)). The method of Boom et al., and all the other extraction methods that involve adsorbing a nucleic acid on a support with a nucleic acid-binding solid phase support (such as silica) and a chaotropic agent basically include the steps of (1) adsorbing a nucleic acid on a nucleic acid-binding solid phase support in the presence of a chaotropic agent (adsorbing step), (2) washing the support and the adsorbed nucleic acid with a washing liquid to remove the non-specifically bound foreign substances and the chaotropic agent (washing step), and (3) eluting the nucleic acid from the support with water or a low salt concentration buffer (eluting step), as described, for example, in JP-A-11-146783, and JP-A-2009-207459.
A problem of such methods, however, is that each support particle is a very fine magnetic particle, and its hydrophilic surface may cause some of the particles to remain in the aqueous solution eluent when the externally applied magnetic procedures are insufficient. The presence of such magnetic particle residues in the eluent may interfere with the subsequent detection of a reaction by blocking the excitation light applied for detecting an amplification reaction.
An advantage according to some aspects of the invention is to provide a device and a method for purifying a target substance whereby magnetic particles can be efficiently removed.
An aspect of the invention is directed to a device for purifying a target substance, including: a mount for installing a capillary containing a liquid plug and having a longitudinal direction; a magnetic force applying mechanism that applies a magnetic force to the capillary to retain a magnetic body detachably retaining the target substance; a control mechanism that restricts movement of the magnetic body along the longitudinal direction of the capillary; and a liquid sending mechanism that moves the liquid along the longitudinal direction of the capillary while the movement of the magnetic body is being restricted by the control mechanism.
Another aspect of the invention is directed to a device for purifying a target substance, including: a capillary containing a liquid plug and having a longitudinal direction; a magnetic force applying mechanism that applies a magnetic force to the capillary to retain a magnetic body detachably retaining the target substance; a control mechanism that restricts movement of the magnetic body along the longitudinal direction of the capillary; and a liquid sending mechanism that moves the liquid along the longitudinal direction of the capillary while the movement of the magnetic body is being restricted by the control mechanism.
In both of the aspects of the invention described above, the target substance may be a nucleic acid, and the capillary may include: a first plug of oil; a second plug of a washing liquid that undergoes phase separation from the oil, and washes the magnetic body detachably retaining the nucleic acid; a third plug of oil; a fourth plug of an eluent that undergoes phase separation from the oil, and elutes the nucleic acid from the magnetic body detachably retaining the nucleic acid; and a fifth plug of oil, the first plug, the second plug, the third plug, the fourth plug, and the fifth plug being contained inside the capillary in this order. The device may include a moving mechanism that moves the magnetic body within a plane that crosses the longitudinal direction of the capillary. The magnetic force applying mechanism may include a permanent magnet. The liquid sending mechanism may include a pressure applying member, and the pressure applying member may be a plunger. Alternatively, the liquid sending mechanism may include a suction member, and the suction member may be a vacuum pump. Alternatively, the capillary may have ends with detachable sealing members.
Still another aspect of the invention is directed to a method for purifying a target substance, including: introducing a magnetic body detachably retaining the target substance into a capillary having a liquid plug; externally applying a magnetic force to the capillary to retain the magnetic body inside the liquid; eluting the target substance from the magnetic body inside the liquid; and moving the liquid with the eluted target substance out of the capillary by moving the liquid along the longitudinal direction of the capillary while restricting movement of the magnetic body.
In the method according to this aspect of the invention, the target substance may be a nucleic acid, and the capillary may include: a first plug of oil; a second plug of a washing liquid that undergoes phase separation from the oil, and washes the magnetic body detachably retaining the nucleic acid; a third plug of oil; a fourth plug of an eluent that undergoes phase separation from the oil, and elutes the nucleic acid from the magnetic body detachably retaining the nucleic acid; and a fifth plug of oil, the first plug, the second plug, the third plug, the fourth plug, and the fifth plug being contained inside the capillary in this order, wherein the magnetic body detachably retaining the target substance may be introduced into the capillary from an end closer to the first plug, wherein the magnetic body may move into the eluent under a magnetic force externally applied to the capillary, wherein the target substance may elute from the magnetic body in the eluent, and wherein the eluent with the eluted target substance may move to an end closer to the fifth plug, and may discharge from the capillary at the end closer to the fifth plug. The method may include moving the magnetic body within a plane that crosses the longitudinal direction of the capillary. The liquid with the eluted target substance may be moved along the longitudinal direction of the capillary by applying pressure inside the capillary, and the pressure may be applied with a plunger. Alternatively, the liquid with the eluted target substance may be moved along the longitudinal direction of the capillary by creating suction inside the capillary, and the suction may be created with a vacuum pump. Alternatively, the capillary may have detachable sealing members at the both ends, and the capillary may be vertically held, and the liquid with the eluted target substance may be moved along the longitudinal direction of the capillary by opening and closing the sealing members.
