JPH11236506A - Magnetic particle, production thereof, and carrier for nonspecific nucleic acid bonding - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide silicone particles having magnetism and particularly a carrier for nonspecific nucleic acid bonding which is used for extracting and purifying nucleic acid from a material contg. nucleic acid or for purifying a nucleic acid amplification product. SOLUTION: The objective magnetic particles comprise (A) a magnetic material, (B) a polydimethylsiloxane compd. having hydrophilic groups, and (C) silica. The process for producing the magnetic particles comprises adding a polydimethylsiloxane compd. having hydrophilic groups to an aq. dispersion of a magnetic material and adding, to the dispersion, a tetraalkoxysilane and/or its partial condensate of hydrolyzate. The carrier for nonspecific nucleic acid bonding comprises the magnetic particles.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a silicon-based particle having magnetic properties, and more particularly to a method for extracting and purifying nucleic acid from a nucleic acid-containing material or purifying a nucleic acid amplification product. Carrier.

[0002]

2. Description of the Related Art Conventionally, as a method for isolating a nucleic acid using a magnetic carrier for binding a nucleic acid, silica-based magnetic particles made of superparamagnetic iron oxide covered with a polymerizable silane film to which a nucleic acid or the like can be covalently bonded are known. Have been. Also, Japanese Patent Application Laid-Open No. 6-472
Japanese Patent Application Laid-Open No. Hei 9-19292 discloses an almost perfect spherical colored spherical fine particle in which an organic or inorganic pigment whose surface is coated with a hydrated metal compound is covered with an inorganic porous wall material. The gazette includes the above-mentioned Japanese Patent Application Laid-Open No. 6-4742.
No. 3 discloses a nucleic acid binding carrier using the magnetic silica particles. However, conventional magnetic silica particles have a problem that those having good static stability are inferior in magnetic separability, and those having good magnetic separability have poor static instability. Magnetic silica particles with good magnetic separation properties have been demanded.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide magnetic particles which are excellent in static stability, magnetic separability and dispersibility and suitable for extracting or purifying nucleic acids.

[0004]

According to the present invention, there are provided magnetic particles comprising (A) a magnetic material, (B) a polydimethylsiloxane compound having a hydrophilic group and (C) silica, and a dispersion of the magnetic material. Particles comprising a magnetic substance, a polydimethylsiloxane compound having a hydrophilic group, and silica after adding a polydimethylsiloxane compound having a hydrophilic group to the body, and then adding a tetraalkoxysilane and / or a partially hydrolyzed condensate thereof. And a method for producing a non-specific nucleic acid comprising the magnetic particles.

Next, the present invention will be described specifically. The magnetic material (A) constituting the magnetic particles of the present invention is preferably a superparamagnetic material. Materials that can constitute such a superparamagnetic material include, for example, ferric oxide (Fe3O4), γ-iron sesquioxide (γ-Fe3O4), various ferrites, metals such as iron, manganese, cobalt, and chromium. Metal alloys and the like can be mentioned. The particle size of the magnetic material used in the present invention is:
Usually, it is 0.005 to 20 μm, and the saturation magnetization of the magnetic material is usually 100 Gauss or more, more preferably 200 Gauss or more, particularly preferably 300 Gauss or more. The magnetic material used in the present invention may be used by dispersing powder in water or an organic solvent, but it is more preferable that magnetic particles generally called magnetic fluid are dispersed in a solvent using a surfactant. preferable. Such a magnetic fluid has a mean particle size of about 5 to 200 nm using water, kerosene, paraffin or the like as a solvent. Specific examples of such a magnetic material include Marpomagna FW-40, FCN-50, and FPS manufactured by Matsumoto Yushi Seiyaku Co., Ltd.
-40, MA-400, FV-42, ML-405, P
F-3020, FV-200, FLD-10S, ML-
220, FN-40, FV-55, Ferricolloid W-40, HC-50, HC-6 manufactured by Taiho Industry Co., Ltd.
0, PA-40, LS-40, FE-20, TS-50
K and the like. Among these magnetic fluids, those using water as a dispersion medium are particularly preferable, and specific examples thereof include Marpomagna W-40 and ferricolloid W-40. (B) The polydimethylsiloxane compound having a hydrophilic group is a compound having a polydimethylsiloxane structure and a hydrophilic group. Examples of the hydrophilic group include a carboxyl group, a sulfone group, a hydroxyl group, and a polyoxyethylene group, and a carboxyl group and a polyoxyethylene group are particularly preferable. In addition, one kind or two or more kinds of hydrophilic groups can be contained, and those having two or more kinds of hydrophilic groups are more preferable. The polydimethylsiloxane compound having a hydrophilic group is -SiXnR3-n (where X represents a hydroxyl group, a chlorine atom, an alkoxy group, an alkoxycarbonyl group;
Represents a hydrogen atom or an alkyl group, and n represents a number of 1 to 3). Specific examples of such compounds include compounds represented by the following general formulas 1 to 5.

