JP2023144299A - Magnetic separation method - Google Patents

Abstract

To provide a magnetic separation method which enables reduction of amount of liquid adhered to magnetic beads and unrecoverable, even when using magnetic beads having small diameter and high saturation magnetization, and which therefore materializes high separability between the magnetic beads and liquid.SOLUTION: A magnetic separation method is provided, including: a magnetic separation step of applying an external magnetic field to a container in which magnetic beads having a saturation magnetization of 50 emu/g or more and an average particle diameter of 0.5 μm or more and 50 μm or less, and liquid are housed, and fixing the magnetic beads to the inner wall of the container; and a liquid discharge step of bringing the magnetic beads fixed to the inner wall into contact with a liquid suction tool, and discharging liquid adhered to the magnetic beads by the liquid suction tool. When a saturation magnetization [emu/g] of the magnetic beads is M(B), a magnetic field gradient [T/m] formed by the external magnetic field is G, and a mass [kg] of the magnetic beads is m, the external magnetic field is applied to the container in such a manner that a magnetic attraction force Fmag[pN] calculated by the following formula (1) is 80 pN or more. Fmag=M(B)×G×m (1).SELECTED DRAWING: Figure 1

Description

The present invention relates to a magnetic separation method.

In recent years, there has been an increasing demand for testing biological materials in the medical diagnosis and life science fields. Among biological material testing methods, the PCR (polymerase chain reaction) method is a method for extracting nucleic acids such as DNA and RNA, and specifically amplifying and detecting the nucleic acids. In the process of testing such biological substances, it is first necessary to extract the substance to be tested from the specimen. Magnetic separation methods using magnetic beads are widely used to extract this biological material. The magnetic separation method uses magnetic beads that have the ability to support biological substances to be extracted, and extracts biological substances by applying a magnetic field. Specifically, after dispersing magnetic beads whose surface has the ability to support the substance to be tested in a dispersion medium, the resulting dispersion is attached to a magnetic field generator such as a magnetic stand, and the magnetic field is turned on and off. Repeat multiple times. Thereby, the substance to be tested is extracted. This magnetic separation method is a method in which magnetic beads are separated and recovered using magnetic force, and therefore, rapid separation operations are possible.

In addition, similar magnetic separation methods are used not only in PCR extraction, but also in fields such as protein purification, exosomes, cell separation, and extraction.

For example, Patent Document 1 discloses a nucleic acid extraction method including an adsorption step, a washing step, and an elution step. In the adsorption step, nucleic acids are adsorbed onto a nucleic acid-binding solid support in an adsorption solution. In the washing step, the nucleic acid-binding solid carrier to which nucleic acids have been adsorbed is placed in a washing solution, and the nucleic acid-binding solid carrier is vibrated by magnetic force. In the elution step, the nucleic acid-binding solid carrier to which the nucleic acid has been adsorbed is placed in the eluate, and the nucleic acid is eluted into the eluate by vibrating the nucleic acid-binding solid carrier using magnetic force. According to such a method, washing efficiency and nucleic acid elution efficiency can be improved.

JP 2017-176023 Publication

Coupled with the recent increase in demand for PCR tests and the like, there is a demand for improved testing accuracy for substances to be tested in diagnosis and various tests in the medical field.

In the magnetic beads described in Patent Document 1, a phenomenon in which the magnetic beads are lined up in a row (spike phenomenon) occurs due to high saturation magnetization due to the magnetic field generated by the magnetic field generator. When the spike phenomenon occurs, many gaps are formed between the magnetic beads, and liquid is likely to be retained in these gaps. In this case, the separability between the magnetic beads and the liquid in magnetic separation decreases. In other words, since the liquid held by the magnetic beads cannot be separated, the amount of liquid collected decreases. In particular, when the magnetic beads have a small diameter, while the amount of adsorption of the substance to be tested can be increased, the amount of liquid held by the magnetic beads tends to increase. As a result, for example, impurities, cleaning components, etc. contained in the liquid are brought into the eluate, reducing the test accuracy when testing the substance to be tested in the eluate.

Therefore, even when using magnetic beads with a small diameter and high saturation magnetization, the amount of liquid that cannot be recovered due to adhesion to the magnetic beads can be kept to a low level, thereby realizing a magnetic separation method that has high separation properties between the magnetic beads and the liquid. This has become an issue.

The magnetic separation method according to the application example of the present invention is as follows:
By applying an external magnetic field to a container containing a liquid and magnetic beads having a saturation magnetization of 50 emu/g or more and an average particle size of 0.5 μm or more and 50 μm or less and fixing the magnetic beads to the inner wall of the container, the magnetic beads are fixed to the inner wall of the container. a magnetic separation step of separating beads and the liquid;
a liquid discharging step of bringing a liquid suction tool into contact with the magnetic beads fixed to the inner wall and discharging the liquid adhering to the magnetic beads with the liquid suction tool;
has
When the saturation magnetization [emu/g] of the magnetic beads is M (B), the magnetic field gradient [T/m] formed by the external magnetic field is G, and the mass [kg] of the magnetic beads is m, the following A magnetic separation method characterized in that the external magnetic field is applied so that the magnetic attraction force F _mag [pN] generated in the magnetic beads determined by equation (1) is 80 pN or more.
F _mag = M(B)×G×m (1)

FIG. 2 is a process diagram for explaining a biological material extraction method including a magnetic separation method according to an embodiment. 2 is a cross-sectional view showing magnetic beads used in the magnetic separation method of FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. 2 is a schematic diagram for explaining the biological material extraction method shown in FIG. 1. FIG. FIG. 7 is a schematic diagram for explaining a biological substance extraction method including a magnetic separation method according to a modification. FIG. 7 is a schematic diagram for explaining a biological substance extraction method including a magnetic separation method according to a modification.

Hereinafter, preferred embodiments of the magnetic separation method of the present invention will be described in detail based on the accompanying drawings.
The magnetic separation method according to the embodiment applies an external magnetic field to a container containing a solid phase containing magnetic beads and a liquid phase containing a liquid, and magnetically attracts the magnetic beads, thereby separating the solid phase and the liquid phase magnetically. This is a method of separation. Such magnetic separation methods are used, for example, in methods for extracting biological substances from specimen samples. In the following description, a biological material extraction method including a magnetic separation method according to an embodiment will be described.

1. Biological Substance Extraction Method FIG. 1 is a process diagram for explaining a biological substance extraction method including a magnetic separation method according to an embodiment. FIG. 2 is a cross-sectional view showing magnetic beads used in the magnetic separation method of FIG. 1. 3 to 9 are schematic diagrams for explaining the biological material extraction method shown in FIG. 1.

The biological material extraction method shown in FIG. 1 includes a dissolution/adsorption step S102, a magnetic separation step S104, a liquid discharge step S106, a washing step S108, and an elution step S110. Of these, the magnetic separation step S104 and the liquid discharge step S106 constitute the magnetic separation method according to the embodiment. In the magnetic separation step S104, an external magnetic field is applied to the container 1 containing the magnetic beads 2 having a saturation magnetization of 50 emu/g or more and the liquid 3, thereby fixing the magnetic beads 2 to the inner wall of the container 1. Thereby, the magnetic beads 2 and the liquid 3 are separated. In the liquid discharge step S106, a pipette 6 (liquid suction tool) is brought into contact with the magnetic beads 2 fixed to the inner wall, and the liquid 3 adhering to the magnetic beads 2 is discharged by the pipette 6.

The biological substances to be extracted by the biological substance extraction method refer to substances such as nucleic acids such as DNA and RNA, proteins, sugars, various cells such as cancer cells, peptides, bacteria, and viruses. Note that the nucleic acid may exist, for example, in a state contained in a biological sample such as a cell or a biological tissue, a virus, a bacteria, or the like. The biological material extraction method shown in FIG. 1 extracts such biological materials through the steps of dissolution/adsorption, separation, washing, and elution. Note that the procedure of the extraction method is usually determined for each magnetic bead dispersion provided as a reagent or the target biological substance, and is usually clearly specified by the provider. Such procedures are commonly referred to as "extraction protocols."

Each step will be explained in sequence below. In addition, in the following explanation, the case where a biological substance is a nucleic acid is made into an example, and is demonstrated.

