Magnetic particle composition, use of magnetic particle composition for nucleic acid separation, kit for obtaining magnetic particle composition, magnetic particles, chaotropic salt, and separation and purification method

Abstract

This magnetic particle composition (e) contains: magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A); and a chaotropic salt (D), wherein the weight proportion of the magnetic metal oxide particles (A) contained in the core particles (P) is at least 60 wt% with respect to the weight of the core particles (P), and the coefficient of variation in the particle size distribution of the magnetic particles (c) is 5-50%.

Description

[Technical field]

[0001] The present invention relates to a magnetic particle composition, use of the magnetic particle composition in nucleic acid separation applications, a kit for obtaining the magnetic particle composition, magnetic particles, a chaotropic salt, and a separation and purification method. [Background technology]

[0002] Conventionally, spin column purification has been commonly used as a method for separating genes and the like derived from organisms living in various environments such as water, soil, and air from samples. However, the purification method using spin chromatography requires complicated steps such as adsorption of the target substance to be separated onto a solid phase column and extraction from the solid phase column while performing centrifugation, and is therefore not suitable for quick and simple separation and purification at, for example, farms, livestock farms, airports, etc., and real-time analysis cannot be performed. Therefore, complicated separation and purification using large equipment is unavoidable at analysis centers, and as a result, a time lag of several days occurs in obtaining the analysis results, which can lead to a worsening of the on-site situation, such as the proliferation of pathogenic bacteria, at farms, for example.

[0003] In response to this, a technique for purifying nucleic acids using magnetic silica particles has been disclosed as a technique that enables easy separation and recovery using magnetic force (Non-Patent Document 1). However, the nucleic acids obtained by this method contain a large amount of impurities other than the target nucleic acid, and the production efficiency is also insufficient. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Procedia Environmental Sciences, 2013, Vol. 18, pp. 856-863 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a magnetic particle composition that can be used in a method for separating a substance to be separated, and that can more efficiently produce a highly purified product. [Means for solving the problem]

[0006] The present inventors conducted extensive research to solve the above problems and arrived at the present invention. That is, the present invention provides a magnetic particle composition (e) comprising magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A) and a chaotropic salt (D), wherein the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%; use of the magnetic particle composition (e) in an application for separating nucleic acids from soil, environmental water, plant or animal excrement; and a kit (K) for obtaining the magnetic particle composition (e), comprising a combination of magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A) and a chaotropic salt (D), wherein the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), and the particle size distribution coefficient of the magnetic particles (c) is 5 to 50%. the magnetic particle composition (e) can be obtained by mixing the magnetic particles (c) and the chaotropic salt (D); magnetic particles (c) for obtaining the magnetic particle composition (e) or the kit (K), the magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A), the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) being 60% by weight or more based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the magnetic particles (c) being 5 to 50%; a chaotropic salt (D) for obtaining the magnetic particle composition (e) or the kit (K); and a separation and purification method for separating a substance to be separated (G) in a sample (F) by using the magnetic particle composition (e). Effect of the Invention

[0007] By separating a sample containing a substance to be separated using the magnetic particle composition of the present invention, a highly pure purified product can be efficiently obtained from the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] [Magnetic particle composition] The magnetic particle composition of the present invention is a magnetic particle composition (e) comprising magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A) and a chaotropic salt (D), wherein the weight proportion of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%.

[0009] The magnetic particles (c) are suitable for use in the separation method of the present invention (method for separating a substance to be separated (G) in a sample (F)) described in detail below. The substance to be separated (G) in the present invention means a target substance (G1) or a non-target substance (G2) in a mixture of multiple substances (such as biological substances) contained in the sample (F). The magnetic particles (c) are also suitable for use in the magnetic particle composition (e) of the present invention, which will be described in detail later, for nucleic acid separation.

[0010] The target substance (G1) refers to a substance that is ultimately desired to be obtained as a purified product from the sample (F).

[0011] The non-target substance (G2) ultimately refers to a substance to be removed from the sample (F).

[0012] Here, examples of the sample (F) in the present invention include samples derived from living organisms in the environment [soil, seawater, plant or animal excrement, biological fluids (serum, blood, lymph, ascites, urine, etc.), various types of cells, culture fluids, etc.], as well as mixtures containing a target substance (G1) and / or a non-target substance (G2) described in detail below. The sample (F) may also be soil, environmental water, plant or animal waste. Environmental water is a concept that includes water that makes up rivers, lakes, marshes, sea areas, groundwater, etc. The sample (F) may also contain microorganisms.

[0013] The magnetic particles (c) in the present invention have core particles (P) which are magnetic silica particles. The core particles (P) are particles containing magnetic oxide particles (A). The core particles (P) may contain other components as long as they contain magnetic oxide particles (A). The magnetic particles (c) in the present invention may be particles consisting only of core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A). In addition, the core particle (P) in this specification is the same as the core layer (P) described in Japanese Patent Application No. 2019-170710, which is the application on which this application claims priority.

[0014] The magnetic metal oxide particles (A) in the present invention may be ferrimagnetic, ferromagnetic, or superparamagnetic. Among the above, superparamagnetic particles are preferred because they can be quickly redispersed without residual magnetization after magnetic separation. Here, superparamagnetic particles refer to a temporary magnetic field induced by the alignment of individual atomic magnetic moments of a substance in the presence of an external magnetic field, and when the external magnetic field is removed, the partial alignment is lost and the magnetic field is no longer observed.

[0015] The magnetic metal oxide particles (A) include oxides of iron, cobalt, nickel, and alloys thereof, among which iron oxide is particularly preferred due to its excellent sensitivity to magnetic fields. The magnetic metal oxide particles (A) may be used alone or in combination of two or more kinds.

[0016] As the iron oxide used for the magnetic metal oxide particles (A), various known iron oxides can be used. Among the iron oxides, magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide, and γ-hematite-α-hematite intermediate iron oxide are preferred because of their excellent chemical stability, and magnetite is more preferred because of its large saturation magnetization and excellent sensitivity to an external magnetic field.

[0017] The magnetic metal oxide particles (A) preferably have a volume average particle diameter of 1 to 50 nm, more preferably 1 to 30 nm, and particularly preferably 1 to 20 nm. When the volume average particle diameter of the magnetic metal oxide particles (A) is 1 nm or more, the magnetic metal oxide particles (A) are easily synthesized.

[0018] The volume average particle diameter of the magnetic metal oxide particles (A) in this specification is the volume average value of the particle diameters measured by observing 200 arbitrary magnetic metal oxide particles (A) using a scanning electron microscope (e.g., "JSM-7000F" manufactured by JEOL Ltd.). The volume average particle diameter of the magnetic metal oxide particles (A) can be controlled by adjusting the metal ion concentration during the production of the magnetic metal oxide particles (A) described below. The volume average particle diameter of the magnetic metal oxide particles (A) can also be adjusted to a desired value by a method such as classification.

[0019] In the magnetic particle composition of the present invention, the weight proportion of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P). The lower limit of the weight proportion of the magnetic metal oxide particles (A) based on the weight of the core particles (P) is 60% by weight, preferably 65% ​​by weight, and the upper limit is preferably 95% by weight, more preferably 80% by weight. If the weight ratio of the magnetic metal oxide particles (A) is less than 60% by weight, the magnetic properties of the resulting magnetic particles (c) are insufficient, and the separation operation for practical use takes time. Also, if the weight ratio of the magnetic metal oxide particles (A) is more than 95% by weight, the synthesis may become difficult.

[0020] The manufacturing method of the magnetic metal oxide particles (A) is not particularly limited, but the particles can be synthesized by a coprecipitation method using a water-soluble iron salt and ammonia based on the method reported by Massart (R. Massart, IEEE Trans. Magn. 1981, 17, 1247), and a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.

[0021] The volume average particle size of the magnetic particles (c) is preferably 0.5 to 20 μm, more preferably 1 to 10 μm, and particularly preferably 1.1 to 5 μm. When the volume average particle size of the magnetic particles (c) is 0.5 μm or more, the time required for separation and recovery tends to be shortened. Also, when the average particle size of the magnetic particles (c) is 20 μm or less, the specific surface area can be relatively large, so that the amount of the separation target substance (G) can be increased and the binding efficiency tends to increase. Furthermore, when the volume average particle diameter of the magnetic particles (c) is 1.1 μm or more and the coefficient of variation of the particle size distribution of the magnetic particles (c) described in detail below is 21 to 35%, the separation property of the separation target substance (G) is improved. In this specification, the term "separability" will be described in terms of two cases: when the substance to be separated (G) is a target substance (G1) and when the substance to be separated (G) is a non-target substance (G2). When the substance to be separated (G) is a target substance (G1), "improved separation ability" means that the purity (proportion) of the target substance (G1) in the components extracted from the sample (F) using magnetic particles (c) is increased. Furthermore, when the substance to be separated (G) is a non-target substance (G2), "improved separation ability" means that the proportion of non-target substance (G2) in the components after removing the non-target substance (G2) from the sample (F) using magnetic particles (c) is reduced.

[0022] The volume average particle diameter of the magnetic particles (c) in the present invention is, for example, the volume average particle diameter measured with a laser diffraction / scattering type particle size distribution measuring device (Microtrac MT3300 manufactured by Microtrac-Bell Corporation).

[0023] The volume average particle size of the magnetic particles (c) can be adjusted to a desired value by changing the conditions of the water washing step during production and by methods such as classification.

[0024] In the magnetic particle composition of the present invention, the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%. If the coefficient of variation exceeds 50%, the separability of the substance to be separated (G) deteriorates. In order to further improve the separation ability of the substance to be separated (G), the lower limit of the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5% or more, preferably 13% or more, more preferably 20% or more, and particularly preferably 21% or more. From the viewpoint of further enhancing the separability of the separation target substance (G), the upper limit of the coefficient of variation of the particle size distribution of the magnetic particles (c) is preferably 35% or less. The coefficient of variation of the particle size distribution of the magnetic particles (c) can be measured by the following measurement method. <Method for measuring coefficient of variation> The coefficient of variation in the present invention is a value obtained by applying the volume average particle diameter (d) and standard deviation (SD) obtained by measurement using, for example, a laser diffraction / scattering type particle size distribution measurement device (Microtrac MT3300 manufactured by Microtrac-Bell Co., Ltd.) to mathematical formula (1). The volume average particle diameter (d) is on the order of nm to μm. Coefficient of variation (%) = SD / d × 100 (1)

[0025] The coefficient of variation of the particle size distribution of the magnetic particles (c) can be adjusted by classifying the magnetic particles (c). For example, magnetic particles (c) having a relatively large particle size can be removed by settling them by centrifugation, while magnetic particles (c) having a relatively small particle size can be removed by removing the supernatant liquid containing the fine particles that have not been settled after centrifugation.

[0026] The magnetic particle composition of the present invention contains magnetic particles (c) containing magnetic metal oxide particles (A) and a chaotropic salt (D). By containing the magnetic particles (c) and the chaotropic salt (D), a highly purified product can be efficiently obtained.

[0027] The magnetic particles (c) can bind the substance to be separated (G) to their surfaces. For example, a complex between the magnetic particles (c) and the substance to be separated (G), that is, a nucleotide chain of DNA, can be bound via a chaotropic salt (D) (guanidine thiocyanate, guanidine hydrochloride, sodium perchlorate, etc.).

[0028] The chaotropic salt (D) in the present invention is a substance used to reduce the interaction of water molecules existing between the surface of magnetic particles and nucleic acid substances such as DNA, and to destabilize the binding state. In other words, it refers to a solute that increases the entropy of water. By using the chaotropic salt (D), it is possible to pull out water molecules from the hydrated state between the surface of magnetic particles and nucleic acid substances, and to adsorb DNA onto the surface of magnetic particles.