Formula 1

Embedded image

Wherein R1 is an alkoxysilyl group such as a trimethoxysilyl group or a triethoxysilyl group, R2 is a reactive organic functional group such as an epoxy group, a carboxyl group, or a carbinol group, and R3 is-(R4O) nR5 (Where R
4 represents an alkylene group such as a methylene group, an ethylene group or a propylene group, and R5 represents an alkyl group such as a methyl group, an ethyl group or a propyl group), an alkyl group or an alkylene group. ]

Formula 2

Embedded image

(Wherein R6 is a methyl group or-(R4
O) nR5 (R4 and R5 are the same as in general formula 1)
Wherein R7 represents an alkylene group, an arylene group or the like, and q and r each represent a number of 0 to 100. )

General formula 3

Embedded image (In the formula, R6 and R7 are the same as in the general formula 2, and s represents a number from 0 to 100.)

General formula 4

Embedded image (In the formula, t and u indicate a number of 0 to 100.)

[0012]

Embedded image

Wherein R8 is a hydroxyl group, -R9COOH
(R9 represents an alkylene group such as a methylene group, an ethylene group or a propylene group); a hydroxyalkyl group such as a hydroxymethyl group or a hydroxyethyl group; and v represents a number of 0 to 100. )

(C) Silica hydrolyzes tetraalkoxysilane and / or its partial hydrolysis / polycondensate.
It is polycondensed. The tetraalkoxysilane is preferably tetraethoxysilane, and the hydrolysis / polycondensate of tetraethoxysilane is ethyl silicate 4
0, ethyl silicate 45 and methyl silicate 50 are preferred.

The magnetic particles of the present invention comprise (A) a magnetic substance and (B) a polydimethylsiloxane compound having a hydrophilic group, and further comprise (C) silica as a component forming the particles. Is a state in which (1) the surface of the magnetic material is coated with a polydimethylsiloxane compound having a hydrophilic group and further coated with silica, and (2) the magnetic materials are bound with a polydimethylsiloxane compound having a hydrophilic group. The surface of the binder is coated with a polydimethylsiloxane compound having a hydrophilic group and silica in this order. (3) The magnetic substances are bound with the polydimethylsiloxane compound having a hydrophilic group. A state in which the surface of the binder is coated with silica; (4)
A state in which the magnetic substances are bound with a polydimethylsiloxane compound having a hydrophilic group, and the surface of the bound substance is further covered with a composite of a polydimethylsiloxane compound having a hydrophilic group and silica. . In the magnetic particles of the present invention, the ratio of each component is usually 1 to 150 parts by weight of component (B), 10 to 500 parts by weight of component (C), and preferably 5 to 5 parts by weight of component (C) per 100 parts by weight of component (A). ~ 80
Parts by weight, 30 to 300 parts by weight of component (C), particularly preferably 10 to 50 parts by weight of component (B), and 50 to 20 parts of component (C).
0 parts by weight.

The magnetic particles of the present invention are prepared by adding a polydimethylsiloxane having a hydrophilic group to a dispersion of a magnetic substance, preferably a magnetic fluid, and then adding tetraalkoxysilane and / or a partial hydrolysis / condensate thereof. It is obtained by performing a hydrolysis / polycondensation reaction. The magnetic substance is usually 0.1 to 20% by weight, more preferably 1 to 10% by weight.
Use in the concentration range of Particles comprising a magnetic substance and a polydimethylsiloxane compound having a hydrophilic group are formed by gradually adding the dimethylpolysiloxane compound (B) to the magnetic fluid under forced stirring. The particle size and particle size distribution of such particles vary depending on the magnetic particle concentration and the shear rate during stirring. If a conventionally known high-dispersion machine such as an ultrasonic disperser or a homogenizer is used, a small particle size can be obtained, and if a low-shear-rate disperser such as a mechanical stirrer is used, particles having a large particle size can be obtained. Examples of the ultrasonic disperser include, for example, a disperser having a horn-type oscillator or a plate-type oscillator, a continuous type disperser for flowing a liquid through an oscillating unit, and a high shear rate disperser. Homo mixer. Colloid yl, a jet homogenizer, a high-pressure homogenizer and the like can be mentioned.