1.1. Dissolution/Adsorption Step In the dissolution/adsorption step S102, first, a specimen sample containing a nucleic acid is placed in the container 1 shown in FIG. A dispersion liquid containing magnetic beads 2 and a dissolving and adsorbing liquid are further placed in this container 1. Then, the contents of container 1 are mixed. Thereby, the magnetic beads 2 are dispersed in the liquid 3 as shown in FIG. Since nucleic acids are usually contained in cell membranes and nuclei, the so-called outer shells of cell membranes and nuclei are first dissolved and removed by the lytic action of the lysing and adsorbing solution to extract the nucleic acids. Thereafter, the nucleic acid is adsorbed to the magnetic beads 2 by the adsorption action of the lysis/adsorption liquid.

As the dissolving and adsorbing liquid, for example, a liquid containing a chaotropic substance is used. The chaotropic substance generates chaotropic ions in an aqueous solution, has the effect of reducing interactions between water molecules, thereby destabilizing the structure, and contributes to the adsorption of nucleic acids to the magnetic beads 2. Examples of chaotropic substances that exist as chaotropic ions in an aqueous solution include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, and sodium perchlorate. Among these, guanidine thiocyanate or guanidine hydrochloride, which have a strong protein denaturing effect, are preferably used.

The concentration of the chaotropic substance in the dissolved adsorption liquid varies depending on the chaotropic substance, but is preferably, for example, 1.0M or more and 9.0M or less. Moreover, especially when using guanidine thiocyanate, it is preferable that it is 5.0M or more and 7.0M or less. Furthermore, especially when using guanidine hydrochloride, it is preferably 4.0M or more and 8.0M or less.

The dissolving and adsorbing liquid may contain a surfactant. Surfactants are used for the purpose of destroying cell membranes or denaturing proteins contained in cells. The surfactant is not particularly limited, but includes, for example, nonionic surfactants such as polyoxyethylene sorbitan monolaurate, Triton surfactants and Tween surfactants, and anionic surfactants such as sodium N-lauroylsarcosine. Examples include surfactants. Among these, nonionic surfactants are preferably used. The nonionic surfactant suppresses the influence of the ionic surfactant when analyzing extracted nucleic acids. As a result, analysis by electrophoresis becomes possible, and the options for analysis techniques can be expanded.

The concentration of the surfactant in the dissolved adsorption liquid is not particularly limited, but is preferably 0.1% by mass or more and 15.0% by mass or less.

Further, the dissolving and adsorbing liquid may contain at least one of a reducing agent and a chelating agent. Examples of the reducing agent include 2-mercaptoethanol and dithiothreitol. Examples of the chelating agent include ethylenediaminetetraacetic acid dihydrogen disodium dihydrate (EDTA).

The concentration of the reducing agent in the dissolved adsorption solution is not particularly limited, but is preferably 0.2M or less. The concentration of the chelating agent in the dissolved adsorption solution is not particularly limited, but is preferably 50 mM or less.

The pH of the dissolved and adsorbed liquid is not particularly limited, but is preferably neutral in the range of 6 or more and 8 or less. Additionally, tris(hydroxy)aminomethane, HCl, or the like may be added as a buffer solution to adjust the pH.

In the dissolution/adsorption step S102, the content in the container 1 is stirred by a vortex mixer, manual shaking, pipetting, etc., as necessary. The stirring time is not particularly limited, but is preferably 5 seconds or more and 40 minutes or less.

1.1.1. Magnetic Beads For the magnetic beads 2, magnetic particles having a saturation magnetization of 50 emu/g or more are used. Saturation magnetization is the value of magnetization when a magnetic material exhibits a constant magnetization regardless of the magnetic field when a sufficiently large magnetic field is applied from the outside. The higher the saturation magnetization of the magnetic beads, the more fully they can function as a magnetic material. Specifically, since the moving speed of the magnetic beads 2 in the magnetic field can be improved, the time required for magnetic separation can be shortened. Moreover, the saturation magnetization of the magnetic beads 2 influences the attraction force when they are fixed by an external magnetic field. If the saturation magnetization is within the above range, a sufficiently high adsorption force can be obtained, so that when the liquid 3 is discharged in the liquid discharge step S106 described later, the magnetic beads 2 are prevented from being discharged together with the liquid 3. I can do that. Thereby, it is possible to suppress a decrease in the yield of nucleic acid due to a decrease in the number of magnetic beads 2.

Further, the saturation magnetization of the magnetic beads 2 is preferably 100 emu/g or more. The upper limit of the saturation magnetization of the magnetic beads 2 is not particularly limited, but is preferably 220 emu/g or less from the viewpoint of ease of selecting materials suitable for a balance between performance and cost.

The saturation magnetization of the magnetic beads 2 can be measured using a vibrating sample magnetometer (VSM) or the like. Examples of the vibrating sample magnetometer include TM-VSM1230-MHHL manufactured by Tamagawa Seisakusho Co., Ltd. The maximum applied magnetic field when measuring saturation magnetization is, for example, 0.5 T or more.

The magnetic beads 2 are not particularly limited as long as they are particles with saturation magnetization as described above, but they are preferably particles having the magnetic metal powder 22 and the coating layer 24 shown in FIG. 2. A particle group, which is an aggregate of such magnetic beads 2, is used in the container 1, and by taking advantage of the large specific surface area of the particle group, efficient adsorption of nucleic acids is possible.

Further, the average particle diameter of the magnetic beads 2 is 0.5 μm or more and 50 μm or less, preferably 2 μm or more and 20 μm or less. If the average particle size of the magnetic beads 2 is within the above range, the specific surface area of the magnetic beads 2 can be made sufficiently large, and the magnetic beads 2 can generate attractive force and adsorption force suitable for immobilization. can. Moreover, aggregation of the magnetic beads 2 can be suppressed and dispersibility can be improved. Note that when the average particle size of the magnetic beads 2 is below the lower limit value, the magnetization value of the magnetic beads 2 becomes small and they tend to aggregate, which may result in a decrease in nucleic acid extraction efficiency. Furthermore, the moving speed of the magnetic beads 2 may decrease, and the time required for magnetic separation may become longer. On the other hand, if the average particle size of the magnetic beads 2 exceeds the above upper limit, the specific surface area of the magnetic beads 2 will become smaller, making it impossible to adsorb a sufficient amount of nucleic acid, which may reduce the amount of nucleic acid extracted. be. In addition, the magnetic beads 2 tend to sediment, and the number of magnetic beads 2 that can contribute to nucleic acid extraction decreases, which may reduce the efficiency of nucleic acid extraction.

Note that the average particle diameter of the magnetic beads 2 can be determined by measuring the volume-based particle size distribution using a laser diffraction/dispersion method, and from an integrated distribution curve obtained from this particle size distribution. Specifically, in the integrated distribution curve, the particle diameter D50 (median diameter) where the cumulative value is 50% from the small diameter side is the average particle diameter of the magnetic beads 2. An example of an apparatus for measuring particle size distribution using a laser diffraction/dispersion method is the MT3300 series manufactured by Microtrac Bell. Note that not only the laser diffraction/dispersion method but also methods such as image analysis may be used.

Further, when the average thickness of the coating layer 24 is t and the average particle size of the magnetic beads 2 is D50, the ratio t/D50 of t to D50 is preferably 0.0001 or more and 0.05 or less, More preferably, it is 0.001 or more and 0.01 or less. If t/D50 is below the lower limit value, the ratio of the thickness of the coating layer 24 to the size of the magnetic metal powder 22 becomes too small, resulting in collisions between the magnetic beads 2 or the inner wall of the container 1 between the magnetic beads 2 and the magnetic beads 2. When a collision occurs, the coating layer 24 may be destroyed or peeled off. Therefore, the amount of nucleic acid that is adsorbed onto the surface of the coating layer 24 and extracted is reduced, and there is a possibility that the extraction efficiency will be reduced. In addition, fragments of the peeled coating layer 24 and magnetic metal powder 22 will be present in the dispersion liquid, and there is a risk that they will be mixed in as contaminants when the nucleic acid is taken out. Furthermore, if the coating layer 24 is destroyed or peeled off, the magnetic metal powder 22 will be exposed, and if it comes into contact with an acidic solution, iron ions, etc. will be eluted, which may result in a decrease in nucleic acid extraction efficiency. . On the other hand, if t/D50 exceeds the above-mentioned upper limit, the volume ratio of the coating layer 24 to the entire volume of the magnetic beads 2 will increase, and the magnetization per volume of the magnetic beads 2 may decrease. As a result, the moving speed when an external magnetic field acts on the magnetic beads 2 in the magnetic separation step S104, which will be described later, may decrease, and the time required for magnetic separation may become longer.