[0029] The weight ratio (c / D) of the magnetic particles (c) to the chaotropic salt (D) is preferably 2 / 98 to 16 / 84, and more preferably 2 / 98 to 10 / 90, from the viewpoint of increasing the purity of the nucleic acid substance.

[0030] The chaotropic salt (D) may be selected from the "Hofmeister series." The Hofmeister series is a series of ions arranged in order of increasing ability to salt out proteins, also known as the lyotropic series. An example of a chaotropic anion is NO 3 - , ClO 4 - , SCN - and N.C.S. - Examples of chaotropic cations include Na + , B.A. 2+and guanidine cation. Preferred chaotropic salts (D) are salts formed from a combination of the above anions and cations, and include guanidine thiocyanate, guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium perchlorate, guanidine thiocyanate, and sodium iodide. Among these, it is preferable to use guanidine thiocyanate from the viewpoint of the yield of nucleic acid material.

[0031] Here, when the substance to be separated (G) does not directly bind to the magnetic particle (c), a substance (J) that binds to the substance to be separated (G) may be immobilized on the surface of the magnetic particle (c). By immobilizing the substance (J) on the surface, the substance to be separated (G) can be bound to the magnetic particle (c) via the substance (J). Hereinafter, the magnetic particle having the substance (J) immobilized on its surface in this manner will also be referred to as a "magnetic particle (c1)". Furthermore, the substance to be separated (G) may be a target substance (G1) or a non-target substance (G2), and the substance (J) is not particularly limited as long as it is a substance that binds to the target substance (G1) or the non-target substance (G2). The bond between the substance (J) and the substance to be separated (G) may be specific or non-specific, but it is preferable that the bond between the substance to be separated (G) and the substance (J) is specific. When the binding between the substance to be separated (G) and the substance (J) is specific, the separation property of the substance to be separated (G) is improved in the separation and purification method of the present invention using the magnetic particles (c).

[0032] Examples of the substance (J) that specifically binds to the substance to be separated (G) include substances that bind to the substance to be separated (G) through mutual reactions such as "gene"-"gene" reactions. In each of the above combinations, when one of them is a substance to be separated (G), the other is a substance (J) that specifically binds to the substance to be separated (G). For example, when the substance to be separated (G) is a "gene", the substance (J) is a "gene".

[0033] The magnetic particle composition of the present invention is preferably used for separating nucleic acids from soil, environmental water, plant or animal waste. Until now, it has not been known to use the magnetic particle composition of the present invention for separating nucleic acids from soil, environmental water, plant or animal waste, and there has been no suggestion that the magnetic particle composition of the present invention is suitable for such applications.

[0034] The present invention also includes use in isolating nucleic acids from soil, environmental water, plant or animal waste.

[0035] The magnetic particle composition of the present invention is preferably a mixture (E) comprising the core-shell particles (C) having a core particle (P) which is a magnetic silica particle containing a magnetic metal oxide particle (A) and a shell layer (Q) which is a silica layer having an average thickness of 3 to 3000 nm formed on the surface of the core particle (P), and comprising the core-shell particles (C) and a chaotropic salt (D). Below, as an example of an embodiment of the magnetic particle composition of the present invention, a case will be described in which the magnetic particles (c) are core-shell particles (C) and the magnetic particle composition (e) is a mixture (E) of the core-shell particles (C) and a chaotropic salt (D).

[0036] The mixture (E) is a mixture containing the core-shell particles (C) and a chaotropic salt (D).

[0037] The core-shell particles (C) are core-shell particles having a core particle (P), which is a magnetic silica particle containing a magnetic metal oxide particle (A), and a shell layer (Q), which is a silica layer having an average thickness of 3 to 3,000 nm formed on the surface of the core particle (P). The weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60 to 95% by weight based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the core-shell particles (C) is 5 to 50%.

[0038] The core-shell particles (C) are particularly suitable for use in the separation and purification method of the present invention (method for separating a substance to be separated (G) in a sample (F)) described in detail below. The substance to be separated (G) in the present invention means a target substance (G1) or a non-target substance (G2) in a mixture of multiple substances (such as biological substances) contained in the sample (F).

[0039] The target substance (G1) refers to a substance that is ultimately desired to be obtained as a purified product from the sample (F).

[0040] The non-target substance (G2) ultimately refers to a substance to be removed from the sample (F).

[0041] Examples of the sample (F) include samples derived from living organisms in the environment [soil, seawater, plant or animal excrement, biological fluids (serum, blood, lymph, ascites, urine, etc.), various types of cells, culture fluids, etc.], as well as mixtures containing a target substance (G1) and / or a non-target substance (G2) described in detail below. The sample (F) may be soil, environmental water, plant or animal waste, or may contain microorganisms.

[0042] As described above, the core-shell particle (C) has a core particle (P) which is a magnetic silica particle containing a magnetic metal oxide particle (A) and a shell layer (Q) which is a silica layer having an average thickness of 3 to 3000 nm formed on the surface of the core particle (P). The core particle (P) is preferably a sphere in which the magnetic metal oxide particle (A) is dispersed in a silica matrix. The shell layer (Q) may contain other components other than silica.

[0043] The magnetic metal oxide particles (A) contained in the core particles (P) preferably have a volume average particle diameter of 1 to 50 nm, more preferably 1 to 30 nm, and particularly preferably 1 to 20 nm. When the volume average particle diameter of the magnetic metal oxide particles (A) is 1 nm or more, the magnetic metal oxide particles (A) are easy to synthesize, and when the volume average particle diameter is 50 nm or less, the magnetic metal oxide particles (A) are easy to uniformly disperse in a silica matrix.

[0044] In the present invention, the lower limit of the weight proportion of the magnetic metal oxide particles (A) based on the weight of the core particles (P) is 60% by weight, preferably 65% ​​by weight, and the upper limit is 95% by weight, preferably 80% by weight. If the weight ratio of the magnetic metal oxide particles (A) is less than 60% by weight, the magnetic properties of the obtained core-shell particles (C) are insufficient, and the separation operation for practical use takes time. Also, if the weight ratio of the magnetic metal oxide particles (A) is more than 95% by weight, the synthesis is difficult.

[0045] As described above, the core-shell particles (C) are particles having a core-shell shape, and a shell layer (Q) is formed on the surface of a core particle (P).

[0046] The average thickness of the shell layer (Q) can be measured by image analysis of an image obtained by observing a cross section of a core-shell particle (C) embedded in a resin (such as an epoxy resin) and cut with a microtome using a transmission electron microscope. The average thickness of the shell layer (Q) is the average value of the thicknesses of the shell layer (Q) of any 100 core-shell particles (C) observed and measured using a transmission electron microscope (e.g., Hitachi's "H-7100"). The thickness of the shell layer (Q) is the average value of the thicknesses of the thinnest and thickest parts of a single core-shell particle (C), and is obtained by calculating the average thickness of the shell layer (Q) of each particle for 100 particles according to the same method, and then calculating the average thickness of the 100 particles.

[0047] The average thickness of the shell layer (Q) is 3 to 3000 nm, preferably 10 to 800 nm, more preferably 50 to 800 nm, particularly preferably 50 to 500 nm, and most preferably 50 to 200 nm. If the average thickness is less than 3 nm, the effect of the shell layer (Q) is not obtained and the amount of separation of the substance to be separated (G) decreases, and if the average thickness exceeds 3000 nm, it is difficult to synthesize the shell layer.

[0048] It may be difficult to distinguish between the shell layer (Q) and the core particle (P) from the image analysis of the image obtained by observation with the above-mentioned transmission electron microscope. The particles of the present invention containing magnetic metal oxide particles can be used for separation and purification by forming a complex with a target substance or a non-target substance, and it is sufficient that the particle surface has a moderate number of silanol groups, and it is preferable that the metal atoms derived from the magnetic metal oxide particles (A) are 10 mol % or less relative to the total atoms on the particle surface. The ratio (mol %) can be measured by X-ray photoelectron spectroscopy (XPS method).

[0049] The volume average particle size of the core-shell particles (C) is preferably from 0.5 to 20 μm, more preferably from 1 to 10 μm, and particularly preferably from 1.1 to 5 μm. When the volume average particle size of the core-shell particles (C) is 0.5 μm or more, the time required for separation and recovery tends to be shortened. Also, when the average particle size of the core-shell particles (C) is 20 μm or less, the specific surface area can be relatively large, so that the amount of the separation target substance (G) can be increased, and the binding efficiency tends to increase. Furthermore, when the volume average particle diameter of the core-shell particles (C) is 1.1 μm or more and the coefficient of variation of the particle size distribution of the core-shell particles (C) described in detail below is 21 to 35%, the separability of the separation target substance (G) is improved.

[0050] The volume average particle diameter of the core-shell particles (C) is, for example, a volume average particle diameter measured with a laser diffraction / scattering type particle size distribution measuring device (Microtrac MT3300 manufactured by Microtrac-Bell Corporation).

[0051] The volume average particle size of the core-shell particles (C) can be controlled by controlling the volume average particle size of the core particles (P) and the average thickness of the shell layer (Q). The volume average particle size of the core particles (P) can be controlled by adjusting the mixing conditions (shear force, etc.) when preparing the oil-in-water emulsion described below to adjust the particle size of the oil-in-water emulsion, and the average thickness of the shell layer (Q) can be controlled by adjusting the amount of (alkyl)alkoxysilane, the amount of catalyst, the reaction time, etc. when forming the shell layer (Q) described below. The volume average particle diameter of the core particles (P) and the core-shell particles (C) can also be adjusted to a desired value by changing the conditions of the water washing step during production and by a method such as classification.

[0052] In the present invention, the coefficient of variation of the particle size distribution of the core-shell particles (C) is 50% or less, as described above. If the coefficient of variation exceeds 50%, the separability of the separation target substance (G) deteriorates. From the viewpoint of further improving the separability of the substance to be separated (G), the lower limit of the coefficient of variation of the particle size distribution of the core-shell particles (C) is 5% or more, preferably 13% or more, more preferably 20% or more, and particularly preferably 21% or more. From the viewpoint of further enhancing the separability of the substance to be separated (G), the upper limit of the coefficient of variation of the particle size distribution of the core-shell particles (C) is preferably 35% or less. The coefficient of variation of the particle size distribution of the core-shell particles (C) can be measured by the following measurement method. <Method for measuring coefficient of variation> The coefficient of variation in the present invention is a value obtained by applying the volume average particle diameter (d) and standard deviation (SD) obtained by measurement using, for example, a laser diffraction / scattering type particle size distribution measurement device (Microtrac MT3300 manufactured by Microtrac-Bell Co., Ltd.) to mathematical formula (1). The volume average particle diameter (d) is on the order of nm to μm. Coefficient of variation (%) = SD / d × 100 (1)

[0053] The coefficient of variation of the particle size distribution of the core-shell particles (C) can be adjusted by classifying the core-shell particles. For example, core-shell particles having a relatively large particle size can be removed by settling them by centrifugation, while core-shell particles having a relatively small particle size can be removed by removing the supernatant liquid containing fine particles that have not been settled after centrifugation.

[0054] In the core-shell particle (C) of the present invention, the ratio of the average thickness of the shell layer (Q) to the particle diameter of the core particle (P) [average thickness of shell layer (Q) / particle diameter of core particle (P)] is preferably 0.001 to 10, more preferably 0.02 to 1.5, and particularly preferably 0.04 to 1.5. When the ratio is 0.001 or more, the separation property of the substance to be separated (G) is improved. When the ratio is 10 or less, the separation property of the substance to be separated (G) is improved. In calculating the ratio, the average thickness of the shell layer (Q) is determined by the method described above. In calculating the ratio, the particle diameter of the core particle (P) can be determined by the following formula using the values ​​of the "volume average particle diameter of the core-shell particle (C)" and the "average thickness of the shell layer (Q)" described above. Particle size of core particles (P) = Volume average particle size of core-shell particles (C) - 2 × Average thickness of shell layer (Q)

[0055] Furthermore, when the ratio of the average thickness of the shell layer (Q) in the core-shell particle (C) to the particle diameter of the core particle (P) [average thickness of the shell layer (Q) / particle diameter of the core particle (P)] is 0.02 to 1.5 and the coefficient of variation of the particle size distribution of the core-shell particle (C) is 21 to 35%, the separability of the separation target substance (G) is greatly improved.