Here, the stirring time is, for example, about 0.1 to 2 hours at about room temperature, and then about 2 to 12 hours at 40 to 80 ° C. Under stirring the obtained particle dispersion, tetraalkoxysilane such as ethyl silicate is gradually added,
Stir by the same means as above. Here, the stirring time is, for example, about 0.1 to 4 hours at about room temperature, and then about 2 to 12 hours at 40 to 80 ° C.

In the specific magnetic particle dispersion of the present invention, a partially hydrolyzed condensate of a polydimethylsiloxane compound, silica, and tetraalkoxysilane in an aqueous phase, and alcohol such as methanol and ethanol as a by-product of the hydrolysis reaction,
It can be used as it is. It can be removed by repeated magnetic precipitation and redispersion in water.

The specific magnetic particles 1 and the specific magnetic particles 2 of the present invention are suitably used as non-specific nucleic acid carriers (hereinafter simply referred to as “carriers”) for isolating nucleic acids. The nucleic acid isolation method comprises mixing the carrier of the present invention, a nucleic acid-containing material, and a nucleic acid extraction solution, separating the nucleic acid-bound carrier from the liquid using a magnetic field, and separating the nucleic acid-bound carrier from the nucleic acid-bound carrier. Eluting. The step of separating the nucleic acid-bound carrier from the liquid using a magnetic field may be performed using a magnet. This magnet has, for example, a magnetic flux density of about 30.
A 0 Gaussian magnet may be used. Specifically, the magnet is brought close to the side wall of the tube containing the carrier, the sample containing the nucleic acid, and the solution for nucleic acid extraction, and the carrier is collected on the side wall of the tube.
There is a method of separating from a solution such as a nucleic acid extraction solution. In the step of eluting the nucleic acid from the carrier that binds the nucleic acid, the carrier to which the nucleic acid is bound is washed several times with, for example, about 70% ethanol, and then a solution having a low ionic concentration such as sterile water or TE buffer is added. Thereby, the nucleic acid bound to the carrier is eluted from the solution having a low ionic concentration.

Materials from which nucleic acids can be removed using the carrier of the present invention are biological materials containing proteins, membranes, DNA or RNA, low molecular weight nucleic acids and the like. Examples of such biological materials include proteins, membranes, bacteriophages containing DNA or RNA, low molecular weight nucleic acids, viruses, bacteria, and combinations thereof. In addition, for the purpose of purification, the nucleic acid may be a nucleic acid in a plasmid or a nucleic acid amplification reaction product.

The nucleic acid isolated using the carrier of the present invention is
Further, if necessary, it can be amplified by a nucleic acid amplification method and detected with a detection probe. For nucleic acid amplification, PC
The R method, the NASBA method, and the like can be applied. The carrier of the present invention can be used as a reagent kit in combination with a nucleic acid extraction solution.

[0022]

The present invention will be described below in more detail with reference to examples. In addition, each measurement and evaluation were performed as follows. The particle size of the magnetic particles (after staining with lauryl acetate), take an electron micrograph of the magnetic particles with a transmission electron microscope, measure the particle size of 500 or more randomly selected magnetic particles, and determine the number average particle size. The diameter was determined. Dispersibility The magnetic particles were diluted to a solids concentration of 0.5% by weight, the absorbance (A0) at 550 nm immediately after ultrasonically dispersing the diluted solution, and the sample bottle was left to shake for 1 hour, and immediately after gentle shaking. Was measured at 550 nm, and the value calculated by (A0 / A) × 100 was determined as the dispersibility index. Stationary stability The magnetic particles are diluted to a solid concentration of 1% by weight, and the diluted liquid is accommodated in a sample bottle having a capacity of 20 ml so that the liquid phase has a height of 2 cm, and the magnetic particles are uniformly dispersed. After shaking the sample bottle to stand, let it stand for 10 minutes, take out only the upper layer non-portion from the liquid level to 1 cm below the liquid level, measure the solid content concentration (c), and calculate by (c / c0) × 100 The value obtained was determined as a static stability index (%).