The thickness of the coating layer 24 can be measured, for example, from a cross-sectional image of the magnetic beads 2 observed using a transmission electron microscope, a scanning electron microscope, or the like. Further, the average thickness t of the coating layer 24 can be calculated by acquiring a plurality of observation images and averaging the measured values from image processing and the like. For example, the average thickness t can be determined by measuring the thickness of the coating layer 24 at five or more locations for one magnetic bead 2, calculating the average value, and then averaging the average value for ten or more magnetic beads 2. It is a value.

Further, the coercive force Hc of the magnetic beads 2 is preferably 1500 A/m or less, more preferably 800 A/m or less, and even more preferably 200 A/m or less. The coercive force Hc refers to the value of an external magnetic field in the opposite direction necessary to return a magnetized magnetic body to an unmagnetized state. In other words, the coercive force Hc means a resistance force against an external magnetic field. The smaller the coercive force Hc of the magnetic beads 2, the more difficult it is for the magnetic beads 2 to aggregate with each other even when switching from a state where a magnetic field is applied to a state where no magnetic field is applied, and the magnetic beads 2 are more uniformly dispersed in the dispersion liquid. be able to. Furthermore, even when switching the application of the magnetic field is repeated, the smaller the coercive force Hc is, the better the redispersibility of the magnetic beads 2 is, so that aggregation of the magnetic beads 2 can be further suppressed. The lower limit of the coercive force Hc of the magnetic beads 2 is not particularly limited, but is preferably 1 A/m or more from the viewpoint of ease of selecting materials suitable for a balance between performance and cost.

The coercive force Hc of the magnetic beads can be measured using a vibrating sample magnetometer or the like, similar to the saturation magnetization described above.

Moreover, it is preferable that the relative magnetic permeability of the magnetic beads 2 is 5 or more. If the relative magnetic permeability of the magnetic beads 2 is below the lower limit, the moving speed of the magnetic beads 2 may decrease, and the time required for magnetic separation may become longer. Note that the upper limit of the relative magnetic permeability of the magnetic beads 2 is not particularly limited, but since the magnetic beads 2 are in powder form, the relative magnetic permeability should substantially take a value of 100 or less due to the influence of the demagnetizing field. There are many.

1.1.1.1. Magnetic Metal Powder The magnetic metal powder 22 is a particle having magnetism, and preferably contains at least one of Fe, Co, and Ni as a constituent element. In particular, from the viewpoint of obtaining high saturation magnetization, the composition of the magnetic metal powder 22 is preferably an alloy containing Fe as a main component (Fe-based alloy). Specifically, the atomic ratio of Fe is preferably 50% or more, more preferably 70% or more. Such a Fe-based alloy can realize magnetic metal powder 22 that has high saturation magnetization and high magnetic permeability even if the particle size is small.

Fe-based alloys include elements such as Co and Ni that exhibit ferromagnetism alone, as well as Cr, Nb, Cu, Al, Mn, Mo, Si, Sn, B, C, and P, depending on the target properties. , Ti, and Zr. Further, the Fe-based alloy may contain unavoidable impurities to the extent that the effect of the magnetic metal powder 22 is not impaired.

As an example of a Fe-based alloy, the content of Si is 2.0% by mass or more and 9.0% by mass or less, the content of B is 1.0% by mass or more and 5.0% by mass or less, and the content of Cr is Examples include alloys in which the percentage is 1.0% by mass or more and 3.0% by mass or less, and the balance is Fe and impurities. In this case, impurities, regardless of whether they are unavoidably mixed or intentionally added, do not impair the effects of the Fe-based alloy as long as the total mass of all elements is 1.0 or less. Permissible.

The metal structure constituting the magnetic metal powder 22 can take various forms such as a crystalline structure, an amorphous structure, and a nanocrystalline structure. In particular, by forming the magnetic beads 2 into an amorphous structure or a nanocrystalline structure, the coercive force Hc of the magnetic beads 2 becomes a low value, and the dispersibility of the magnetic beads 2 in the liquid can be improved. Note that the volume fraction of the amorphous structure or nanocrystalline structure in the magnetic metal powder 22 is preferably 40% or more, and more preferably 60% or more. This volume fraction is determined from the results of crystal structure analysis using X-ray diffraction. Further, each of the crystalline structure, amorphous structure, and nanocrystalline structure may exist alone, or two or more of these may exist in a mixed structure.

A specific alloy system suitable for forming an amorphous structure or a nanocrystalline structure includes Fe and one or more selected from the group consisting of Cr, Si, B, C, P, Nb, and Cu. Examples include added compositions.

1.1.1.2. Coating Layer The coating layer 24 covers the particle surface of the magnetic metal powder 22, as shown in FIG. The coating layer 24 may be formed on at least a portion of the particle surface of the magnetic metal powder 22, but it is preferable that the coating layer 24 covers the entire particle surface.

The main function of the covering layer 24 is to adsorb biological materials. From this point of view, it is preferable that the coating layer 24 has the following substance or chemical structure on its surface.

A first preferred material constituting the covering layer 24 is an oxide such as silicon oxide. Silicon oxide is a substance particularly suitable for extracting nucleic acids, and its compositional formula is preferably SiOx (0<x≦2), and specifically _SiO2 . Silicon oxide specifically adsorbs nucleic acids in an aqueous solution containing chaotropic substances, thereby making it possible to extract and recover nucleic acids. In addition to silicon oxide, examples of oxides include composite oxides or composites of silicon and one or more selected from the group consisting of Al, Ti, V, Nb, Cr, Mn, Sn, and Zr. Examples include things.

The second preferred substance constituting the coating layer 24 is a substance that has a functional group on the surface of the coating layer for increasing the bondability with biological materials. Examples of functional groups that increase bonding include OH group, COOH group, NH ₂ group, epoxy group, trimethylsilyl group, NHS group, etc., although it depends on the target substance.

Preferred substances constituting the coating layer 24 include proteins such as streptavidin, protein A, and protein G, and carbon. Furthermore, when nucleic acids are to be extracted, preferred substances include nucleic acids having complementary properties to the target nucleic acids, specifically oligo (dT) primer cDNAs.

1.2. Magnetic Separation Step In the magnetic separation step S104, an external magnetic field is applied to the magnetic beads 2 to which nucleic acids have been adsorbed to cause magnetic attraction. This moves the magnetic beads 2 to the inner wall of the container 1 and fixes them. As a result, the magnetic beads 2 as a solid phase and the liquid 3 as a liquid phase can be separated.

The magnetic separation step S104 and the liquid discharging step S106 described below are performed not only after the dissolution/adsorption step S102 but also in the cleaning step S108 and the elution step S110 described later, if necessary.

Before performing magnetic attraction, the contents in the container 1 are stirred as necessary. This increases the probability that the nucleic acid will be adsorbed to the magnetic beads 2. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. are used.

Here, when the saturation magnetization of the magnetic beads 2 is high, when an external magnetic field acts on the magnetic beads 2, a phenomenon in which the magnetic beads 2 are lined up in a row (spike phenomenon) is likely to occur. FIG. 4 shows how a spike phenomenon occurs in the magnetic beads 2 housed in the container 1. In the example shown in FIG. 4, the rows of magnetic beads 2 extend radially along unillustrated lines of magnetic force extending from the magnet 5. In the example shown in FIG. When the spike phenomenon occurs, many gaps are formed between the magnetic beads 2, and the liquid 3 is easily held in these gaps. Conventionally, it has been difficult to discharge this retained liquid 3, so there has been a problem that the amount of liquid 3 that can be discharged is reduced.