[0056] The core-shell particles (C) in the present invention have a shell layer (Q) which is a silica layer, and therefore have silanol groups on the particle surface, which allows a predetermined type of separation target substance (G) to be bound to the surface.

[0057] As described above, the core-shell particles (C) can bind the substance to be separated (G) to their surfaces by the silanol groups of the core-shell particles (C). Specifically, a method of binding a complex between the silanol groups of the core-shell particles (C) and the nucleotide chain of DNA, which is the substance to be separated (G), via a chaotropic salt (D) (guanidine thiocyanate, guanidine hydrochloride, sodium perchlorate, etc.) can be mentioned.

[0058] In the case where the substance to be separated (G) does not directly bind to the core-shell particle (C), a substance (J) that binds to the substance to be separated (G) may be immobilized on the surface of the core-shell particle (C). By immobilizing the substance (J) on the surface, the substance to be separated (G) can be bound to the core-shell particle (C) via the substance (J). Hereinafter, the core-shell particle having the substance (J) immobilized on the surface in this manner is also referred to as a "core-shell particle (C1)". Furthermore, the substance to be separated (G) may be a target substance (G1) or a non-target substance (G2), and the substance (J) is not particularly limited as long as it is a substance that binds to the target substance (G1) or the non-target substance (G2). The bond between the substance (J) and the substance to be separated (G) may be specific or non-specific, but it is preferable that the bond between the substance to be separated (G) and the substance (J) is specific. When the bond between the substance to be separated (G) and the substance (J) is specific, the separation property of the substance to be separated (G) is improved in the separation and purification method of the present invention using the core-shell particles (C).

[0059] Examples of the substance (J) that specifically binds to the substance to be separated (G) include substances that bind to the substance to be separated (G) through mutual reactions such as "gene"-"gene" reactions. In each of the above combinations, when one of them is a substance to be separated (G), the other is a substance (J) that specifically binds to the substance to be separated (G). For example, when the substance to be separated (G) is a "gene", the substance (J) is a "gene".

[0060] Next, a method for producing the magnetic particles (c) of the present invention will be described using a method for producing core-shell particles (C) as the magnetic particles (c) as a representative example. The core-shell particles (C) can be produced by a production method that includes at least the following two steps. (Step 1) A step of preparing an oil-in-water emulsion of (alkyl)alkoxysilane containing magnetic metal oxide particles (A), and carrying out a hydrolysis polycondensation reaction of the (alkyl)alkoxysilane to produce core particles (P) in which the magnetic metal oxide particles (A) are encapsulated in silica. (Step 2) A step of forming a shell layer (Q) by subjecting (alkyl)alkoxysilane to a hydrolysis and polycondensation reaction on the surface of the core particle (P). The above steps will be described below.

[0061] First, step 1 will be described. Examples of methods for producing core particles (P) include a method in which a dispersion (B1) (hereinafter also simply referred to as "dispersion (B1)") containing magnetic metal oxide particles (A) and 30 to 1000% by weight of (alkyl)alkoxysilane based on the weight of the magnetic metal oxide particles (A) is mixed with a solution (B2) (hereinafter also simply referred to as "solution (B2)") containing water, a nonionic surfactant and a catalyst for hydrolysis of the (alkyl)alkoxysilane to prepare an oil-in-water emulsion, and a hydrolysis polycondensation reaction of the (alkyl)alkoxysilane is carried out to produce particles in which the magnetic metal oxide particles (A) are encapsulated in silica. After the hydrolysis and polycondensation reaction of the (alkyl)alkoxysilane, the core particles (P) are obtained by solid-liquid separation using centrifugation and a magnet or the like. Above and below, (alkyl)alkoxysilane means alkylalkoxysilane and / or alkoxysilane.

[0062] The (alkyl)alkoxysilane to be used includes a compound represented by the following general formula (1). R 1(4-n) Si(OR 2 )n (1) In general formula (1), R 1 and R2 represents a monovalent hydrocarbon group having 1 to 10 carbon atoms. Some of the hydrogen atoms of the hydrocarbon group may be substituted with an amino group, a carboxyl group, a hydroxyl group, a mercapto group, or a glycidyloxy group.

[0063] Examples of the monovalent hydrocarbon group having 1 to 10 carbon atoms include aliphatic hydrocarbon groups having 1 to 10 carbon atoms (such as a methyl group, an ethyl group, an n- or iso-propyl group, an n- or iso-butyl group, an n- or iso-pentyl group, and a vinyl group), aromatic hydrocarbon groups having 6 to 10 carbon atoms (such as a phenyl group), and aromatic aliphatic groups having 7 to 10 carbon atoms (such as a benzyl group).

[0064] In general formula (1), n ​​represents an integer of 1 to 4. However, when an alkylalkoxysilane in which n is 1 is used, it is necessary to use it in combination with an (alkyl)alkoxysilane in which n is 2 to 4. From the viewpoint of the strength of the particles after the reaction and the amount of silanol groups on the particle surface, n is preferably 4.

[0065] Specific examples of the compound represented by the general formula (1) include alkoxysilanes (tetramethoxysilane, tetraethoxysilane, tetraisopropoxysilane, tetrabutoxysilane, etc.); alkylalkoxysilanes (methyltrimethoxysilane, methyltriethoxysilane, etc.); alkylalkoxysilanes having an alkyl group substituted with an amino group [3-aminopropyltrimethoxysilane, 3-aminopropylethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, etc.]; and alkylalkoxysilanes having an alkyl group substituted with a carboxyl group. (7-carboxyheptyltriethoxysilane, 5-carboxypentyltriethoxysilane, etc.); alkylalkoxysilanes having an alkyl group substituted with a hydroxyl group (3-hydroxypropyltrimethoxysilane, 3-hydroxypropylethoxysilane, etc.); alkylalkoxysilanes having an alkyl group substituted with a mercapto group (3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, etc.); alkylalkoxysilanes having an alkyl group substituted with a glycidyloxy group (3-glycidyloxypropyltrimethoxysilane, 3-glycidyloxypropyltriethoxysilane, etc.). The (alkyl)alkoxysilanes may be used alone or in combination of two or more kinds.

[0066] The amount of (alkyl)alkoxysilane used is preferably 30 to 1,000% by weight, more preferably 40 to 500% by weight, based on the weight of the magnetic metal oxide particles (A). When the amount of (alkyl)alkoxysilane used is 30% by weight or more based on the weight of the magnetic metal oxide particles (A), the surfaces of the magnetic metal oxide particles (A) tend to be uniformly coated. When the amount of (alkyl)alkoxysilane used is 1,000% by weight or less based on the weight of the magnetic metal oxide particles (A), the recovery time by magnetic force can be shortened.

[0067] The amount of water used is preferably 500 to 50,000% by weight, and more preferably 1,000 to 10,000% by weight, based on the weight of the magnetic metal oxide particles (A).

[0068] Furthermore, in the synthesis of the core particles (P), a water-soluble organic solvent or the like may be contained in the solution (B2) or the like. Examples of the water-soluble organic solvent include monohydric alcohols having 1 to 4 carbon atoms (e.g., methanol, ethanol, and n- or iso-propanol), glycols having 2 to 9 carbon atoms (e.g., ethylene glycol and diethylene glycol), amides (e.g., N-methylpyrrolidone), ketones (e.g., acetone), cyclic ethers (e.g., tetrahydrofuran and tetrahydropyran), lactones (e.g., γ-butyrolactone), sulfoxides (e.g., dimethyl sulfoxide), and nitriles (e.g., acetonitrile), all of which have a solubility in water at 25° C. of 100 g / 100 g of water or more. Among these, from the viewpoint of uniformity of particle diameter of the core-shell particles (C), monohydric alcohols having 1 to 4 carbon atoms are preferred. The water-soluble organic solvents may be used alone or in combination of two or more kinds.

[0069] The amount of the water-soluble organic solvent used is preferably 100 to 500% by weight based on the weight of water.

[0070] Examples of the nonionic surfactant include alkylene oxide (hereinafter, alkylene oxide is abbreviated as AO) adducts of monohydric alcohols having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.); AO adducts of dihydric to octahydric alcohols having 3 to 36 carbon atoms (glycerin, trimethylolpropane, pentaerythritol, sorbitol, sorbitan, etc.); AO adducts of alkylphenols having an alkyl group having 6 to 24 carbon atoms (octylphenol, nonylphenol, etc.); ethylene oxide adducts of polypropylene glycol and polyethylene glycol. propylene oxide adducts of choline; AO adducts of fatty acids having 8 to 24 carbon atoms (such as decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and coconut oil fatty acid); fatty acid esters of the above-mentioned dihydric to octahydric alcohols having 3 to 36 carbon atoms and AO adducts thereof [TWEEN (registered trademark) 20, TWEEN (registered trademark) 80, and the like]; alkyl glucosides (N-octyl-β-D-maltoside, n-dodecanoylsucrose, n-octyl-β-D-glucopyranoside, and the like); and fatty acid esters of sucrose, fatty acid alkanolamides, and AO adducts thereof (such as polyoxyethylene fatty acid alkanolamides). These may be used alone or in combination of two or more. In the description of the nonionic surfactant, AO may be ethylene oxide, propylene oxide, butylene oxide, etc., and the addition form may be block addition or random addition. The number of moles of AO added is preferably 1 to 50 moles, more preferably 1 to 20 moles, per mole of alcohol, phenol, or fatty acid.

[0071] Among these nonionic surfactants, from the viewpoints of solubility in water and viscosity, 1 to 50 mol (preferably 1 to 20 mol) ethylene oxide adducts of monohydric alcohols having 8 to 24 carbon atoms (polyoxyethylene alkyl ethers, polyoxyethylene alkenyl ethers, etc.) are preferred. Among these nonionic surfactants, from the viewpoint of improving the separability of the substance to be separated (G) when it is separated and purified using the finally obtained core-shell particles (C), an adduct of 1 to 50 moles (preferably 1 to 20 moles) of ethylene oxide to a monohydric alcohol (e.g., oleyl alcohol) having an alkenyl group having 8 to 24 carbon atoms is preferred.

[0072] The amount of the nonionic surfactant used is preferably 10 to 1,000% by weight, more preferably 100 to 500% by weight, based on the weight of the magnetic metal oxide particles (A). When the amount of the nonionic surfactant used is 10% by weight or more or 1,000% by weight or less based on the weight of the magnetic metal oxide particles (A), the emulsion becomes stable and the particle size distribution of the generated particles tends to become narrow.

[0073] The amount of the solution (B2) used in step 1 is preferably 1,000 to 10,000% by weight, and more preferably 1,500 to 4,000% by weight, based on the weight of the magnetic metal oxide particles (A) contained in the dispersion liquid (B1). When the amount of the aqueous solution containing a nonionic surfactant used is 1,000% by weight or more or 10,000% by weight or less relative to the weight of the magnetic metal oxide particles (A), the emulsion becomes stable and the particle size distribution of the resulting particles tends to be narrow.

[0074] The catalyst for hydrolysis of the (alkyl)alkoxysilane may be Lewis acid, hydrochloric acid, etc., and specifically may be inorganic acid (hydrochloric acid, etc.), organic acid (acetic acid, etc.), inorganic base compound (ammonia, etc.), amine compound (ethanolamine, etc.), etc. The amount of the hydrolysis catalyst used is preferably 1 to 1000% by weight, more preferably 2 to 500% by weight, based on the weight of the (alkyl)alkoxysilane.