Example 1 35 parts by weight of ion-exchanged water was added to an aqueous dispersion of ferric tetroxide having an average particle size of 0.01 μm (ferricolloid W40, manufactured by Taiho Kogyo KK, solid content: 40% by weight), and a homogenizer was added. And stirred at 8000 rpm for 1 minute at room temperature.
While further stirring at 8000 rpm, 2 parts by weight of a 10% aqueous solution of the silane coupling agent represented by the general formula 2 was added, and the mixture was further stirred at 8000 rpm. The obtained dispersion was transferred to a separable flask equipped with a reflux condenser and a stirrer, and heated at 60 ° C. for 4 hours while stirring at 300 rpm to obtain an average particle size of 1.2 μm and a carboxyl group content of 68 mmol /
g of magnetic particles were obtained. The obtained magnetic particles had a dispersibility index of 95% and a stationary stability index of 97%. Example 2 In Example 1, after completion of stirring at 60 ° C. for 4 hours, the mixture was cooled to room temperature, and partially hydrolyzed condensate of tetraethoxysilane (ethyl silicate 40,
5 parts by weight (Tama Chemical Co., Ltd.) and 300 rpm at room temperature
Then, the mixture was stirred at 60 ° C. for 1 hour and subsequently at 60 ° C. for 4 hours to obtain magnetic particles having an average particle diameter of 1.3 μm and a carboxyl group content of 47 mmol / g. The dispersibility index (%) of the obtained magnetic particles is 9
The static stability index (%) was 3%, and the static stability index (%) was 96%. Example 3 In Example 1, after completion of stirring at 60 ° C. for 4 hours, the mixture was cooled to room temperature and partially hydrolyzed condensate of tetraethoxysilane (ethyl silicate 40,
2.5 parts by weight of Tama Chemical Co., Ltd.
Stirring at rpm for 1 hour, followed by stirring at 60 ° C. for 4 hours,
Magnetic particles having an average particle size of 1.4 μm and a carboxyl group content of 35 mmol / g were obtained. Dispersibility index (%) of the obtained magnetic particles
Was 92%, and the static stability index (%) was 92%.

Example 4 Magnetic particles (solid content: 20% by weight) obtained in Example 1
Take 0 microliter and add Salmon sperm DNA
0.1 μg / ml, 1 ml of a 1 wt% BSA solution, and 1 ml of a 5 M guanidinium thiocyanate solution were mixed, shaken at room temperature for 30 minutes, and then magnetic particles were collected by magnetic separation. The collected magnetic particles
M Guanidium thiocyanate / 0.1% by weight Trito
After washing with an nX-100 solution and subsequently with a 70% by weight ethanol solution, it was dispersed in 1 ml of distilled water. After shaking the obtained dispersion at room temperature for 10 minutes, the magnetic particles were removed by magnetic separation, and the supernatant was measured at an absorbance of 260 nm / 280 nm. Table 1 shows the results. Examples 5 to 7 Nucleic acids were extracted in the same manner as in Example 4 except that the amount of Salmon sperm DNA used was as shown in Table 1. Table 1 shows the results.

[0025]

[Table 1]

Examples 8 to 11 Nucleic acids were extracted in the same manner as in Example 4 except that human whole blood was used in the amount shown in Table 2 instead of Salmon sperm DNA. Table 2 shows the results.

[0027]

[Table 2]

[0028]

The magnetic particles of the present invention are excellent in dispersibility and stationary stability. The non-specific nucleic acid carrier of the present invention can non-specifically adsorb a large amount of nucleic acid, is excellent in recovery efficiency and operability, and can efficiently extract DNA and / or RNA. Can also be used as a pretreatment for The non-characteristic nucleic acid carrier of the present invention also has an advantage that it is easy to automate.

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Claims (2)

[Claims] 1. A magnetic particle comprising: (A) a magnetic substance; (B) a polydimethylsiloxane compound having a hydrophilic group; and (C) silica. 2. The method according to claim 1, wherein a polydimethylsiloxane compound having a hydrophilic group is added to the aqueous dispersion of the magnetic substance, and tetraalkoxysilane and / or a partial hydrolysis condensate thereof are added. Method for producing magnetic particles. 3. A non-specific nucleic acid binding carrier comprising the magnetic particles according to claim 1.