Therefore, in the present embodiment, the above problem is solved by optimizing the external magnetic field applied in the magnetic separation step S104 and performing an operation to increase the discharge amount of the liquid 3 in the liquid discharge step S106, which will be described later. .

In the magnetic separation step S104, as described above, an external magnetic field is applied to the contents of the container 1, thereby causing the external magnetic field to act on the magnetic beads 2. To apply this external magnetic field, for example, as shown in FIG. 4, a magnet 5 placed on the side of the container 1 is used. The side of the container 1 refers to a position on an axis perpendicular to the vertical axis. The magnet 5 may be an electromagnet or a permanent magnet. When there is a gradient in the strength of the external magnetic field, a magnetic attraction force is generated on the magnetic beads 2 according to the gradient. When the magnetic attraction force generated in one of the magnetic beads 2 is F _mag , the magnetic attraction force F _mag is determined by the following formula (1).

F _mag = M(B)×G×m (1)
In the above formula (1), M(B) is the saturation magnetization [emu/g] of the magnetic beads 2, G is the magnetic field gradient [T/m] formed by the external magnetic field, and m is the magnetic bead The mass of 2 is [kg].

In the magnetic separation step S104, an external magnetic field is applied to the magnetic beads 2 so that the magnetic attraction force F _mag determined by the above equation (1) becomes 80 pN or more. That is, the saturation magnetization M(B) of the magnetic beads 2 and the mass m of the magnetic beads 2 are both values specific to the magnetic beads 2. Therefore, the magnetic attraction force F _mag generated on the magnetic beads 2 is proportional to the magnetic field gradient G formed by the external magnetic field. Therefore, in order to generate the magnetic attraction force F _mag within the above range, the applied magnetic field gradient G may be adjusted according to the saturation magnetization M(B) and mass m of the magnetic beads 2 .

When an external magnetic field acts on the magnetic beads 2, the magnetic beads 2 move toward the magnet 5. When the magnetic attraction force F _mag generated on the magnetic beads 2 is within the above range, the moving speed of the magnetic beads 2 can be sufficiently increased. Thereby, the time required for magnetic separation can be shortened. As a result, the time required for nucleic acid testing can be shortened.

When the magnetic beads 2 reach the inner wall of the container 1, the magnetic beads 2 are fixed to the inner wall. Further, when the magnetic attraction force F _mag generated on the magnetic beads 2 is within the above range, the stability of the fixed magnetic beads 2 can also be improved. On the other hand, when the magnetic beads 2 are fixed to the inner wall, most of the liquid 3 accumulates at the bottom of the container 1.

In the liquid discharge step S106, which will be described later, not only the liquid 3 accumulated at the bottom of the container 1 but also the liquid 3 attached to the magnetic beads 2 can be discharged by the liquid suction operation. At this time, if the magnetic attraction force F _mag generated on the magnetic beads 2 is within the above range, even if a liquid attraction force is applied to the magnetic beads 2, the probability that the fixation of the magnetic beads 2 will be released can be reduced. Thereby, it is possible to increase the probability that only the liquid 3 is attracted and discharged without attracting the magnetic beads 2.

Note that when the point on the surface of the magnet 5 that is closest to the fixed magnetic beads 2 is defined as measurement point a, and the point closest to the magnet 5 on the surface of the fixed magnetic beads 2 is defined as measurement point b, The magnetic field gradient G is the difference in magnetic field between measurement point a and measurement point b. This magnetic field gradient G is determined as follows.

First, the distance [m] between measurement point a and measurement point b is assumed to be Δs. Further, the magnetic field [T] at measurement point a is Ba, and the magnetic field [T] at measurement point b is Bb. At this time, the magnetic field gradient G is determined by the following equation (2).
G=(Ba-Bb)/Δs (2)

In addition, when the circumference is π, the average particle diameter of the magnetic beads 2 divided by 2 is the average radius r [cm], and the true specific gravity of the magnetic beads 2 is d [g/cm ³ ], the magnetic The mass m of the beads 2 is determined by the following formula (3).
m={(4/3)×π× ^r3 ×d}/1000 (3)

The magnetic attraction force F _mag is set to be 80 pN or more, preferably 100 pN or more, and more preferably 1000 pN or more. Although the upper limit of the magnetic attraction force F _mag is not particularly set, it is preferably 12,000,000 pN or less from the viewpoint of ease of selecting materials suitable for the balance between performance and cost.

Note that after magnetic attraction is performed, acceleration may be applied to the container 1 as necessary. Thereby, the liquid 3 adhering to the magnetic beads 2 can be shaken off, so that the amount of liquid 3 adhering to the magnetic beads 2 can be reduced. The acceleration may be centrifugal acceleration. A centrifugal separator may be used to apply centrifugal acceleration.

1.3. Liquid Discharge Step In the liquid discharge step S106, the liquid 3 in the container 1 is discharged by sequentially performing the following two operations.

1.3.1. First Operation In the first operation, first, as shown in FIG. 5, the pipette 6 is inserted into the container 1. Next, as shown in FIG. 5, the liquid 3 accumulated at the bottom of the container 1 is sucked out with a pipette 6 and discharged. In the magnetic separation step S104 described above, since the magnetic beads 2 are fixed to the inner wall of the container 1, the liquid 3 can be selectively discharged in this first operation.

Note that if there is no liquid 3 accumulated at the bottom of the container 1, the first operation may be omitted. Further, depending on the shape of the container 1, the liquid 3 may accumulate in a place other than the bottom of the container 1, and in that case, the operation of discharging the accumulated liquid 3 becomes the first operation.

1.3.2. Second Operation In the second operation, first, as shown in FIG. 6, the pipette 6 is brought close to the fixed magnetic beads 2. Then, as shown in FIG. 7, the tip of the pipette 6 is brought into contact with the fixed magnetic beads 2. Next, the pipette 6 is brought into contact with the magnetic beads 2, and a suction operation is performed using the pipette 6. As a result, the liquid 3 adhering to the magnetic beads 2 is sucked into the pipette 6. Since the liquid 3 attached to the magnetic beads 2 is attached to the magnetic beads 2 due to its surface tension, it cannot be separated from the magnetic beads 2 by gravity or the inertial force accompanying the acceleration described above. However, if the magnetic beads 2 are suctioned with the pipette 6 in contact with them, the liquid 3 adhering to the magnetic beads 2 can be suctioned. Thereby, even if a spike phenomenon occurs in the magnetic beads 2, the amount of liquid 3 remaining in the container 1 (residual liquid amount) can be sufficiently reduced. As a result, it is possible to prevent the liquid 3 adhering to the magnetic beads 2 from being brought into the cleaning step S108 and subsequent steps described below, and thereby reducing the purity of the nucleic acid that is finally extracted.

Furthermore, as described above, the magnetic beads 2 are fixed with sufficient magnetic attraction force. This magnetic attraction force exceeds the attraction force of a liquid suction tool such as the pipette 6. Therefore, when the liquid 3 adhering to the magnetic beads 2 is aspirated with the pipette 6, the magnetic beads 2 are prevented from being aspirated together with the liquid 3. Thereby, it is possible to suppress a decrease in the amount of extracted nucleic acid due to a decrease in the number of magnetic beads 2.

Therefore, if the magnetic attraction force F _mag determined by the above formula (1) is less than the lower limit value, there is a possibility that the number of magnetic beads 2 will decrease due to the second operation. When the magnetic beads 2 are reduced in this step, the nucleic acids adsorbed to the magnetic beads 2 are also lost, resulting in a decrease in the final amount of extracted nucleic acids.

Note that the state in which the pipette 6 and the magnetic beads 2 are in contact includes not only a state in which the pipette 6 and the magnetic beads 2 are in direct contact, but also a state in which they are in indirect contact through a thin film of the liquid 3. include. Even in the latter case, the liquid 3 adhering to the magnetic beads 2 can be aspirated by the pipette 6 using the surface tension of the liquid 3, as in the former case. Note that the thickness of the thin film is not particularly limited, but may be, for example, 1 mm or less.