[0075] The method for mixing the dispersion liquid (B1) and the solution (B2) is not particularly limited, and they can be mixed all at once using the equipment described below. From the viewpoint of the uniformity of the particle size of the core-shell particles (C), however, a method in which the dispersion liquid (B1) is dropped while stirring the solution (B2) is preferred.

[0076] Equipment for mixing the dispersion (B1) and the solution (B2) is not particularly limited as long as it is a commercially available emulsifier or disperser, and examples thereof include batch-type emulsifiers such as Homogenizer (manufactured by IKA), Polytron (manufactured by Kinematica), and TK Auto Homo Mixer (manufactured by Primix Corporation), Ebara Milder (manufactured by Arihara Manufacturing Co., Ltd.), TK Filmix, TK Pipeline Homo Mixer (manufactured by Primix Corporation), Colloid Mill (manufactured by Kobelco Eco-Solutions Co., Ltd.), and Clearmix (manufactured by M Technique Co., Ltd.). Examples of such emulsifiers include continuous emulsifiers such as a Thrasher, a Trigonal Wet Fine Mill (manufactured by Nippon Coke and Engineering Co., Ltd.), a Capitron (manufactured by Eurotech Co., Ltd.), and a Fine Flow Mill (manufactured by Pacific Machinery Co., Ltd.); high-pressure emulsifiers such as a Microfluidizer (manufactured by Mizuho Kogyo Co., Ltd.), a Nanomizer (manufactured by Nanomizer Co., Ltd.), and a APV Gaulin (manufactured by Gaulin Co., Ltd.); membrane emulsifiers such as a Membrane Emulsifier (manufactured by Reika Kogyo Co., Ltd.); vibration emulsifiers such as a Vibro Mixer (manufactured by Reika Kogyo Co., Ltd.); and ultrasonic emulsifiers such as an Ultrasonic Homogenizer (manufactured by Branson). Among these, from the viewpoint of uniformity of particle size, APV Gaulin, Homogenizer, TK Auto Homo Mixer, Ebara Milder, TK Filmix, TK Pipeline Homo Mixer and Clear Mix (manufactured by M Technique Co., Ltd.) are preferred.

[0077] The temperature for the hydrolysis and polycondensation reaction of the (alkyl)alkoxysilane is preferably 10 to 100° C., more preferably 25 to 60° C. The reaction time is preferably 0.5 to 5 hours, more preferably 1 to 2 hours.

[0078] Next, a description will be given of step 2. Examples of a method for forming the shell layer (Q) include a method in which the core particles (P) obtained in step 1, an (alkyl)alkoxysilane, a catalyst for hydrolysis of the (alkyl)alkoxysilane, water, and, if necessary, a water-soluble organic solvent are mixed together to carry out a hydrolysis polycondensation reaction of the (alkyl)alkoxysilane, thereby forming a shell layer (Q) containing silica on the surface of the core particles (P).

[0079] As the (alkyl)alkoxysilane used in step 2, the (alkyl)alkoxysilanes exemplified in the explanation of step 1 can be mentioned, and the preferred ones are also the same.

[0080] In the reaction for forming the shell layer (Q), the concentration of the core particles (P) is preferably less than 50% by weight, more preferably less than 20% by weight, based on the weight of the reaction solution. When the concentration of the core particles (P) is less than 50% by weight, the core particles (P) are uniformly dispersed in the solution, making it easier to form a uniform shell layer (Q), and agglomeration of the core particles (P) via the silica can also be suppressed.

[0081] In the reaction for forming the shell layer (Q), the concentration of the (alkyl)alkoxysilane is preferably less than 50% by weight, more preferably less than 20% by weight, based on the weight of the reaction solution. When the concentration of the (alkyl)alkoxysilane in the solution is less than 50% by weight, the core particles (P) can be prevented from agglomerating together via the silica, and the formation of particles consisting of only silica, their aggregates, and aggregates consisting of these particles and the core particles (P) can also be prevented.

[0082] As the catalyst for hydrolysis of the (alkyl)alkoxysilane used in step 2, the catalysts for hydrolysis exemplified in the explanation of step 1 can be used. The amount of the hydrolysis catalyst used is preferably 1 to 2000% by weight, and more preferably 2 to 1000% by weight, based on the weight of the (alkyl)alkoxysilane.

[0083] The amount of water used is preferably 0.01 to 99.9% by weight, more preferably 0.1 to 99.9% by weight, based on the weight of the reaction solution [the total weight of the core particles (P), (alkyl)alkoxysilane, hydrolysis catalyst, water, water-soluble organic solvent, etc. used in the reaction]. When the amount of water used is 0.01% by weight or more relative to the weight of the (alkyl)alkoxysilane, the reaction rate of the hydrolysis of the (alkyl)alkoxysilane does not become too slow, and the reaction time for forming a shell layer (Q) of the desired average thickness can be shortened.

[0084] A water-soluble organic solvent may or may not be used, and when used, one type may be used alone or two or more types may be used in combination. Examples of the water-soluble organic solvent include the water-soluble organic solvents exemplified in the explanation of step 1, and the preferred ones are also the same.

[0085] In addition to the above, a non-ionic surfactant or the like can be used to improve the dispersibility of the core particles (P) during the reaction. Examples of the nonionic surfactant include the nonionic surfactants exemplified in the explanation of step 1, and the preferred ones are also the same.

[0086] The temperature for the hydrolysis and polycondensation reaction of the (alkyl)alkoxysilane in step 2 is preferably 0 to 90° C., and more preferably 15 to 50° C. The reaction time for the hydrolysis and polycondensation reaction of the (alkyl)alkoxysilane in step 2 is preferably 1 to 5 hours, and more preferably 1 to 3 hours.

[0087] When producing magnetic particles that are not core-shell particles (C) as the magnetic particles (c) of the present invention, only step 1 (step of producing core particles (P)) of the above-mentioned method for producing core-shell particles (C) is carried out, and step 2 (step of producing shell layer (Q)) is not carried out. By carrying out only step 1, magnetic particles (c) that are not core-shell particles (C) can be obtained.

[0088] Next, a kit for obtaining the magnetic particle composition (e) of the present invention will be described. The kit (K) of the present invention is a kit (K) for obtaining a magnetic particle composition (e), The magnetic particle (c) has a core particle (P) which is a magnetic silica particle containing a magnetic metal oxide particle (A), and a chaotropic salt (D). the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%, The kit (K) is for obtaining the magnetic particle composition (e), characterized in that the magnetic particle composition (e) can be obtained by mixing the magnetic particles (c) and the chaotropic salt (D).

[0089] In kit (K), the magnetic particles (c) and the chaotropic salt (D) for obtaining the magnetic particle composition (e) are present in a separated state. The magnetic particle composition (e) can be obtained by mixing the magnetic particles (c) with the chaotropic salt (D) immediately before use.

[0090] Furthermore, it is preferable that the magnetic particles (c) are core-shell particles (C), and the kit is capable of obtaining a mixture (E) by mixing the core-shell particles (C) with a chaotropic salt (D).

[0091] The magnetic particle (c) of the present invention is a magnetic particle (c) for obtaining the magnetic particle composition (e) of the present invention or the kit (K) of the present invention, The magnetic particles (c) have core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A), The weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), and the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%. By using the magnetic particles (c) of the present invention, the magnetic particle composition (e) of the present invention or the kit (K) of the present invention can be obtained. In addition, the magnetic particles (c) are preferably core-shell particles (C).

[0092] The chaotropic salt (D) of the present invention is a chaotropic salt (D) for obtaining the magnetic particle composition (e) of the present invention or the kit (K) of the present invention. By using the chaotropic salt (D) of the present invention, the magnetic particle composition (e) of the present invention or the kit (K) of the present invention can be obtained.

[0093] Next, the separation and purification method of the present invention will be described. The separation and purification method of the present invention is a method for separating a substance to be separated (G) from a sample (F) by using the magnetic particle composition (e) of the present invention. A separation and purification method for separating a target substance (G) from a sample (F) using magnetic particles (c) will be described below. The sample (F) used in the separation and purification method of the present invention is preferably soil, environmental water, plant or animal excrement. The separation and purification method of the present invention is suitable for separating a target substance from such a sample.

[0094] A first form of the method for separating and purifying a substance to be separated (G) of the present invention is a method for separating and purifying a substance to be separated (G) which is a target substance (G1), comprising a complex formation step (1) of contacting a sample (F1) containing the target substance (G1) with a magnetic particle composition (e) of the present invention to form a complex (H1) between the magnetic particles (c) and the target substance (G1), a complex separation step (2) of separating the complex (H1) from the sample (F1) by magnetic force, and a target substance dissociation step (3) of obtaining the target substance (G1) from the complex (H1) by adding a dissociation solution (I).

[0095] (Separation and purification method) The first embodiment of the separation and purification method of the present invention is a method in which a substance to be separated (G) is a target substance (G1), and the target substance (G1) is extracted and purified from a sample (F1) containing the target substance (G1). A first embodiment of the separation and purification method of the present invention includes (1) a complex formation step, (2) a complex separation step, and (3) a target substance dissociation step. Each step will be described below. (1) Complex formation process In this step, a sample (F) containing a target substance (G1) and a non-target substance (G2) is contacted with a magnetic particle composition (e) containing magnetic particles (c) and a chaotropic salt (D) to form a complex (H1) between the magnetic particles (c) and the target substance (G1). The chaotropic salt (D) may be present in a state of being bound to the complex (H1) as a counter ion, or may be present in a solution containing the complex (H1). The method for removing the non-target substance (G2) will be described later. In the complex formation step, it is preferable to contact the sample (F1) containing the target substance (G1) with the magnetic particle composition (e) in the presence of ethanol, which promotes the adsorption of the target substance (G1) to the magnetic particles (c).

[0096] (2) Complex separation process Next, the complex (H1) is separated from the sample (F1) by magnetic force. Since the complex (H1) contains the magnetic particles (c), and the magnetic particles (c) contain the magnetic metal oxide particles (A), the complex (H1) can be collected by magnetic force. Thereafter, the remaining sample (F1) is removed, so that the complex (H1) can be separated from the sample (F1). An example of such a method is a method in which the complex (H1) is collected from the outside of the reaction vessel by the magnetic force of a magnet or the like, the supernatant is discharged, and the complex (H1) is separated.

[0097] (3) Target substance dissociation process Next, the target substance (G1) is dissociated from the complex (H1) to obtain the target substance (G1). The method of dissociating the target substance (G1) from the complex (H1) is not particularly limited, but includes a method of dissociating the target substance (G1) by adding a substance that inhibits the binding between the magnetic particles (c) and the target substance (G1). The substance that inhibits the binding between the magnetic particles (c) and the target substance (G1) varies depending on the types of the target substance (G1) and the substance (G), and includes substances that inhibit binding due to pH difference, salt concentration difference, temperature difference, and the action of a surfactant. Examples of substances that can inhibit binding (dissociation solution I) include water, ethanol, IPA, and TE-HCl Buffer aqueous solution.

[0098] Furthermore, when the substance to be separated (G) is a non-target substance (G2), a separation and purification method can be used which includes a complex formation step of contacting a sample (F2) containing the target substance (G1) and the non-target substance (G2) with the magnetic particle composition (e) to form a complex (H2) between the magnetic particles (c) and the non-target substance (G2), and a non-target substance removal step of removing the non-target substance (G2) from the sample (F2) by separating the complex (H2) from the sample (F2) using a magnetic force, thereby obtaining the sample (F3) containing the target substance (G1).