The invention has thus enabled providing a device and a method for purifying a target substance whereby magnetic particles can be efficiency removed.
The invention will be described with reference to the accompanying drawings, wherein like numbers reference like elements.
The objects, features, advantages, and ideas of the invention will be clearly understood by a skilled person from the descriptions of the invention, and it would be easy for a skilled person to reproduce the invention from the following descriptions. The embodiments and concrete examples of implementation discussed in the following detailed explanation serve solely to illustrate or describe the preferred embodiments of the invention, and do not limit the invention in any ways. It will be understood that various modifications and alterations may be made to the invention by a skilled person from the following descriptions, provided such modifications and alterations do not exceed the intent and the scope of the invention disclosed below.
As illustrated in
The liquid plug 10 is preferably an aqueous solution, more preferably a salt solution, particularly a buffer. The capillary 12 may have more than one plug. In this case, it is preferable to provide a wax plug or an oil plug between the liquid plugs so that the liquid plugs can independently exist.
The capillary 12 is hollow inside, and has a tubing portion (also called “tube portion”) that allows a liquid to longitudinally travel inside the hollow space. The tube portion has a longitudinal direction, but may be bent. The size and the shape of the hollow space inside the tube portion are not particularly limited, as long as the liquid can maintain the form of a plug inside the tube portion. The size of the hollow space inside the tube portion, and the shape of the cross section perpendicular to the longitudinal direction may vary along the longitudinal direction of the tube portion. Whether the liquid can maintain a plug shape inside the tube portion depends on conditions such as the material of the tube portion, and the type of the liquid, and as such the shape of the cross section perpendicular to the longitudinal direction of the tube portion is appropriately designed to allow the liquid to maintain a plug shape inside the tube portion. The outer cross sectional shape of the tube portion perpendicular to the longitudinal direction is not limited either. The thickness of the tube portion is not particularly limited. When the cross section of the hollow space inside the tube portion perpendicular to the longitudinal direction is circular in shape, the tube portion may have an inner diameter (the diameter of the circle of the cross section of the inner hollow perpendicular to the longitudinal direction) of, for example, 0.5 mm to 3 mm. Such an inner diameter of the tube portion is preferable in terms of ease of forming a liquid plug over a wide range of tube portion materials and liquid types. The material of the tube portion is not particularly limited, and may be, for example, glass, polymer, or metal. The materials selected for the tube portion are preferably materials that are transparent to visible light, such as glass and polymer, because such materials provide visual access to inside of the tube portion (the hollow space) from outside. It is also preferable to use a magnetically transparent material or a non-magnetic material for the tube portion because such materials make it easier to externally apply a magnetic force to the tube portion such as when passing magnetic particles M through the tube portion.
The target substance is not limited, and may be a macromolecule such as a nucleic acid (e.g., DNA, RNA), and a protein, or a low-molecular substance such as a compound. The shape of the magnetic body M is not particularly limited, and is preferably particulate. In order to detachably retain the target substance, the magnetic body M either detachably binds to the target substance, or has a target substance-binding substance that detachably binds to the magnetic body M. For example, when the target substance is a nucleic acid, the magnetic body M may have nucleic acid binding molecules such as silica particles. The magnetic body M may have an antibody binding molecule such as protein A when the target substance is an antibody.
The purification device 80 according to the invention includes a magnetic force applying section 20 provided with the magnetic force applying mechanism 14 and the control mechanism 16. The magnetic force applying mechanism 14 externally applies a magnetic force to the capillary 12 to retain the magnetic body M inside the capillary 12, and control the movement of the magnetic body M. The magnetic force applying mechanism 14 can be designed by a skilled person with ease. For example, the magnetic force applying mechanism 14 has a magnetic force applying body such as a permanent magnet, and can retain the magnetic body M with the magnetic force of the magnetic force applying body. The control mechanism 16 controls the longitudinal movement of the magnetic body M inside the capillary 12, and can stop the magnetic body M as desired. This is made possible, for example, with a mechanism by which the magnetic force applying mechanism 14 can be moved, and stopped at the desired position. Preferably, the control mechanism 16 is adapted to move the magnetic body M within a plane that crosses the longitudinal direction of the capillary 12, particularly a plane orthogonal to the longitudinal direction of the capillary 12. Specifically, it is preferable that the control mechanism 16 swings the magnetic body M. For example, because the magnetic body M is attracted by whichever of the magnetic force applying bodies is closer to the magnetic body M, the magnetic force applying body that is more proximate than the other to the capillary attracts the magnetic body M when two magnetic force applying bodies are provided. As the magnetic force applying body is moved away from the capillary 12, the magnetic body M is attracted by the other magnetic force applying body approaching the capillary 12 from the opposite side. In this way, the magnetic body M can be moved sideways. The magnetic body M can thus be moved back and forth sideways by swinging the pair of magnetic force applying bodies sideways. This makes it easier for the magnetic body M to contact the liquid as it moves in the liquid plug 10, and improves the washing and eluting effects.
