CN112779245B - High-load magnetic bead for nucleic acid extraction and preparation method and application thereof - Google Patents
High-load magnetic bead for nucleic acid extraction and preparation method and application thereof Download PDFInfo
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- CN112779245B CN112779245B CN201911089465.8A CN201911089465A CN112779245B CN 112779245 B CN112779245 B CN 112779245B CN 201911089465 A CN201911089465 A CN 201911089465A CN 112779245 B CN112779245 B CN 112779245B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
- H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
- H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
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- H—ELECTRICITY
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- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F41/00—Apparatus or processes specially adapted for manufacturing or assembling magnets, inductances or transformers; Apparatus or processes specially adapted for manufacturing materials characterised by their magnetic properties
Abstract
The invention discloses a high-load magnetic bead for nucleic acid extraction and a preparation method and application thereof. The magnetic bead comprises a magnetic core and a coating layer coated on the surface layer of the magnetic core, wherein the ratio R/D of the particle size R of the magnetic core to the thickness D of the coating layer is 0.5-8, and the coating layer is provided with hydroxyl, carboxyl, amino, sulfydryl, phenolic hydroxyl or tosyl groups. During preparation, the magnetic cores with the coating layers are activated by the treatment liquid to prepare magnetic beads; the treatment liquid is an aqueous solution containing at least one of inorganic salt, inorganic base, water-soluble organic base and surfactant; the activation treatment includes ultrasonic treatment with stirring of a treatment liquid containing magnetic cores with coating layers. The activation treatment can form microstructures such as folds and the like on the coating material on the surface of the magnetic beads, and the specific surface area of the magnetic beads is increased under the condition of keeping the magnetic strength of the magnetic beads unchanged, so that the loading capacity of the magnetic beads for extracting nucleic acid is increased.
Description
Technical Field
The invention belongs to the field of magnetic materials, and relates to a high-load magnetic bead for nucleic acid extraction, and a preparation method and application thereof.
Background
The gene detection is an important one of in vitro diagnosis and detection, and is mainly applied to a plurality of fields of pregnancy detection, genetic disease detection, tumor detection, agriculture and the like. The gene detection mode is that the qualitative and quantitative detection is carried out after nucleic acid is extracted and enriched from a complex sample, so as to obtain a corresponding data conclusion; on the other hand, the emergence of the second generation sequencing technology has further expanded the application of the gene detection technology. However, the complexity of the sample and the microscale of the target nucleic acid increase the difficulty of gene extraction, and a method for rapidly and efficiently enriching and recovering nucleic acid samples is the basis of a gene detection technology.
The traditional methods for extracting, enriching and recovering nucleic acid comprise the following two methods: 1. a solution precipitation method, in which other substances such as proteins are precipitated while cell membranes are destroyed by using anionic (cationic) surfactants and salts, and after separation and purification, a purified nucleic acid sample is obtained by adjusting the salt concentration, the most common methods in the method are a CTAB method for a plant sample and an SDS method for a microorganism sample, but the solution precipitation method is difficult to be used in series with a high-throughput screening technique, and the method uses an organic solvent for extraction and a centrifugation method which is time-consuming for separation for a plurality of times; 2. the membrane/column separation method mainly utilizes a surface modified membrane or a packed column material to enrich nucleic acid, and separates and purifies a nucleic acid sample through an elution process.
The magnetic bead is a nano particle with superparamagnetism, and the main structure of the magnetic bead is a central magnetic core with superparamagnetism and an outer coating material with functional modification. The magnetic beads have good biocompatibility and are widely applied to various in-vitro diagnostic tests, and the performance of the magnetic beads is mainly determined by the particle size and shape of the magnetic beads, the type and thickness of an outer coating material, the type of an outermost layer modification material and other factors. Among the existing methods for preparing magnetic beads, there are two main methods for controlling the particle size of magnetic beads: one method is mainly characterized in that a central magnetic core with a certain size is prepared firstly, and then the particle size and the uniformity of the magnetic bead are controlled by controlling the adding speed and the adding amount of a coating material, so that the magnetic bead prepared by the method has a smooth surface, no wrinkles and a small specific surface area; another method for controlling the particle size of the magnetic bead is to prepare an inner core in advance by using high polymer materials such as Polystyrene (PS) and the like, form a fine pore channel structure in a swelling mode, then use the precipitation of a ferrite compound in the pore channel to realize the normalization of the particle size of the magnetic bead, and finally modify the outer layer of the magnetic bead.