[0099] A second embodiment of the method for separating and purifying a substance to be separated (G) of the present invention is a method for separating and purifying a substance to be separated (G) which is a non-target substance (G2), comprising the steps of: contacting a sample (F2) containing a target substance (G1) and the non-target substance (G2) with a magnetic particle composition (e) of the present invention to form a complex (H2) between the magnetic particles (c) and the non-target substance (G2); a non-target substance removing step of removing the non-target substance (G2) from the sample (F2) by magnetic force to obtain the sample (F3) containing the target substance (G1); The separation and purification method includes the steps of:

[0100] The second embodiment of the separation and purification method of the present invention is a method in which the separation target substance (G) is a non-target substance (G2), and the non-target substance (G2) is removed from a sample (F2) containing the non-target substance (G2). The separation and purification method of the present invention comprises (1) a complex formation step and (2) a non-target substance removal step. Each step will be described below.

[0101] (1) Complex formation process In this step, a sample (F) containing a target substance (G1) and a non-target substance (G2) is contacted with a magnetic particle composition (e) containing magnetic particles (c) and a chaotropic salt (D) to form a complex (H1) of the magnetic particles (c) and the target substance (G1), and a complex (H2) of the magnetic particles (c) and the non-target substance (G2). The complex (H2) may be formed by directly binding the non-target substance (G2) to the magnetic particle (c). Alternatively, the magnetic particle (c) may have a substance (G) that binds to the non-target substance (G2), and the complex (H2) may be formed by binding the non-target substance (G2) to the magnetic particle (c) via the substance (G). The chaotropic salt (D) may be present by binding to the complex (H1) and the complex (H2) as a counter ion, or may be present in a solution containing the complex (H1) and the complex (H2).

[0102] (2) Non-target substance removal process Next, the complex (H1) and the complex (H2) are separated from the sample (F2) by magnetic force. The complex (H1) and the complex (H2) contain magnetic particles (c), and the magnetic particles (c) contain magnetic metal oxide particles (A), so the complex (H1) and the complex (H2) can be collected by magnetic force. In the complex (H2), the non-target substance (G2) has a weaker adsorption to the magnetic particles (c) than the target substance (G1), and the non-target substance (G2) dissociates from the complex (H2) during magnetic collection and moves into the sample liquid. The complex (H1) and the complex (H2) are collected from the outside of the reaction vessel by the magnetic force of a magnet or the like, stirred, and the supernatant liquid is discharged, so that the complex (H1) is collected by the magnet from the outside of the reaction vessel and remains in the reaction vessel as a residue, while the non-target substance (G2) that formed the complex (H2) is contained in the supernatant liquid. By this method, the non-target substance (G2) can be separated.

[0103] Examples of the target substance (G1) in the separation and purification method of the present invention include single-stranded DNA, double-stranded DNA, single-stranded RNA, and double-stranded RNA.

[0104] In the present invention, the non-target substance (G2) means at least one of the substances contained in the sample (F) excluding the target substance (G1). That is, the type of the non-target substance (G2) may be multiple. For example, if the sample (F) is soil and the target substance (G1) is single-stranded DNA derived from microorganisms contained in the soil, at least one of the other components contained in the soil [proteins (albumin, etc.), lipids, inorganic matter, etc.] is a non-target substance (G2).

[0105] In the present invention, the substance to be separated (G) is preferably a nucleic acid, and more preferably at least one selected from the group consisting of single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA viruses, bacteria, and proteins. Moreover, the substance to be separated (G) is preferably at least one selected from the group consisting of DNA and RNA.

[0106] In the separation and purification method of the present invention, it is preferable that the magnetic particles (c) are core-shell particles (C), and it is preferable to separate the substance to be separated (G) in the sample (F) using a mixture (E) containing the core-shell particles (C) and a chaotropic salt (D). A separation and purification method for separating a target substance (G) from a sample (F) using core-shell particles (C) will be described below.

[0107] In this case, the first form of the method for separating and purifying a substance to be separated (G) is a method for separating and purifying a substance to be separated (G) when the substance to be separated (G) is a target substance (G1), the method comprising: a complex formation step (1) of contacting a sample (F1) containing the target substance (G1) with the mixture (E) to form a complex (H1) between the core-shell particle (C) and the target substance (G1); a complex separation step (2) of separating the complex (H1) from the sample (F1) by magnetic force; and a target substance dissociation step (3) of obtaining the target substance (G1) from the complex (H1) by adding a dissociation solution (I).

[0108] (Separation and purification method) In the separation and purification method, a substance to be separated (G) is a target substance (G1), and the target substance (G1) is extracted and purified from a sample (F1) containing the target substance (G1). The separation and purification method includes (1) a complex formation step, (2) a complex separation step, and (3) a target substance dissociation step. Each step will be described below. (1) Complex formation process In this step, a sample (F) containing a target substance (G1) and a non-target substance (G2) is contacted with a mixture (E) containing a core-shell particle (C) and a chaotropic salt (D) to form a complex (H1) between the core-shell particle (C) and the target substance (G1). The chaotropic salt (D) may be present in a state of being bound to the complex (H1) as a counter ion, or may be present in a solution containing the complex (H1). The method for removing the non-target substance (G2) will be described later.

[0109] (2) Complex separation process Next, the complex (H1) is separated from the sample (F1) by magnetic force. Since the complex (H1) contains the core-shell particles (C), which contain the magnetic metal oxide particles (A), the complex (H1) can be collected by magnetic force. Thereafter, the remaining sample (F1) is removed, whereby the complex (H1) can be separated from the sample (F1). An example of such a method is a method in which the complex (H1) is collected from the outside of the reaction vessel by the magnetic force of a magnet or the like, the supernatant is discharged, and the complex (H1) is separated.

[0110] (3) Target substance dissociation process Next, the target substance (G1) is dissociated from the complex (H1) to obtain the target substance (G1). The method for dissociating the target substance (G1) from the complex (H1) is not particularly limited, but includes a method in which the target substance (G1) is dissociated by adding a substance that inhibits the binding between the core-shell particle (C) and the target substance (G1). The substance that inhibits the binding between the core-shell particle (C) and the target substance (G1) varies depending on the types of the target substance (G1) and the substance (G), and includes substances that inhibit the binding due to pH difference, salt concentration difference, temperature difference, and the action of a surfactant. Examples of substances that can inhibit the binding (dissociation liquid I) include water, ethanol, IPA, and TE-HCl Buffer aqueous solution.

[0111] In addition, when the substance to be separated (G) is a non-target substance (G2), a separation and purification method can be used which includes a complex formation step in which a sample (F2) containing the target substance (G1) and the non-target substance (G2) is contacted with the mixture (E) to form a complex (H2) between the core-shell particle (C) and the non-target substance (G2), and a non-target substance removal step in which the complex (H2) is separated from the sample (F2) by magnetic force to remove the non-target substance (G2) from the sample (F2), thereby obtaining the sample (F3) containing the target substance (G1).

[0112] In this case, the second form of the separation and purification method is a method in which the separation target substance (G) is a non-target substance (G2), and the non-target substance (G2) is removed from a sample (F2) containing the non-target substance (G2). The separation and purification method includes (1) a complex formation step and (2) a non-target substance removal step. Each step will be described below.

[0113] (1) Complex formation process In this process, a sample (F) containing a target substance (G1) and a non-target substance (G2) is contacted with a mixture (E) containing core-shell particles (C) and a chaotropic salt (D) to form a complex (H1) of the core-shell particle (C) and the target substance (G1), and a complex (H2) of the core-shell particle (C) and the non-target substance (G2). The complex (H2) may be formed by directly binding the non-target substance (G2) to the core-shell particle (C). Alternatively, the core-shell particle (C) may have a substance (G) that binds to the non-target substance (G2), and the complex (H2) may be formed by binding the non-target substance (G2) to the core-shell particle (C) via the substance (G). The chaotropic salt (D) may be present by binding to the complex (H1) and the complex (H2) as a counter ion, or may be present in a solution containing the complex (H1) and the complex (H2).

[0114] (2) Non-target substance removal process Next, the complex (H1) and the complex (H2) are separated from the sample (F2) by magnetic force. The complex (H1) and the complex (H2) contain core-shell particles (C), and the core-shell particles (C) contain magnetic metal oxide particles (A), so the complex (H1) and the complex (H2) can be collected by magnetic force. In the complex (H2), the non-target substance (G2) has a weaker adsorption to the core-shell particles (C) than the target substance (G1), and the non-target substance (G2) dissociates from the complex (H2) during magnetic collection and moves into the sample liquid. The complex (H1) and the complex (H2) are collected from the outside of the reaction vessel by the magnetic force of a magnet or the like, stirred, and the supernatant liquid is discharged, so that the complex (H1) is collected by the magnet from the outside of the reaction vessel and remains in the reaction vessel as a residue, while the non-target substance (G2) that formed the complex (H2) is contained in the supernatant liquid. By this method, the non-target substance (G2) can be separated. EXAMPLES

[0115] Production Example 1: Preparation of core-shell type core-shell particles (C1-1) <Preparation of magnetic metal oxide particles (A)> A reaction vessel was charged with 186 parts of iron (III) chloride hexahydrate, 68 parts of iron (II) chloride tetrahydrate, and 1288 parts of water, which were dissolved and heated to 50°C. While maintaining the temperature at 50-55°C under stirring, 280 parts of 25% by weight ammonia water were dropped over 1 hour to obtain magnetite particles in water. 64 parts of oleic acid, a dispersant, were added to the obtained magnetite particles, and stirring was continued for 2 hours. After cooling to room temperature, the magnetite particles with oleic acid adsorbed thereon obtained by solid-liquid separation by decantation were washed three times with 1000 parts of water, and further washed twice with 1000 parts of acetone, and dried at 40°C for two days to obtain magnetic metal oxide particles (A-1) with a volume average particle diameter of 15 nm.

[0116] <Preparation of core particles (P)> 240 parts of tetraethoxysilane was added to 80 parts of the magnetic metal oxide particles (A-1) obtained in Production Example 1 and dispersed to prepare a dispersion liquid (B1). Next, 5050 parts of water, 3500 parts of 25% by weight aqueous ammonia solution, and 400 parts of Emulmin 200 (manufactured by Sanyo Chemical Industries, Ltd.) were added to a reaction vessel and mixed using Clearmix (manufactured by M Technique Co., Ltd.) to obtain a solution (B2). After heating to 50°C, the above dispersion liquid (B1) was dropped into the solution (B2) over 1 hour while stirring the Clearmix at a rotation speed of 6,000 rpm, and then reacted at 50°C for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant liquid containing the fine particles, and core particles (P-1) containing 83% by weight of magnetic metal oxide particles (A-1) were obtained.

[0117] <Preparation of core-shell particles (PC)> 80 parts of core particles (P-1), 2500 parts of deionized water, 260 parts of 25% by weight aqueous ammonia, 2500 parts of ethanol, and 1200 parts of tetraethoxysilane were added to a reaction vessel and mixed using Clearmix (manufactured by M Technique Co., Ltd.), and reacted for 2 hours while stirring at a rotation speed of 6,000 rpm. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant containing fine particles. After centrifugation, 4000 parts of deionized water was added to the particles that had precipitated, and the dispersed particles were collected by contacting a magnet from the outside of the vessel, and the operation of removing the supernatant was repeated 10 times to obtain core-shell particles (PC-1).

[0118] <Classification process of core-shell particles (PC)> 5,000 parts of water was added to the solid phase containing the obtained core-shell particles (PC-1) to disperse the particles, and the mixture was centrifuged at 2,800 rpm for 1 minute, after which the supernatant liquid containing the fine particles was removed four times (centrifugation step 1). Next, 5,000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 600 rpm for 1 minute to recover the supernatant, thereby removing the settled particles with large particle sizes (centrifugation process 2). Furthermore, the particles were collected using a magnet and the supernatant was removed. Then, 5,000 parts of water was added to disperse the core-shell particles, and the particles were collected using a magnet and the supernatant was removed 10 times (washing step 1) to obtain core-shell particles (C-1).