Furthermore, when considering the realistic range of the average radius r and mass m of the magnetic beads 2, it is preferable that the magnetic field gradient formed by the external magnetic field during the second operation is 30 T/m or more and 1000 T/m or less. It is preferably 50 T/m or more and 500 T/m or less, and even more preferably 100 T/m or more and 400 T/m or less. By setting the magnetic field gradient within the above range, the necessary magnetic attraction force F _mag can be secured, and the moving speed of the magnetic beads 2 can be sufficiently increased to sufficiently shorten the time required for magnetic separation. I can do it.

The pipette 6 is an example of a liquid suction tool, and includes a cylindrical portion 62 and a pressure reducing portion 64, as shown in FIG. 7, for example. The cylindrical portion 62 has a cylindrical shape and is open at both ends. The tip of the cylindrical portion 62 serves as a suction port for sucking the liquid 3. The pressure reducing part 64 is connected to the base end of the cylindrical part 62 and has a function of making the inside of the cylindrical part 62 negative pressure. The pressure reducing section 64 is composed of, for example, a rubber ball, a piston, an electric pump, or the like.

In the second operation, with the cylindrical part 62 of the pipette 6 in contact with the magnetic beads 2, the pressure reducing part 64 is operated to create a negative pressure inside the cylindrical part 62, thereby causing the cylindrical part 62 to adhere to the magnetic beads 2. This is an operation for sucking the liquid 3 into the cylindrical portion 62.

Since such a second operation can be performed quickly and easily, the time required for discharging the liquid 3 can be easily shortened. Further, such a second operation is suitable for electrification and automation, and is therefore useful in that it facilitates labor saving.

The negative pressure generated by the pipette 6 may be increased if the above-described magnetic attraction force F _mag of the magnetic beads 2 is large, and may be decreased if the magnetic attraction force F _mag of the magnetic beads 2 is small.

When the magnetic field gradient formed by the external magnetic field is within the above-mentioned range, the negative pressure generated by the pipette 6 is preferably 3 Pa or more and 35 Pa or less, more preferably 5 Pa or more and 30 Pa or less. Thereby, while suppressing the attraction of the magnetic beads 2 together with the liquid 3, it is possible to sufficiently reduce the amount of the liquid 3 that cannot be attracted and remains attached to the magnetic beads 2. Further, such negative pressure contributes to a quick suction operation, so that the time required for magnetic separation can be shortened.

Note that the negative pressure generated by the pipette 6 is, for example, the maximum value of the negative pressure when a suction operation is performed with the pipette 6, as measured by a high-precision micromanometer. Examples of high-precision micropressure gauges include Manostar Gauge manufactured by Yamamoto Electric Manufacturing Co., Ltd., and the like.

On the other hand, the liquid suction tool may be other than the pipette 6. Another example of the liquid suction tool is a liquid suction member 6A including a porous body 66 shown in FIG. The liquid absorbing member 6A shown in FIG. 8 includes a support rod 68 and a porous body 66 attached to the tip of the support rod 68. The porous body 66 has a porous shape, and includes, for example, a sponge made of synthetic resin, a natural sponge such as a corpus cavernosum, a rubber sponge, a cellulose sponge, and the like. Examples of the constituent material of the porous body 66 include polypropylene, polystyrene, polyester, cellulose, cotton, linen, wool, and the like. Since the porous body 66 has a large number of pores, it sucks the liquid 3 using the capillary phenomenon caused by the surface tension of the liquid 3 as a driving force. Therefore, the second operation may be an operation in which the liquid 3 adhering to the magnetic beads 2 is sucked into the porous body 66 by bringing the porous body 66 of the liquid absorbing member 6A into contact with the magnetic beads 2. .

Such a second operation can be performed simply by bringing the liquid absorbing member 6A into contact with the magnetic beads 2, so it is useful in that it can be performed easily and quickly, and it is also easy to reduce the cost of the liquid absorbing member 6A. Useful. Further, since the porous body 66 is usually highly flexible, the liquid absorbing member 6A is useful in that even if the porous body 66 comes into contact with the magnetic beads 2, the fixation of the magnetic beads 2 is difficult to be released. It is.

In addition, in the second operation, the pipette 6 or the like may be brought into contact with any position of the magnetic beads 2 as long as the pipette 6 or the liquid suction member 6A comes into contact with the fixed magnetic beads 2.

After sequentially performing the first operation and second operation as described above, the application of the external magnetic field is turned off. Then, the fixation of the magnetic beads 2 is released, and the magnetic beads 2 fall to the bottom of the container 1, as shown in FIG. Thereby, the nucleic acid adsorbed to the magnetic beads 2 can be purified.

1.4. Washing Step In the washing step S108, the magnetic beads 2 to which nucleic acids have been adsorbed are washed. Washing refers to an operation in which contaminants adsorbed to the magnetic beads 2 are removed by bringing the magnetic beads 2 to which nucleic acids have been adsorbed into contact with a washing solution and then separating them again. .

Specifically, the cleaning liquid is supplied into the container 1 using a pipette or the like. Then, the magnetic beads 2 and the washing liquid are stirred. As a result, the washing liquid comes into contact with the magnetic beads 2, and the magnetic beads 2 to which nucleic acids are adsorbed are washed. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. are used. Further, at this time, the external magnetic field may be temporarily removed. Thereby, the magnetic beads 2 are dispersed in the washing liquid, so that the washing efficiency can be further improved.

Next, an external magnetic field is applied to the magnetic beads 2 again to fix them on the inner wall of the container 1, and then the cleaning liquid is discharged. By repeating the supply and discharge of the washing liquid as described above one or more times, the magnetic beads 2 are washed, thereby making it possible to accurately remove impurities other than nucleic acids.

The washing liquid is not particularly limited as long as it does not promote the elution of nucleic acids and the binding of contaminants to the magnetic beads 2, but examples thereof include organic solvents such as ethanol, isopropyl alcohol, and acetone, or aqueous solutions thereof; Examples include low salt concentration aqueous solutions. Examples of low salt concentration aqueous solutions include buffer solutions. The salt concentration of the low salt concentration aqueous solution is preferably 0.1 mM or more and 100 mM or less, more preferably 1 mM or more and 50 mM or less. The salt used to form the buffer solution is not particularly limited, but salts of TRIS, HEPES, PIPES, phosphoric acid, and the like are preferably used.

The cleaning liquid may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), SDS, or the like. Further, the cleaning liquid may contain a chaotropic substance such as guanidine hydrochloride. The pH of the cleaning solution is not particularly limited.

Note that the cleaning step S108 may be performed as necessary, and may be omitted if cleaning is not necessary.

Also in the cleaning step S108, operations similar to those in the magnetic separation step S104 and the liquid discharge step S106 described above, that is, the magnetic separation method according to the embodiment can be performed. Thereby, it is possible to prevent a large amount of washing liquid from remaining on the fixed magnetic beads 2. As a result, it is possible to prevent the cleaning liquid or its components from moving to the elution step S110. Furthermore, the magnetic beads 2 are prevented from being discharged together with the washing solution, and the amount of extracted nucleic acid can be prevented from decreasing due to the decrease in the number of magnetic beads 2.

1.5. Elution Step In the elution step S110, the nucleic acids adsorbed on the magnetic beads 2 are eluted into an eluate. Elution is an operation in which the magnetic beads 2 to which nucleic acids are adsorbed are brought into contact with an eluate and then separated again to transfer the nucleic acids to the eluate.

Specifically, first, an eluate is supplied into the container 1 using a pipette or the like. Then, the magnetic beads 2 and the eluate are stirred. Thereby, the eluate comes into contact with the magnetic beads 2, and the nucleic acid can be eluted. For stirring, for example, a vortex mixer, manual shaking, pipetting, etc. are used. Further, at this time, the external magnetic field may be temporarily removed. Thereby, the magnetic beads 2 are dispersed in the eluate, so that the elution efficiency can be further improved.

Next, an external magnetic field is applied to the magnetic beads 2 again to fix them on the inner wall of the container 1, and then the eluate containing the nucleic acid is discharged. Thereby, the nucleic acid can be recovered.

The eluate is not particularly limited as long as it is a liquid that promotes the elution of nucleic acids from the magnetic beads 2 to which nucleic acids are adsorbed. An aqueous solution containing 10 mM Tris-HCl buffer and 1 mM EDTA and having a pH of about 8 is preferably used.