In recent years, magnetic beads are widely used in the fields of scientific research, medical diagnosis, second-generation sequencing and the like, the process mainly comprises the steps of enriching and extracting nucleic acid in a sample by using the magnetic beads with special modifications on the surfaces, carrying out magnetic separation in a magnetic absorption mode, and eluting to obtain purified nucleic acid. However, the current method for extracting nucleic acid using magnetic beads has a limited load, and cannot extract a large amount of nucleic acid. The conventional methods for increasing the loading of magnetic beads mainly include two methods, one is to increase the specific surface area of the magnetic beads by reducing the particle size of the magnetic beads, but the magnetic performance of the magnetic beads is reduced along with the reduction of the particle size, so that the magnetic recovery efficiency is affected. The other method for increasing the loading capacity of the magnetic beads is to modify special complex groups on the surfaces of the magnetic beads so as to increase the loading capacity of the magnetic beads, but the production cost of the method is usually high, so that the method is not beneficial to large-scale use.
Disclosure of Invention
The invention provides a magnetic bead, which comprises a magnetic core and a coating layer coated on the surface layer of the magnetic core, wherein the ratio R/D of the particle size R of the magnetic core to the thickness D of the coating layer is 0.5-8, and the coating layer is provided with hydroxyl, carboxyl, amino, sulfhydryl, phenolic hydroxyl or tosyl and other groups;
wherein R represents the diameter of the magnetic core.
Preferably, the value of R/D is 0.75 to 5; more preferably 1-2.
According to the technical scheme of the invention, the magnetic core is a superparamagnetic material, such as gamma-ferric oxide, ferroferric oxide, ferrous oxide or an alloy of other metals and iron. Wherein the magnetic core has a particle size of 10nm to 10 μm, for example 50nm to 1 μm, further for example 80 to 300nm; illustratively, the particle size of the magnetic core is 100nm, 150nm, 200nm. The shape of the magnetic core may be spherical, cubic, rectangular parallelepiped, cylindrical, rod-like, amorphous, etc., and is preferably spherical.
According to a solution of the invention, the thickness of the coating is 10-500nm, for example 15-200nm, such as 20-100nm; illustratively, the thickness of the cladding layer is 20nm, 25nm, 30nm, 40nm, 50nm. Wherein, the raw material for forming the coating layer can be at least one of silicon dioxide, high molecular polymer and biological macromolecule material; for example, the high molecular polymer may be at least one of polyethylene, polypropylene, polystyrene, and the like; the biomacromolecule material can be at least one of dopamine, sucrose, dextran and the like.
According to the technical scheme of the invention, the saturation magnetization of the magnetic beads is 25-80emu/g, preferably 30-60emu/g.
According to the technical scheme of the invention, the quantity of the bindable nucleic acid per milligram of magnetic beads is 10-100 mug, and preferably 30-80 mug.
The second aspect of the present invention provides a method for preparing the magnetic bead, wherein the magnetic bead is prepared by activating the magnetic core with the coating layer with a treatment liquid;
the treatment liquid is an aqueous solution containing at least one of inorganic salt, inorganic base, water-soluble organic base and surfactant;
the activation treatment comprises ultrasonic treatment under stirring of a treatment liquid containing magnetic cores with coating layers. The ultrasonic treatment can make the microstructure on the surface of the magnetic bead uniform, thereby not only increasing the specific surface area, but also obtaining the magnetic bead with the optimal R/D microstructure.
The magnetic core with the coating layer is activated by the treatment liquid, so that a folded microstructure appears on the surface of the coating layer, and the specific surface area of the magnetic bead is greatly increased on the premise of not influencing the magnetic performance of the magnetic bead.
According to the technical scheme of the present invention, the inorganic salt may be selected from at least one of inorganic salts known in the art, such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium fluoride, potassium hydrogen fluoride, cesium fluoride, sodium chloride, potassium chloride, sodium bromide, and the like; preferably, the inorganic salt is selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium fluoride, sodium chloride or sodium bromide. Further, the concentration of the inorganic salt in the treatment liquid is 0 to 10wt%, for example, 0.01 to 8wt%, 0.05 to 5wt%, 0.1 to 2.5wt%; illustratively, the concentration is 0.073wt%.
According to the technical scheme of the invention, the inorganic base can be selected from inorganic bases known in the art, such as at least one of sodium hydroxide, potassium hydroxide, ammonia water, calcium hydroxide and the like; further, the source of the inorganic base may be a metal simple substance (such as metallic sodium), a metal oxide (sodium oxide, sodium peroxide, or potassium oxide), an alkali solution obtained by mixing a metal hydroxide with water. Further, the concentration of the inorganic base in the treatment liquid is 0 to 10wt%, for example, 1 to 8wt%, 2 to 5wt%.