[0119] Production Examples 2 to 5: Preparation of core-shell type core-shell particles (C-2) to (C-5) The same operations as in Production Example 1 were carried out except that in the "classification process of core-shell particles (PC)" in Production Example 1, the production conditions were changed to those shown in Table 1, thereby obtaining core-shell particles (C-2) to (C-5).

[0120] Production Example 6: Preparation of core-shell particles (C-6) Magnetic metal oxide particles (A-2) having a volume average particle diameter of 2 nm were obtained in the same manner as in Production Example 1, except that in Production Example 1 <Preparation of magnetic metal oxide particles (A)>, 186 parts of iron (III) chloride hexahydrate and 68 parts of iron (II) chloride tetrahydrate were changed to 50 parts of iron (III) chloride hexahydrate and 18 parts of iron (II) chloride tetrahydrate. Thereafter, the same operations as in <Preparation of core particles (P)> and <Preparation of core-shell particles (PC)> of Production Example 1 were performed, except that the preparation conditions in the <Classification process of core-shell particles (PC)> were set to the preparation conditions shown in Table 1, and the same operations as in Production Example 1 were performed to obtain core-shell particles (C-6).

[0121] Production Example 7: Preparation of core-shell type core-shell particles (C-7) Magnetic metal oxide particles (A-3) having a volume average particle diameter of 47 nm were obtained in the same manner as in Production Example 1, except that in Production Example 1 <Preparation of magnetic metal oxide particles (A)>, 186 parts of iron (III) chloride hexahydrate and 68 parts of iron (II) chloride tetrahydrate were changed to 583 parts of iron (III) chloride hexahydrate and 213 parts of iron (II) chloride tetrahydrate. Thereafter, the same operations as in <Preparation of core particles (P)> and <Preparation of core-shell particles (PC)> of Production Example 1 were performed, except that the preparation conditions in the <Classification process of core-shell particles (PC)> were set to the preparation conditions shown in Table 1, and the same operations as in Production Example 1 were performed to obtain core-shell particles (C-7).

[0122] Production Example 8: Preparation of core-shell type core-shell particles (C-8) In the <Preparation of core-shell particles (PC)> of Production Example 1, 1,200 parts of tetraethoxysilane was changed to 20 parts of tetraethoxysilane, and the preparation conditions in the <Classification process of core-shell particles (PC)> were changed to the preparation conditions shown in Table 1. Except for this, the same operation as in Production Example 1 was performed to obtain core-shell particles (C-8).

[0123] Production Example 9: Preparation of core-shell type core-shell particles (C-9) In the <Preparation of core-shell particles (PC)> of Production Example 1, 1,200 parts of tetraethoxysilane was changed to 30,000 parts of tetraethoxysilane, and the preparation conditions in the <Classification process of core-shell particles (PC)> were changed to the preparation conditions shown in Table 1. Except for this, the same operation as in Production Example 1 was performed to obtain core-shell particles (C-9).

[0124] Production Example 10: Preparation of magnetic particles (c1) consisting of core particles only <Preparation of magnetic particles (Pc)> 240 parts of tetraethoxysilane was added to 80 parts of the magnetic metal oxide particles (A-1) obtained in Production Example 1 and dispersed to prepare a dispersion (B1). Next, 5050 parts of water, 3500 parts of 25% by weight aqueous ammonia solution, and 400 parts of Emulmin 200 (manufactured by Sanyo Chemical Industries, Ltd.) were added to a reaction vessel and mixed using Clearmix (manufactured by M Technique Co., Ltd.) to obtain a solution (B2). After heating to 50°C, the above dispersion (B1) was dropped into the solution (B2) over 1 hour while stirring the Clearmix at a rotation speed of 6,000 rpm, and then reacted at 50°C for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 20 minutes to remove the supernatant liquid containing the fine particles, and a solid phase containing magnetic particles (Pc1) containing 83% by weight of magnetic metal oxide particles (A-1) was obtained.

[0125] <Classification process of magnetic particles (Pc)> 5,000 parts of water was added to the solid phase containing the obtained magnetic particles (Pc1) to disperse the particles, and the mixture was centrifuged at 2,800 rpm for 1 minute, after which the supernatant liquid containing the fine particles was removed four times (centrifugation step 1). Next, 5,000 parts of water was added to the obtained solid phase to disperse the particles, and the mixture was centrifuged at 600 rpm for 1 minute to recover the supernatant, thereby removing the settled particles with large particle sizes (centrifugation process 2). Furthermore, the particles were collected using a magnet and the supernatant was removed. After that, 5,000 parts of water was added to disperse the core-shell particles, and the particles were collected using a magnet and the supernatant was removed 10 times (washing step 1), to obtain non-core-shell magnetic particles (c1) consisting of only core particles.

[0126] Comparative Manufacturing Example 1: Preparation of Comparative Particles (C'-1) Comparative particles (C'-1) were obtained by carrying out the same operations as in Production Example 1, except that in the <Preparation of core particles (P)> of Production Example 1, NSA-17 (manufactured by Sanyo Chemical Industries, Ltd.) was used instead of Emulmin 200, and in the <Classification process of core-shell particles (PC)> of Production Example 1, the preparation conditions were changed to those shown in Table 1.

[0127] Comparative Manufacturing Example 2: Preparation of Comparative Particles (C'-2) Comparative particles (C'-2) were obtained by carrying out the same operation as in Production Example 10 except that in the <Classification step of magnetic particles (Pc)> of Production Example 10, the conditions were as shown in Table 1.

[0128] The core-shell particles (C-1) to (C-9), non-core-shell type magnetic particles (c1), and comparative particles (C'-1) to (C'-2) obtained in Production Examples 1 to 10 and Comparative Production Examples 1 and 2 were evaluated as follows.

[0129] <Method for measuring the volume average particle size of magnetic metal oxide particles (A)> Using a scanning electron microscope (model number: JSM-7000F, manufacturer: JEOL Ltd.), 200 randomly selected magnetic metal oxide particles (A) were observed to measure their particle diameters and obtain the volume average particle diameter. The results are shown in Table 1.

[0130] <Method for measuring the weight ratio of magnetic metal oxide particles (A) in core particles (P)> For Production Examples 1 to 9 and Comparative Production Example 1, which are core-shell particles, any 20 core particles (P) obtained by the procedure of <Preparation of Core Particles (P)> were observed with a scanning electron microscope (model number: JSM-7000F, manufacturer: JEOL Ltd.), and the content of magnetic metal oxide particles (A) in the core particles (P) was measured with an energy dispersive X-ray spectrometer (model number: INCA Wave / Energy, manufacturer: Oxford University), and the average value was taken as the content S. In addition, the content of silica was measured by the same measurement, and the average value was taken as the content T. The weight percentage of the magnetic metal oxide particles (A) was calculated using the following formula. The results are shown in Table 1. Weight percentage of magnetic metal oxide particles (A) (% by weight) = [(S) / (S+T)] x 100 For Production Example 10 and Comparative Production Example 2, which are non-core-shell type magnetic particles consisting only of core particles, the content of magnetic metal oxide particles (A) in the magnetic particles (Pc) was measured in the same manner for any 20 magnetic particles (Pc1) obtained by the procedure of <Preparation of magnetic particles (Pc)>, and the results are shown in the column of "Weight ratio of magnetic metal oxide particles (A) in core particles (P)" in Table 1. In Production Example 10 and Comparative Production Example 2, the magnetic particles (Pc) are the core particles (P).

[0131] <Method of measuring volume average particle size and coefficient of variation of core-shell particles (C-1) to (C-9), non-core-shell type magnetic particles (c1), and comparative particles (C'-1) to (C'-2)> The core-shell particles (C-1) to (C-9), the non-core-shell type magnetic particles (c1), and the comparative particles (C'-1) to (C'-2) were each dispersed in a phosphate buffer solution, and the resulting dispersion of the magnetic particles was used as a sample to measure the particle size distribution using a laser diffraction / scattering type particle size distribution analyzer (Microtrack MT3300, manufactured by Microtrack Bell Co., Ltd.), and the volume average particle size and coefficient of variation were calculated. The results are shown in Table 1.

[0132] <Method for measuring the average thickness of the shell layer (Q) of the core-shell particles (C-1) to (C-9) and the comparative particle (C'-1)> The core-shell particles (C-1) to (C-9) or comparative particle (C'-1) obtained in the <Classification process of core-shell particles (PC)> of Production Examples 1 to 9 and Comparative Production Example 1 were embedded in epoxy resin and cut with a microtome. The cross section was observed with a transmission electron microscope ([Model number "H-7100", manufactured by Hitachi, Ltd.]) and the film thickness was calculated from the average value of the thickest and thinnest parts of one core-shell particle (C) [or comparative particle (C')]. Furthermore, the thickness of 99 random core-shell particles (C) [or comparative particles (C')] was measured in the same manner as above, and the average thickness of the total of 100 particles was taken as the average thickness of the shell layer (Q). The results are shown in Table 1. In addition, the average particle diameter of the core particles (P) was calculated using the following formula from the average thickness of the obtained shell layer (Q) and the volume average particle diameter of the core-shell particles (C) [or comparative particles (C')], and the ratio of the average thickness of the shell layer (Q) to the particle diameter of the core particles (P) is shown in Table 1. Particle size of core particles (P) = Volume average particle size of core-shell particles (C) - 2 × Average thickness of shell layer (Q)

[0133] [Table 1]

[0134] <Examples 1 to 13 and Comparative Examples 1 to 3: Preparation of magnetic particle composition (e)> (1) For each of the core-shell particles (C-1) to (C-9), non-core-shell magnetic particles (c1), and comparative particles (C'-1) to (C'-2) obtained in Production Examples 1 to 10 and Comparative Production Examples 1 to 2, 296 mg was placed in a glass container, 10 mL of pure water was added to a sample bottle, and then the mixture was stirred with a vortex mixer to obtain an aqueous dispersion containing magnetic particles.

[0135] (2) 600 μL of the aqueous dispersion containing the magnetic particles (weight of magnetic particles contained: 177.6 mg) was collected in a 15 mL microtube. A magnet was applied from the outside of the microtube to fix the magnetic particles to the wall of the microtube, and only the dispersion medium (water) of the aqueous dispersion was discarded. 900 μL of a chaotropic salt aqueous solution was added to the microtube in which only the magnetic particles remained, to prepare magnetic particle compositions (e1) to (e13) according to Examples 1 to 13 and comparative magnetic particle compositions (e'1) to (e'2) according to Comparative Examples 1 and 2. In addition, 600 μL of the aqueous dispersion of the core-shell particles (C-3) prepared in (1) was collected in a 15 mL microtube, a magnet was applied from the outside of the microtube to fix the magnetic particles to the wall of the microtube, and only the dispersion medium (water) of the aqueous dispersion was discarded. 900 μL of pure water was added to the microtube with only the magnetic particles remaining, and this was used as a comparative magnetic particle composition (e'3) according to Comparative Example 3. The chaotropic salt aqueous solution used was prepared by dissolving the chaotropic salt (D) shown in Table 2 in Tris-EDTA Buffer [(tris(hydroxymethyl)aminomethane: 10 mM, tetrasodium ethylenediaminetetraacetate: 2 mM, pH 7.86] to a concentration of 6 M.