The eluate may contain a surfactant such as Triton (registered trademark), Tween (registered trademark), SDS, and the like. Moreover, sodium azide may be contained as a preservative.

Further, in the elution step S110, the eluate may be heated. Thereby, elution of the nucleic acid can be promoted. The heating temperature of the eluate is not particularly limited, but is preferably 70°C or more and 200°C or less, more preferably 80°C or more and 150°C or less, and even more preferably 95°C or more and 125°C or less.

Examples of the heating method include a method of supplying a preheated eluate, a method of supplying an unheated eluate to a container and then heating it, and the like. The heating time is not particularly limited, but is preferably 30 seconds or more and 10 minutes or less.

Note that the elution step S110 may be performed as necessary, and may be omitted, for example, if the purpose is only to separate the magnetic beads 2 and the liquid 3 in the magnetic separation step S104.

Also in the elution step S110, the same operations as the above-described magnetic separation step S104 and liquid discharge step S106, that is, the magnetic separation method according to the embodiment can be performed. This can prevent a large amount of nucleic acid from remaining on the immobilized magnetic beads 2. As a result, it is possible to suppress a decrease in the yield of nucleic acids. Furthermore, the magnetic beads 2 are prevented from being discharged and recovered together with the eluate. Thereby, it is possible to suppress the magnetic beads 2 from becoming contaminants in the analysis of the nucleic acids eluted into the eluate.

2. Effects of the Embodiment As described above, the magnetic separation method according to the embodiment includes a magnetic separation step S104 and a liquid discharge step S106. In the magnetic separation step S104, an external magnetic field is applied to the container 1 containing the liquid 3 and the magnetic beads 2 having a saturation magnetization of 50 emu/g or more and an average particle size of 0.5 μm or more and 50 μm or less, and the magnetic beads 2 are separated from the container 1. be fixed to the inner wall of the Thereby, the magnetic beads 2 and the liquid 3 are separated. In the liquid discharge step S106, the liquid 3 adhering to the magnetic beads 2 is discharged by the pipette 6 while the pipette 6 (liquid suction tool) is in contact with the magnetic beads 2 fixed to the inner wall of the container 1.

In this magnetic separation method, the saturation magnetization [emu/g] of the magnetic beads 2 is M (B), the magnetic field gradient [T/m] formed by the external magnetic field is G, and the mass of the magnetic beads 2 [kg] An external magnetic field is applied so that the magnetic attraction force F _mag [pN] generated in the magnetic beads 2, which is determined by the following formula (1), is 80 pN or more, where is m.
F _mag = M(B)×G×m (1)

According to such a configuration, the magnetic beads 2 having a small diameter and high saturation magnetization are used, and even if a spike phenomenon occurs in the magnetic beads 2 due to the action of an external magnetic field, the liquid 3 attached to the magnetic beads 2 can be discharged. be able to. Thereby, the amount of residual liquid 3 in the container 1 can be sufficiently reduced. In other words, even when using magnetic beads 2 with a small diameter and high saturation magnetization, the separability between the magnetic beads 2 and the liquid 3 can be improved. As a result, it is possible to prevent impurities contained in the liquid 3 adhering to the magnetic beads 2 from being brought into the washing step S108 or the elution step S110, thereby reducing the purity of the nucleic acid that is finally extracted. can.

Furthermore, it is possible to prevent the magnetic beads 2 from being discharged together with the liquid 3 in the liquid discharge step S106. Thereby, it is possible to suppress the amount of nucleic acid finally extracted from decreasing as the number of magnetic beads 2 decreases. As a result, more nucleic acids can be subjected to analysis, thereby increasing the reliability of analysis results.

Furthermore, by using magnetic beads 2 with high saturation magnetization and a small average particle size, the time required for magnetic separation can be shortened and more nucleic acids can be adsorbed. Thereby, the time required to subject the nucleic acid to analysis can be shortened, and the reliability of the analysis results can be increased.

In addition, in the washing step S108, by performing the same operations as the magnetic separation step S104 and the liquid discharging step S106, components contained in the washing solution are brought to the elution step S110, and the purity of the nucleic acid to be finally extracted is improved. It is possible to suppress a decrease in the reliability of the analysis results and a decrease in the reliability of the analysis results. Further, in the liquid discharge step S106, it is possible to prevent the magnetic beads 2 from being discharged together with the cleaning liquid.

Furthermore, in the elution step S110, by performing the same operations as the magnetic separation step S104 and the liquid discharge step S106, the nucleic acid eluted into the eluate can be efficiently recovered. This allows more nucleic acids to be subjected to analysis, thereby increasing the reliability of the analysis results. Furthermore, it is possible to prevent the magnetic beads 2 from being collected together with the eluate in the liquid discharge step S106. This suppresses the negative influence of the magnetic beads 2 on nucleic acid analysis.

Moreover, it is preferable that the magnetic beads 2 include an amorphous structure or a nanocrystalline structure. As a result, the coercive force Hc of the magnetic beads 2 becomes a low value. As a result, the dispersibility of the magnetic beads 2 in the liquid can be improved. As a result, when the application of the external magnetic field is turned off, the contact area between the magnetic beads 2 and the liquid 3 can be increased, so that the adsorption efficiency of nucleic acids to the magnetic beads 2, the washing efficiency of the magnetic beads 2, This makes it easier to increase the elution efficiency of nucleic acids adsorbed to each other.

Moreover, it is preferable that the coercive force Hc of the magnetic beads 2 is 1500 A/m or less. Thereby, aggregation of the magnetic beads 2 can be suppressed, and the magnetic beads 2 can be more uniformly dispersed in the liquid.

Further, the pipette 6 (liquid suction tool) includes a cylindrical portion 62 having a cylindrical shape, and a pressure reducing portion 64 that makes the inside of the cylindrical portion 62 a negative pressure. The second operation included in the liquid discharge step S106 is to remove the liquid 3 attached to the magnetic beads 2 by creating a negative pressure inside the cylindrical portion 62 while the cylindrical portion 62 is in contact with the magnetic beads 2. It may also be an operation of sucking into the cylindrical portion 62.

Since such a second operation using the pipette 6 can be performed quickly and easily, the time required for discharging the liquid 3 can be easily shortened. Further, such a second operation is suitable for electrification and automation, and is therefore useful in that it facilitates labor saving.

The negative pressure generated by the pipette 6 is preferably 3 Pa or more and 35 Pa or less. Thereby, while suppressing the attraction of the magnetic beads 2 together with the liquid 3, it is possible to sufficiently reduce the amount of the liquid 3 that cannot be attracted and remains attached to the magnetic beads 2.

On the other hand, the liquid suction member 6A (liquid suction tool) includes a porous body 66 having a porous shape. The second operation included in the liquid discharge step S106 may be an operation of bringing the porous body 66 into contact with the magnetic beads 2 to suck the liquid 3 attached to the magnetic beads 2 into the porous body 66. good.

Such a second operation using the liquid absorbing member 6A can be performed simply by bringing the liquid absorbing member 6A into contact with the magnetic beads 2, and is therefore useful in that it can be performed easily and quickly. The liquid absorbing member 6A is also useful in that it is easy to reduce costs.

Furthermore, it is preferable that the magnetic beads 2 include magnetic metal powder 22 made of an alloy containing Fe as a main component. Even if the Fe-based alloy has a small particle size, it is possible to realize the magnetic metal powder 22 that has high saturation magnetization, low coercive force, and high magnetic permeability. Therefore, the magnetic beads 2 having such magnetic metal powder 22 have a high movement speed due to the action of an external magnetic field, can shorten the time required for magnetic separation, and have excellent dispersibility in a liquid.

Furthermore, the liquid 3 to be subjected to the magnetic separation step S104 may contain nucleic acids, proteins, saccharides, cells, peptides, bacteria, or viruses. In extracting these biological substances, high purity biological substances can be extracted by passing through the magnetic separation step S104 and the liquid discharge step S106. Thereby, when analyzing the extracted biological material, it is possible to suppress the influence of contaminants on the analysis results.

3. Modified Example Next, a biological substance extraction method including a magnetic separation method according to a modified example will be described.