The liquid sending mechanism 18 of the purification device 80 is provided to move the liquid plug 10 along the longitudinal direction of the capillary 12, and eject the liquid through a capillary end portion 24. For example, the liquid sending mechanism 18 may be a pressure applying member that applies pressure to the liquid inside the capillary 12, and moves the liquid under the applied pressure, or a suction member that moves the liquid by evacuating the capillary 12 and creating a negative pressure therein. The pressure applying member may be, for example, a piston or a plunger as shown in
The purification device 80 of the foregoing configuration may be used to purify a target substance. For example, a target substance of interest for purification is obtained from a sample such as cells and viruses containing the target substance, for example, by lysing or extracting the sample with a lysing solution or an extractant. The magnetic body M that can detachably retain the target substance is then added, and bound to the target substance. The magnetic body M detachably retaining the target substance may be washed with a buffer or the like with a centrifuge tube or the like. The magnetic body M detachably retaining the target substance is then introduced into the capillary 12 having the liquid plug 10. The method for introducing the magnetic body M is not particularly limited, and the magnetic body M may be introduced into the capillary 12 through the end portion 24 after being suspended in the same liquid used for the liquid plug 10 so that the liquids coalesce inside the capillary 12.
Simultaneously, a magnetic force is applied from outside of the capillary 12 to retain the magnetic body M inside the liquid. For example, as illustrated in
With the magnetic body M retained inside the liquid, the target substance is eluted from the magnetic body Min the liquid. The elution method is not particularly limited, and may be appropriately decided according to the manner in which the target substance binds to the target substance-binding substance of the magnetic body M. For example, in the case of a nucleic acid and silica, the liquid may be heated by heating the capillary. In the case of an antibody and an antigen, the antibody and the antigen may be separated from each other by making the pH acidic by addition of an acid to the liquid. Here, the liquid effect on the magnetic body M can be improved by swinging the magnetic body M on a plane that crosses the longitudinal direction of the capillary 12, particularly a plane orthogonal to the longitudinal direction of the capillary 12, as described above.
Thereafter, as illustrated in
More than one liquid plug may be provided, one being a washing liquid for washing the magnetic body M detachably retaining the target substance, and the other being an eluent for eluting the target substance from the magnetic body M. Specifically, in this case, the capillary 12 may include a first plug of oil, a second plug of a washing liquid that undergoes phase separation from the oil, and washes the magnetic body detachably retaining the target substance, a third plug of oil, a fourth plug of an eluent that undergoes phase separation from the oil, and elutes the target substance from the magnetic body M detachably retaining the target substance, and a fifth plug of oil, in this order inside the capillary 12. As with the case of a single plug, the magnetic body M detachably retaining the target substance is introduced into the washing liquid. The magnetic body M is then moved to the eluent with the control mechanism 16 that controls the movement of the magnetic force applying mechanism 14. The target substance is then eluted in the eluent, and is ejected and collected through the end of the capillary as described above. Here, the washing effect can be further improved by providing more than one washing liquid plug.
As illustrated in
The following describes how the nucleic acid purification device is used to extract a nucleic acid from human blood.
First, as illustrated in
Fifty microliters of blood collected from human was pipetted into the adsorption vessel 150 through the opening. After capping the opening, the adsorption vessel 150 was agitated for 30 seconds by shaking it with hand to adsorb the blood nucleic acid to the magnetic particles M (see
The liquids inside the tube are moved by pushing the plunger 130 toward the opening 140 while moving the permanent magnets 320A and 320B reciprocally along the axis orthogonal to the tube longitudinal direction. Under the applied pressure of the plunger 130 inside the tube, the second plug 210 of a washing liquid moves to the level that lies on the axis along which the magnetic force applying section is undergoing reciprocal movement. This moves the magnetic particles M to the second plug relative to the first plug of oil. The magnetic particles M are washed by the washing liquid, and the foreign substances around the magnetic particles M are removed as the permanent magnets 320A and 320B make reciprocal movement while the plunger 130 sends the liquid (see
The entire disclosure of Japanese Patent Application No. 2014-038183, filed Feb. 28, 2014 is expressly incorporated by reference herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-038183 | Feb 2014 | JP | national |