[0136] [Table 2]

[0137] <Examples 14 to 30 and Comparative Examples 4 to 6: DNA Separation> [Complex formation process] (1) As a sample (F1) containing DNA as the target substance (D1) and BSA as an impurity, a mixture of 150 μL of an aqueous DNA solution and 150 μL of an aqueous BSA solution was prepared. The DNA aqueous solution was prepared by dissolving DNA (deoxyribonucleic acid, derived from salmon sperm, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) in the above Tris-EDTA buffer at a concentration of 2.40 mg / ml, and the BSA aqueous solution was prepared by dissolving BSA in the above Tris-EDTA buffer at a concentration of 2.40 mg / ml.

[0138] (2-1) 300 μL of the above-mentioned sample (F1) was added to each of the microtubes containing the magnetic particle compositions (e1) to (e13) obtained in Examples 1 to 13 and the comparative magnetic particle compositions (e'1) to (e'3) obtained in Comparative Examples 1 to 3. The microtubes were then shaken in a shaking incubator at 37°C, 350 rpm, and for 2.0 hours to promote contact between the magnetic particles and the DNA and BSA, thereby forming complexes between the magnetic particles and the DNA and BSA, thereby obtaining dispersions containing the DNA separation complexes (1-1) to (1-13) and (H1-1) to (H1-3) according to Examples 14 to 26 and Comparative Examples 4 to 6 listed in Table 3.

[0139] (2-2) 300 μL of the sample (F1) was added to the microtube containing the magnetic particle composition (e11) obtained in Example 11, and 900 μL of 99.5% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added, and the microtube was then shaken using a shaking incubator at 37° C., 350 rpm, and 2.0 hr to promote contact between the magnetic particles and the DNA and BSA, thereby forming complexes between the magnetic particles and the DNA and BSA, thereby obtaining a dispersion containing the DNA separation complex (1-14) according to Example 27. The concentration (volume fraction) of ethanol in the contents of the microtube in Example 27 was 43 vol% based on the volume of the liquid component among the components constituting the contents in the microtube.

[0140] (2-3) 300 μL of the sample (F1) was added to the microtube containing the magnetic particle composition (e3) obtained in Example 3, and 900 μL, 300 μL, and 1800 μL of 99.5% ethanol were added, respectively, to prepare a dispersion. The microtube was then shaken in a shaking incubator at 37° C., 350 rpm, and 2.0 hr to promote contact between the magnetic particles and DNA and BSA, thereby forming complexes between the magnetic particles and DNA and BSA, and thus obtaining dispersions containing the DNA separation complexes (1-15) to (1-17) according to Examples 28 to 30. The concentrations (volume fractions) of ethanol in the contents of the microtubes in Examples 28 to 30 were 43, 20, and 60% by volume, respectively, based on the volume of the liquid components among the components constituting the contents of the microtubes.

[0141] (3) After the complexes for DNA separation (1-1) to (1-17) and (H1-1) to (H1-3) were formed, the microtubes were allowed to stand to separate into a precipitate and a supernatant, and the supernatant was removed to recover the precipitate containing the complexes for DNA separation.

[0142] [Complex separation process] (4) 900 μL of 70% by volume ethanol aqueous solution was added to each of the collected precipitates to prepare a dispersion, and a magnet was placed on the outside of the container containing the dispersion to collect the complex for DNA separation on the magnet side. With the complex for DNA separation collected on the magnet side, the supernatant was removed, and 900 μL of 70% by volume ethanol aqueous solution was added again to prepare a dispersion. Dispersion in 70% by volume ethanol aqueous solution and removal of the supernatant were repeated a total of 10 times, and the complex for DNA separation was collected in a microtube.

[0143] [Target substance dissociation process] (5) 400 μL of the above Tris-EDTA buffer was added to the microtube containing the recovered DNA isolation complex obtained in (4) above, and the mixture was stirred in a vortex mixer for 15 seconds every 5 minutes three times to dissociate the DNA and BSA from the DNA isolation complex. Next, a magnet was applied to the outside of the vessel to cause the magnetic particles to thicken toward the magnet, and the entire amount of the supernatant containing the separated DNA and BSA (total amount of 400 μL) was collected with a pipette.

[0144] <Measurements of the recovered DNA and BSA> [Pre-measurement processing] A 100 μL aliquot was sampled from the supernatant (400 μL) recovered in the [target substance dissociation step] in (5) above, and added dropwise to a gravity column for desalting and buffer exchange (PD-10, GE Healthcare) to adsorb the DNA and BSA in the supernatant onto the adsorbent. After that, 1,100 μL of pure water was added to the column as an eluate for the adsorbed DNA and BSA, and the eluate that naturally fell was collected in a microtube. When 1,100 μL of eluate had been collected, the microtube was replaced, and 1,100 μL of pure water was again added to the column to collect 1,100 μL of eluate in the microtube. The procedure of replacing the microtube, adding 1,100 μL of pure water to the column, and collecting 1,100 μL of eluate in the microtube was repeated four more times (the procedure of collecting 1,100 μL of eluate in the microtube was performed a total of six times). A total of 1,100 μL of eluate was collected into a microtube over six operations. 200 μL each was taken from the eluate collected during the third to sixth operations, and the resulting liquid (800 μL in total) was used as the "DNA and BSA measurement sample."

[0145] [Measurement of DNA recovery amount] A DNA solution of known concentration was prepared as a standard solution, and the absorbance at 260 nm was measured using a spectrophotometer to prepare a calibration curve showing the relationship between absorbance and DNA concentration. The absorbance at 260 nm of the above-mentioned "DNA and BSA measurement sample" was also measured, and the concentration of DNA contained in the eluate obtained by pretreatment was calculated using the calibration curve. From the concentration of DNA contained in the eluate obtained in the pretreatment, the weight of DNA contained in 100 μL of the supernatant recovered in the [target substance dissociation step] that was dropped onto the column was calculated, and further the weight of DNA contained in the total amount of the supernatant recovered in the [target substance dissociation step] was calculated. Since the amount of DNA recovered is affected by the surface area of ​​the magnetic particles, the weight of DNA contained in the total amount of supernatant was divided by the total surface area of ​​the magnetic particles used in each Example and Comparative Example to calculate the weight of DNA recovered per unit surface area of ​​magnetic particles, and this is shown in Table 3.

[0146] [Measurement of BSA recovery amount] Using a standard solution of BSA with a known concentration [albumin, derived from bovine serum, low salt concentration, Fujifilm Wako Pure Chemical Industries, Ltd.], the absorbance at 280 nm was measured using a spectrophotometer, and a calibration curve showing the relationship between absorbance and BSA concentration was prepared. The absorbance at 280 nm of the above-mentioned "DNA and BSA measurement sample" was also measured, and the concentration of BSA contained in the eluate obtained by pretreatment on the column was determined using a calibration curve. Based on this value, in the same manner as in [Measurement of DNA concentration], the weight of BSA contained in the total amount of the supernatant was divided by the total surface area of ​​the magnetic particles used in the example (weight of recovered BSA per unit surface area of ​​magnetic particles) to calculate the value, which is shown in Table 3.

[0147] [Measurement of DNA purity] The absorbance of the DNA and BSA measurement samples at 280 nm (path length 10 mm) and at 260 nm (path length 10 mm) was measured using a UV-1800 ultraviolet-visible spectrophotometer (Shimadzu Corporation). The ratio of absorbance at 260 nm and 280 nm (absorbance ratio = A260 / A280) was calculated and evaluated as an index of DNA purity. The results are shown in Table 3. DNA with 100% purity has an absorbance ratio (A260 / A280) of 1.8, and when the absorbance ratio is in the range of 1.76 to 1.90, the recovered DNA is judged to be highly pure.

[0148] [Table 3]

[0149] <Examples 31 to 41 and Comparative Examples 7 to 9: Isolation of DNA from Environmental Samples> [Complex formation process] (1) DNA recovery solutions were prepared as samples (F2) by extracting DNA from culture soil, collected soil, river water, and animal excrement using the following method. Potting compost and harvested soil are examples of soil. Next, 300 μL of sample (F2) was added to a microtube containing any of the magnetic particle compositions (e1) to (e13) and comparative compositions (e'1) to (e'3) obtained in Examples 1 to 13 and Comparative Examples 1 to 3 in the combinations shown in Table 4, and in the case of Examples 40 and 41, 900 μL of 99.5% ethanol was also added. The microtube containing the contents was shaken in a shaking incubator at 37°C, 350 rpm, and 2.0 hours to form a complex between the magnetic particles and DNA (complex 2 for DNA separation). Thus, complexes 2 for DNA separation (2-1) to (2-11) and (H2-1) to (H2-3) according to Examples 31 to 41 and Comparative Examples 7 to 9 shown in Table 4 were formed.

[0150] (2) After the complex 2 for DNA isolation was formed, the microtube was left to stand to separate into a precipitate and a supernatant, the supernatant was removed, and the precipitate containing the complex 2 for DNA isolation was collected.

[0151] [Complex separation process] (3) 900 μL of 70% by volume ethanol aqueous solution was added to each of the collected precipitates to prepare a dispersion, and a magnet was placed on the outside of the container containing the dispersion to collect the DNA separation complex 2 on the magnet side. With the DNA separation complex 2 still collected on the magnet side, the supernatant was removed, and 900 μL of 70% by volume ethanol aqueous solution was added again to prepare a dispersion. Dispersion in the 70% by volume ethanol aqueous solution and removal of the supernatant were repeated a total of 10 times, and the DNA separation complex 2 was collected in a microtube.

[0152] [Target substance dissociation process] (4) 400 μL of the above Tris-EDTA buffer was added to the microtube containing the recovered DNA isolation complex 2 obtained in (3) above, and the mixture was stirred in a vortex mixer for 15 seconds every 5 minutes three times to dissociate the DNA from the DNA isolation complex 2. Next, a magnet was applied to the outside of the vessel to cause the magnetic particles to thicken on the magnet side, and the entire amount of the supernatant containing the separated DNA (total amount of 400 μL) was collected with a pipette.

[0153] <Analysis of the recovered DNA> The supernatant recovered in the [target substance dissociation step] of DNA separation complex 2 was subjected to the [measurement pretreatment] in the same manner as the supernatant recovered in the [target substance dissociation step] of DNA separation complex 1 described above, to prepare a DNA measurement sample, which was then analyzed in the same manner as the [measurement of DNA concentration] and [measurement of DNA purity] of DNA separation complex 1 described above, and the results are shown in Table 4.

[0154] [Preparation of DNA recovery solution from culture soil] In a Kenis Plant Box [made by AGC Techno Glass] with a hole of 4 mm diameter on the bottom, 100 g of horticultural culture soil (product name: Flower and Vegetable Soil [made by Taiyo Shokusan Co., Ltd.]) that had been heat-sterilized at 130°C for 30 minutes was placed, and two Komatsuna seeds [Takii Co., Ltd.] were sown in three places. The seeds were then covered with 1 cm of soil and placed in an artificial weather machine (LPH-241 / 411SP, made by Nippon Medical Equipment Co., Ltd.) set to the following conditions: temperature 20°C, humidity 55%, light intensity 60%, light / dark: 12hr / 12hr. The seeds were grown by giving them 50 ml of tap water once every two days, and the culture soil was collected from the plant box that had been cultivated for two weeks after germination. A wet weight of 2 g of culture soil was sampled from the collected culture soil, suspended in 2 ml of 10 mM Tris-EDTA-HCl Buffer [tris(hydroxymethyl)aminomethane: 10 mM, tetrasodium ethylenediaminetetraacetate: 1 mM, pH 7.4], and shaken at 5°C for 1 hour. Next, 20 μL of sodium dodecyl sulfate [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.] was added to the suspension, and the mixture was incubated for 30 minutes at 65 to 70° C. After that, the mixture was centrifuged at 3600 G and room temperature for 10 minutes, and the supernatant was used as a DNA recovery solution.