FIGS. 10 and 11 are schematic diagrams for explaining a biological material extraction method including a magnetic separation method according to a modification.

Hereinafter, a magnetic separation method according to a modification will be described. In the following explanation, the differences from the magnetic separation method according to the embodiment will be mainly explained, and the explanation of similar matters will be omitted. Note that in FIGS. 10 and 11, the same components as those in the embodiment described above are denoted by the same reference numerals.

In the magnetic separation method according to the embodiment, as shown in FIG. 4, a magnet 5 is placed on the side of the container 1. On the other hand, in a magnetic separation method according to a modification, a magnet 5 is arranged vertically below the container 1, as shown in FIGS. 10 and 11. The magnetic separation method according to the modification is the same as the magnetic separation method according to the embodiment, except that the arrangement of the magnets 5 is different.

In the magnetic separation step S104, as shown in FIG. 10, magnetic beads 2 are fixed to the bottom of the container 1. Then, the magnetic beads 2 are fixed while being immersed in the liquid 3.

In the liquid discharge step S106, as shown in FIG. 11, the tip of the pipette 6 is brought into contact with the fixed magnetic beads 2. Next, with the pipette 6 in contact with the magnetic beads 2, a suction operation is performed using the pipette 6. As a result, the liquid 3 adhering to the magnetic beads 2 is sucked into the pipette 6.

In the embodiment, the first operation and the second operation are sequentially performed in the liquid discharge step S106, but in this modification, the liquid 3 is discharged in one operation. In other words, the liquid 3 attached to the magnetic beads 2 can also be discharged with one operation.
Even in the above modifications, the same effects as in the embodiment described above can be obtained.

Although the magnetic separation method of the present invention has been described above based on the illustrated embodiments, the present invention is not limited thereto. For example, the magnetic separation method of the present invention may be one in which an arbitrary desired step is added to the above embodiment. Further, the magnetic separation method of the present invention may be used for purposes other than the biological material extraction method shown in the embodiment, for example, for the purpose of extracting substances other than biological materials.

Next, specific examples of the present invention will be described.
4. Preparation of magnetic bead dispersion 4.1. Example 1
First, as magnetic beads, coated magnetic powder comprising magnetic metal powder having an amorphous structure as a main structure and a silicon oxide film covering the surface of the particle was prepared. The alloy composition _of the _magnetic metal powder _{was Fe73Si11Cr2B11C3} , _and the main structure was an amorphous structure. Note that the numerical values in the composition formula represent atomic %.

Next, the prepared magnetic beads and pure water were placed in a 1.5 mL microtube (container) to prepare 500 μL of a magnetic bead dispersion. Note that the content of magnetic beads in the magnetic bead dispersion was 5% by mass.

The characteristics of the magnetic beads, specifically, the average particle diameter D50, saturation magnetization, and coercive force are shown in Table 1.

Table 1 also shows the magnetic attraction force generated on one particle of magnetic beads when the magnetic field gradient formed by the external magnetic field applied to magnetic beads is changed to three levels: 208 T/m, 240 T/m, and 312 T/m. are also shown.

4.2. Examples 2-4
A magnetic bead dispersion was prepared in the same manner as in Example 1, except that the characteristics of the magnetic beads were changed as shown in Table 1.

4.3. Comparative examples 1 to 3
A magnetic bead dispersion was prepared in the same manner as in Example 1, except that magnetic beads containing magnetic metal powder whose main structure was a crystalline structure were used, and the characteristics of the magnetic beads were changed as shown in Table 1. . The alloy composition of the magnetic metal powder was Fe ₈₈ Si ₅ Cr ₇ . Note that the numerical values in the composition formula represent atomic %.

5. Evaluation of magnetic separation using magnetic beads 5.1. Relationship between magnetic attraction force generated in magnetic beads and suction operation using a pipette First, as a magnetic separation step, insert the microtube containing the magnetic bead dispersion liquid of each example and each comparative example into a magnetic stand, and leave it still for 10 seconds. did. As a result, an external magnetic field acted on the magnetic beads, and the magnetic beads were immobilized on the inner wall of the microtube. Note that a magnetic stand was used in which the distance between the microtube and the magnet could be changed.

Next, as the first operation of the liquid discharge step, the tip of the micropipette was inserted into the bottom of the microtube, and the pure water accumulated at the bottom was sucked and discharged. Subsequently, as a second operation in the liquid discharge step, the tip of the micropipette was brought into contact with the magnetic beads fixed to the inner wall of the microtube, and a suction operation was performed using the micropipette. At this time, the magnetic field gradient formed by the magnetic stand is as shown in Table 1.

Then, whether or not the magnetic beads were attracted during the suction operation was evaluated in accordance with the following evaluation criteria.

A: Magnetic beads were not attracted to the micropipette B: Magnetic beads were attracted to the micropipette The above evaluation results are shown in Table 1.

As shown in Table 1, the magnetic beads used in each Example and Comparative Example 1 generated a magnetic attraction force of 80 pN or more. On the other hand, the magnetic attraction force of the magnetic beads used in Comparative Examples 2 and 3 was less than 80 pN.

Then, when a suction operation using a micropipette was performed on the magnetic beads in which this magnetic attraction force was generated, the magnetic beads of each Example and Comparative Example 1 were not attracted. On the other hand, in the magnetic beads used in Comparative Examples 2 and 3, attraction of the magnetic beads was observed.

Based on the above, by using magnetic beads with appropriate saturation magnetization and particle size, and applying an external magnetic field so that the magnetic attraction force generated in the magnetic beads is within a predetermined range, the magnetism generated in the liquid discharging process can be reduced. It was recognized that unintended decrease in beads could be suppressed.

5.2. Relationship between the magnetic attraction force generated in magnetic beads and the suction operation using a liquid suction member The suction operation was performed in the same manner as in 5.1, except that a liquid suction member was used instead of a micropipette as the liquid suction tool. The presence or absence of accompanying attraction of magnetic beads was evaluated. The liquid absorbing member used here was a sponge cotton swab equipped with a porous body made of polyurethane. Further, the evaluation criteria are the same as in 5.1. The evaluation results are shown in Table 2.

As shown in Table 2, even when a liquid suction member is used instead of a micropipette, the magnetic beads of each Example and Comparative Example 1 generate a magnetic attraction force of 80 pN. Also, magnetic beads were not attracted. On the other hand, in the magnetic beads used in Comparative Examples 2 and 3, attraction of the magnetic beads was observed.

5.3. Relationship between magnetic attraction force generated by magnetic beads and negative pressure generated by a pipette The presence or absence of magnetic beads attracted to the micropipette was evaluated, except that the Pipetman P series manufactured by Gilson was used as the micropipette. Note that the magnetic field gradient formed by the external magnetic field applied to the magnetic beads was 208 T/m. Further, the above evaluation was performed while changing the negative pressure generated by the micropipette in six steps from 5 Pa to 50 Pa. Note that the evaluation criteria are the same as in 5.1. The evaluation results are shown in Table 2.

As shown in Table 2, with the magnetic beads used in each Example and Comparative Example 1, attraction of the magnetic beads did not occur if the negative pressure generated by the micropipette was 30 Pa or less. In particular, with the magnetic beads used in Examples 3 and 4 and Comparative Example 1, attraction of the magnetic beads did not occur even when the negative pressure generated by the micropipette was set to 40 Pa. On the other hand, with the magnetic beads used in Comparative Examples 2 and 3, unintended attraction of the magnetic beads occurred regardless of the magnitude of the negative pressure.

Based on the above, by using magnetic beads with appropriate saturation magnetization and particle size, and by applying an external magnetic field so that the magnetic attraction force generated in the magnetic beads is within a predetermined range, it is possible to achieve a sufficiently large negative pressure. It was found that even if a suction operation was performed, it was possible to suppress the unintended decrease in magnetic beads.

5.4. Relationship between presence or absence of contact with magnetic beads and suction operation using a pipette First, only the magnetic beads used in each Example and each Comparative Example were placed in a microtube, and the total weight including the microtube was measured. Let the measurement result be the first weight. Note that the weight of the magnetic beads alone was 25 mg. In addition, two microtubes each containing the same type of magnetic beads were prepared.