[0155] [Preparation of DNA recovery solution from collected soil] Two grams of wet soil was collected from an outdoor environment (Nishikyo Ward, Kyoto City, Kyoto Prefecture, Japan, near the Katsura Laboratory of Sanyo Chemical Industries, Ltd.), suspended in 2 ml of 10 mM Tris-EDTA-HCl Buffer (pH 7.4), and shaken at 5°C for 1 h. Next, 20 μL of sodium dodecyl sulfate was added to the suspension, and the mixture was incubated for 30 minutes at 65 to 70° C. After that, the mixture was centrifuged at 3600 G and room temperature for 10 minutes, and the supernatant was used as a DNA recovery solution.

[0156] [Preparation of DNA recovery solution from river water] 40 mL of river water collected from the Katsura River (Near Katsura Imperial Villa, Nishikyo Ward, Kyoto City, Kyoto Prefecture, Japan) was filtered through a 0.7 μm pore glass fiber filter (GF / F, WhatmanGF / F, Whatman). The filtered filter was transferred to a microtube, and 3.5 mL of 10 mM Tris-EDTA-HCl Buffer (pH 7.4) was added to the microtube, followed by stirring for 5 minutes with a vortex mixer. Next, 50 μg of protease K [Sigma-Aldrich] and 17.5 μL of sodium dodecyl sulfate were added and incubated for 15 minutes at 56° C. Then, the mixture was centrifuged at 3600 G and room temperature for 10 minutes, and the supernatant was used as a DNA recovery solution.

[0157] [Preparation of DNA recovery solution from animal waste] 1 g of feces from a C57BL / 6 mouse (Shimizu Experimental Materials Co., Ltd.) was suspended in 9 ml of phosphate buffer, and after standing, 2 ml of the supernatant was collected and centrifuged at 15,000 rpm for 3 minutes. The supernatant after centrifugation was discarded, and an SDS solution in which sodium dodecyl sulfate was dissolved at a concentration of 1% in 10 mM Tris-EDTA-HCl Buffer (pH 7.4) was added to the remaining precipitate, and incubated at 65 to 70 °C for 30 minutes. Then, it was centrifuged at 3600 G at room temperature for 10 minutes, and the supernatant was used as the DNA recovery solution.

[0158] [Table 4]

[0159] <Examples 42 to 58 and Comparative Examples 10 to 12: RNA isolation> [Complex formation] (1) As a sample (F3) containing the target substance (D2) RNA and the impurity BSA, a mixture of 150 μL of an aqueous RNA solution in which citric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved at a concentration of 25 mM and 150 μL of an aqueous BSA solution was prepared. The RNA aqueous solution was prepared by dissolving RNA (ribonucleic acid, yeast-derived, Fujifilm Wako Pure Chemical Industries, Ltd.) in the same Tris-EDTA buffer as used in Examples 14 to 26 at a concentration of 2.40 mg / ml, and the BSA aqueous solution was the same as used in Examples 14 to 26.

[0160] (2-1) 300 μL of the above-mentioned sample (F3) was added to each of the microtubes containing the magnetic particle compositions (e1) to (e13) obtained in Examples 1 to 13 and the comparative magnetic particle compositions (e′1) to (e′3) obtained in Comparative Examples 1 to 3. The microtubes were then shaken in a shaking incubator at 37° C., 350 rpm, and for 2.0 hours to promote contact between the magnetic particles and the RNA and BSA, thereby forming complexes between the magnetic particles and the RNA and BSA, thereby obtaining dispersions containing the RNA separation complexes (3-1) to (3-13) and (H3-1) to (H3-3) according to Examples 42 to 54 and Comparative Examples 10 to 12 listed in Table 5.

[0161] (2-2) 300 μL of the sample (F3) was added to the microtube containing the magnetic particle composition (e11) obtained in Example 11, and 900 μL of 99.5% ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was then added, and the microtube was then shaken using a shaking incubator at 37° C., 350 rpm, and 2.0 hr to promote contact between the magnetic particles and the RNA and BSA, thereby forming a complex between the magnetic particles and the RNA and BSA, thereby obtaining a dispersion containing the RNA separation complex (3-14) according to Example 55. The concentration (volume fraction) of ethanol in the contents of the microtube in Example 55 was 43 vol% based on the volume of the liquid component among the components constituting the contents in the microtube.

[0162] (2-3) 300 μL of the sample (F3) was added to the microtube containing the magnetic particle composition (e3) obtained in Example 3, and 900 μL, 300 μL, and 1800 μL of 99.5% ethanol were added, respectively, to prepare a solution, and then the microtube was shaken in a shaking incubator under conditions of 37° C., 350 rpm, and 2.0 hr to promote contact between the magnetic particles, RNA, and BSA, thereby forming complexes between the magnetic particles, RNA, and BSA, and obtaining dispersions containing the RNA separation complexes (3-15) to (3-17) according to Examples 56 to 58. The concentrations (volume fractions) of ethanol in the contents of the microtubes of Examples 56 to 58 were 43, 20, and 60 vol%, respectively, based on the volume of the liquid components among the components constituting the contents of the microtubes.

[0163] (3) After the RNA isolation complexes (3-1) to (3-17) and (H3-1) to (H3-3) were formed, the microtubes were allowed to stand to separate into precipitates and supernatants, and the supernatants were removed to recover precipitates containing the RNA isolation complexes.

[0164] [Complex separation process] (4) 900 μL of 70% by volume ethanol aqueous solution was added to each of the collected precipitates to prepare a dispersion, and a magnet was placed on the outside of the container containing the dispersion to collect the RNA separation complexes on the magnet side. With the RNA separation complexes still collected on the magnet side, the supernatant was removed, and another 900 μL of 70% by volume ethanol aqueous solution was added to prepare a dispersion. Dispersion in 70% by volume ethanol aqueous solution and removal of the supernatant were repeated a total of 10 times, and the RNA separation complexes were collected in a microtube.

[0165] [Target substance dissociation process] (5) 400 μL of the above Tris-EDTA buffer was added to the microtube containing the recovered RNA isolation complex obtained in (4) above, and the mixture was stirred in a vortex mixer for 15 seconds every 5 minutes three times to dissociate the RNA and BSA from the RNA isolation complex. Next, a magnet was applied to the outside of the vessel to cause the magnetic particles to thicken toward the magnet, and the entire amount of the supernatant containing the separated RNA and BSA (total amount of 400 μL) was collected with a pipette.

[0166] <Analysis of recovered RNA and BSA> The supernatant recovered in the [target substance dissociation step] of the RNA separation complex was subjected to the [measurement pretreatment] in the same manner as the supernatant recovered in the [target substance dissociation step] of the DNA separation complex 1 described above, to prepare an RNA measurement sample.

[0167] [Measurement of RNA recovery amount] An RNA solution with a known concentration was prepared as a standard solution, and the absorbance at 260 nm was measured using a spectrophotometer to prepare a calibration curve showing the relationship between absorbance and RNA concentration. The absorbance at 260 nm of the above-mentioned "RNA measurement sample" was also measured, and the concentration of RNA contained in the eluate obtained in the pretreatment was determined using a calibration curve. The amount of RNA recovered was calculated from the concentration of RNA contained in the eluate obtained by pretreatment in the same manner as in the case of the above-mentioned DNA separation complex 1, and is shown in Table 5.

[0168] [Measurement of BSA recovery amount] For the "RNA measurement sample", measurements and calculations were carried out in the same manner as in [Measurement of DNA purity] for the DNA separation complex 1, and the amount of BSA recovered was calculated. The results are shown in Table 5.

[0169] [Measurement of RNA purity] For the above-mentioned "RNA measurement sample", measurements and calculations were carried out in the same manner as in [Measurement of BSA recovery amount] for the above-mentioned DNA separation complex 1, and the results are shown in Table 5. Since the absorbance ratio (A260 / A280) of 100% pure RNA is 2.0, RNA purity is determined to be high when the absorbance ratio is in the range of 1.95 to 2.05.

[0170] [Table 5]

Claims

1. A magnetic particle composition (e) comprising magnetic particles (c) having core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A) and a chaotropic salt (D), wherein the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%, and the weight ratio (c / D) of the magnetic particles (c) to the chaotropic salt (D) is 2 / 98 to 16 / 84.

2. The magnetic particle composition (e) according to claim 1, wherein the magnetic metal oxide particles (A) have a volume average particle size of 1 to 50 nm.

3. The magnetic particle composition (e) according to claim 1 or 2, wherein the magnetic metal oxide particles (A) contain iron oxide.

4. The magnetic particle composition (e) according to any one of claims 1 to 3, wherein the magnetic particles (c) consist solely of core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A).

5. the magnetic particles (c) are core-shell particles (C) of a core-shell type having a core particle (P) which is a magnetic silica particle containing a magnetic metal oxide particle (A) and a shell layer (Q) which is a silica layer having an average thickness of 3 to 3000 nm formed on the surface of the core particle (P); The magnetic particle composition (e) according to any one of claims 1 to 3, which is a mixture (E) containing core-shell particles (C) and a chaotropic salt (D).

6. The magnetic particle composition (e) according to claim 5, wherein the core-shell particles (C) have a volume average particle size of 0.5 to 20 μm.

7. The magnetic particle composition (e) according to any one of claims 1 to 6, which is used for separating nucleic acids from soil, environmental water, plant or animal waste.

8. 8. Use of the magnetic particle composition (e) according to any one of claims 1 to 7 in the isolation of nucleic acids from soil, environmental water, plant or animal waste.

9. A kit (K) for obtaining a magnetic particle composition (e), comprising: The magnetic particles (c) have core particles (P) which are magnetic silica particles containing magnetic metal oxide particles (A), and a chaotropic salt (D), the weight ratio of the magnetic metal oxide particles (A) contained in the core particles (P) is 60% by weight or more based on the weight of the core particles (P), the coefficient of variation of the particle size distribution of the magnetic particles (c) is 5 to 50%, and the weight ratio (c / D) of the magnetic particles (c) to the chaotropic salt (D) is 2 / 98 to 16 / 84, A kit (K) for obtaining a magnetic particle composition (e), characterized in that the magnetic particle composition (e) can be obtained by mixing the magnetic particles (c) and the chaotropic salt (D).

10. A separation and purification method for separating a substance to be separated (G) in a sample (F) by using the magnetic particle composition (e) according to any one of claims 1 to 7.

11. 11. The method for separation and purification according to claim 10, wherein the sample (F) is soil, environmental water, or plant or animal waste, and the substance to be separated (G) is a nucleic acid.

12. a complex formation step in which the separation target substance (G) is a target substance (G1), a sample (F1) containing the target substance (G1) is contacted with the magnetic particle composition (e) according to any one of claims 1 to 7, and a complex (H1) between the magnetic particles (c) and the target substance (G1) is formed; a complex separating step of separating the complex (H1) from the sample (F1) by magnetic force; a target substance dissociation step of obtaining the target substance (G1) from the complex (H1) by adding a dissociation solution (I); The method for separation and purification according to claim 10 or 11, comprising:

13. The separation and purification method according to claim 12, wherein in the complex formation step, the sample (F1) containing the target substance (G1) and the magnetic particle composition (e) are contacted in the presence of ethanol.

14. a complex formation step in which the separation target substance (G) is a non-target substance (G2), and a sample (F2) containing a target substance (G1) and the non-target substance (G2) is contacted with the magnetic particle composition (e) according to any one of claims 1 to 7 to form a complex (H2) between the magnetic particles (c) and the non-target substance (G2); a non-target substance removing step of removing the non-target substance (G2) from the sample (F2) by magnetic force to obtain the sample (F3) containing the target substance (G1); The method for separation and purification according to claim 10 or 11, comprising:

15. The separation and purification method according to claim 14, wherein the non-target substance (G2) is of multiple types.

16. The method for separation and purification according to any one of claims 10 to 15, wherein the substance to be separated (G) is at least one species selected from the group consisting of DNA and RNA.

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