Next, 485 mg of pure water was placed in this microtube, and mixed and stirred using a vortex mixer. Thereafter, as a magnetic separation step, the microtube was inserted into a magnetic stand and left standing for 10 seconds. As a result, an external magnetic field acted on the magnetic beads, and the magnetic beads were immobilized on the inner wall of the microtube.

Next, as the first operation of the liquid discharge step, the tip of a micropipette (liquid suction tool) was inserted into the bottom of the microtube, and the pure water accumulated at the bottom was sucked and discharged. Subsequently, as a second operation in the liquid discharge step, the tip of the micropipette was brought into contact with the magnetic beads fixed to the inner wall of the microtube, and a suction operation was performed using the micropipette. At this time, the magnetic field gradient formed by the magnetic stand was 208 T/m. In addition, among the two microtubes, in one, the tip end was brought into contact with the magnetic beads in the second operation, and in the other one, the tip end was not brought into contact with the magnetic beads in the second operation.

Next, the total weight of the microtube after performing the liquid discharge step was measured. Let the measurement result be the second weight. Then, the first weight was subtracted from the second weight, and the subtraction result was taken as the residual liquid amount. Table 2 shows the determined residual liquid amount.

As shown in Table 2, it was found that by bringing the tip of the micropipette into contact with the magnetic beads in the second operation of the liquid discharging step, the amount of residual liquid could be significantly reduced compared to the case where the tips were not brought into contact.

From the above, it was confirmed that the liquid adhering to the magnetic beads can be efficiently discharged by performing a suction operation with a liquid suction tool such as a pipette in contact with the magnetic beads.

Note that when the magnetic beads of Comparative Examples 2 and 3 were used, attraction of the magnetic beads occurred. For this reason, it was not possible to calculate the amount of residual liquid, so it is indicated as "-" in Table 2.

6. Nucleic acid extraction by magnetic separation First, in the dissolution/adsorption step, the dispersion containing HeLa cells, the magnetic bead dispersion prepared in each example and each comparative example, and the dissolution/adsorption solution were placed in a container, and a vortex mixer was used. Stirring was performed for 10 minutes. Note that an aqueous solution containing guanidine hydrochloride was used as the dissolution and adsorption solution.

Next, as a magnetic separation step, magnetic separation (B/F separation) using a magnetic stand was performed. In the first operation of the liquid discharge step, the liquid accumulated at the bottom of the container was discharged with a pipette, and in the second operation, the tip of the pipette was brought into contact with the magnetic beads, and the liquid adhering to the magnetic beads was aspirated. In addition, the magnetic field gradient formed by the external magnetic field applied to the magnetic beads in the liquid discharge step was 208 T/m.

Next, a cleaning process was performed according to the following procedure.
First, the first cleaning liquid was placed in a container and stirred for 5 seconds. Subsequently, as a magnetic separation step, magnetic separation was performed using a magnetic stand. Subsequently, a liquid discharge step similar to that described above was performed. Thereafter, these washing operations were repeated multiple times.

Next, the second cleaning solution was added to the container and stirred for 5 seconds. Subsequently, as a magnetic separation step, magnetic separation was performed using a magnetic stand. Subsequently, a liquid discharge step similar to that described above was performed. Thereafter, these washing operations were repeated multiple times.

Next, an elution step was performed according to the following procedure.
First, sterile water as an eluent was placed in a container and stirred for 10 minutes. Subsequently, as a magnetic separation step, magnetic separation was performed using a magnetic stand. Subsequently, a liquid discharging process similar to the above was performed as a liquid discharging process. A nucleic acid extract was obtained as described above.

7. Evaluation of Nucleic Acid Extracts The nucleic acid extracts obtained using the magnetic beads of each Example and each Comparative Example were evaluated by the following method.

First, the obtained nucleic acid extract was subjected to real-time PCR measurement using a reagent TaqMan RNase P Detection Reagents Kit manufactured by Thermo Fisher Scientific. Real-time PCR measurement is a method of detecting a target substance, ie, a nucleic acid, present in a specimen by polymerase chain reaction. In real-time PCR measurement, an evaluation value called a Ct (cycle threshold) value is obtained. The Ct value is a numerical value indicating how many times amplification is performed until the target substance reaches a detectable threshold. More specifically, the Ct value is the number of cycles when the amplified product reaches a certain amount in PCR and the fluorescence brightness reaches a certain value or more. In other words, the smaller the Ct value, the higher the extraction efficiency of the substance to be tested, indicating that the test time can be shortened. Therefore, the more nucleic acids there are in the nucleic acid extract, the fewer the number of amplifications required to reach the threshold value, and the smaller the Ct value. Table 2 shows the Ct values determined for each nucleic acid extract.

As is clear from Table 2, sufficiently low Ct values were obtained in the nucleic acid extracts obtained using the magnetic beads of each Example.

On the other hand, in the nucleic acid extracts obtained using the magnetic beads of each comparative example, as indicated by ND (not detected) in Table 2, nucleic acids could not be detected, so the Ct value was calculated. I couldn't. The following may be the reason for this result.

The magnetic beads of Comparative Example 1 have a large average particle size and therefore a small specific surface area. Therefore, it is thought that in the nucleic acid extract obtained using the magnetic beads of Comparative Example 1, almost no nucleic acids could be detected.

The magnetic beads of Comparative Examples 2 and 3 have a small magnetic attraction force generated by the action of an external magnetic field. Therefore, it is considered that during the process of extracting the nucleic acids obtained using the magnetic beads of Comparative Examples 2 and 3, the number of magnetic beads decreased, and the amount of extracted nucleic acids decreased accordingly.

DESCRIPTION OF SYMBOLS 1... Container, 2... Magnetic beads, 3... Liquid, 5... Magnet, 6... Pipette, 6A... Liquid absorbing member, 22... Magnetic metal powder, 24... Coating layer, 62... Cylindrical part, 64... Decompression part, 66... Porous body, 68...Support rod, S102...Dissolution/adsorption process, S104...Magnetic separation process, S106...Liquid discharge process, S108...Washing process, S110...Elution process

Claims (8)

By applying an external magnetic field to a container containing a liquid and magnetic beads having a saturation magnetization of 50 emu/g or more and an average particle size of 0.5 μm or more and 50 μm or less and fixing the magnetic beads to the inner wall of the container, the magnetic beads are fixed to the inner wall of the container. a magnetic separation step of separating beads and the liquid;
a liquid discharging step of bringing a liquid suction tool into contact with the magnetic beads fixed to the inner wall and discharging the liquid adhering to the magnetic beads with the liquid suction tool;
has
When the saturation magnetization [emu/g] of the magnetic beads is M (B), the magnetic field gradient [T/m] formed by the external magnetic field is G, and the mass [kg] of the magnetic beads is m, the following A magnetic separation method characterized in that the external magnetic field is applied so that the magnetic attraction force F _mag [pN] generated in the magnetic beads determined by equation (1) is 80 pN or more.
F _mag = M(B)×G×m (1) The magnetic separation method according to claim 1, wherein the magnetic beads include an amorphous structure or a nanocrystalline structure. 3. The magnetic separation method according to claim 1, wherein the magnetic beads have a coercive force of 1500 A/m or less. The liquid suction tool includes a cylindrical part having a cylindrical shape and a pressure reducing part that makes the inside of the cylindrical part negative pressure,
The liquid discharging step includes an operation of drawing the liquid attached to the magnetic beads into the cylindrical portion by creating a negative pressure in the cylindrical portion while the cylindrical portion is in contact with the magnetic beads. The magnetic separation method according to any one of claims 1 to 3. The magnetic separation method according to claim 4, wherein the negative pressure is 3 Pa or more and 35 Pa or less. The liquid suction tool includes a porous body having a porous shape,
4. The liquid discharging step includes an operation of bringing the porous body into contact with the magnetic beads, thereby sucking the liquid attached to the magnetic beads into the porous body. Magnetic separation method described in Section. 7. The magnetic separation method according to claim 1, wherein the magnetic beads include magnetic metal powder made of an alloy containing Fe as a main component. 8. The magnetic separation method according to claim 1, wherein the liquid contains nucleic acids, proteins, saccharides, cells, peptides, bacteria, or viruses.

Images