According to the technical scheme of the invention, the water-soluble organic base can be selected from at least one of DBU (1, 8-diazabicyclo [5.4.0] undec-7-ene), DBN (1, 5-diazabicyclo [4.3.0] non-5-ene), methylamine, ethylamine and the like. Further, the concentration of the water-soluble organic base in the treatment liquid is 0 to 10% by weight, for example, 1 to 8% by weight, 2 to 5% by weight.
According to an aspect of the present invention, the surfactant may be at least one selected from tetrabutylammonium fluoride, tetrabutylammonium bromide, tetrabutylammonium chloride, gleditsia sinensis lam powder, SDS (sodium dodecyl sulfate), morwet EFW (sodium butylnaphthalene sulfonate), and the like. Further, the concentration of the surfactant in the treatment liquid is 0 to 0.5wt%, for example, 0.01 to 0.4wt%, 0.05 to 0.35wt%, 0.1 to 0.3wt%; illustratively, the concentration is 0.33wt%.
Wherein the dosage of the inorganic salt, the non-polar alkali, the water-soluble organic alkali and the surfactant is not 0 at the same time.
According to the technical scheme of the invention, the mass ratio of the solute in the treatment liquid to the magnetic core with the coating layer is not particularly limited, and the proportion of the solute to the magnetic core with the coating layer can be selected by a person skilled in the art according to different solutes. For example, when the solute in the treatment fluid is an inorganic salt, the mass ratio of the inorganic salt to the magnetic core with the coating layer can be 1 (0.5-10), such as 1 (1-5), illustratively 1.36; when the solute in the treatment fluid is a surfactant, the mass ratio of the surfactant to the magnetic core with the coating layer can be (1-20): 1, for example (1-10): 1, and exemplary is 3.33).
According to the technical scheme of the invention, the magnetic core, the coating layer and the ratio R/D of the particle size R of the magnetic core to the thickness D of the coating layer have the meanings as described above.
According to the technical scheme of the invention, the ultrasonic time is 0.5-8h, and the ultrasonic frequency is 20-100kHz; preferably, the ultrasonic time is 1-4h, and the ultrasonic frequency is 60-100kHz; illustratively, the ultrasound time is 2.5 or 3 hours and the ultrasound frequency is 40kHz.
According to the technical scheme of the invention, the preparation method further comprises the process of modifying the magnetic core with the coating layer before the activation treatment. For example, the magnetic core with the coating layer is re-modified with amino, hydroxyl, carboxyl, amino, mercapto, phenolic hydroxyl, or p-toluenesulfonyl.
The specific process is as shown in fig. 1, and comprises coating the magnetic core with a coating material to form a magnetic bead with a certain R/D value, and activating the magnetic bead with a treatment solution to prepare a high-capacity magnetic bead for nucleic acid extraction.
Preferably, the preparation method of the magnetic bead comprises the following steps:
(1) Coating the magnetic core by using a coating material to obtain the magnetic core with a coating layer;
(2) Modifying the magnetism of the coating layer, and then carrying out activation treatment on the magnetic core with the coating layer by using treatment liquid; or, directly using a treatment solution to perform activation treatment on the magnetic core with the coating layer to obtain the magnetic beads;
the treatment liquid is an aqueous solution containing at least one of inorganic salt, inorganic base, water-soluble organic base and surfactant;
the activation treatment includes ultrasonic treatment with stirring of a treatment liquid containing magnetic cores with coating layers.
According to the technical scheme of the invention, the magnetic core can be prepared by a coprecipitation method, a hydrothermal method, a high-temperature thermal decomposition method, a microemulsion method, a polymer deposition method, a biosynthesis method and other magnetic core synthesis methods known in the art.
The third aspect of the present invention provides a magnetic bead obtained by the above-described production method.
The fourth aspect of the present invention provides use of the magnetic bead described above for extracting nucleic acid.
The invention has the beneficial effects that:
the invention relates to a preparation method of a high-loading magnetic bead for nucleic acid extraction, which specifically comprises the steps of utilizing the magnetic bead with the optimal ratio of the magnetic core particle size (R) to the coating layer thickness (D) obtained by screening, carrying out ultrasonic activation treatment on the magnetic bead by adopting a treatment solution, forming microstructures such as folds on a coating material on the surface of the magnetic bead, and increasing the specific surface area of the magnetic bead under the condition of keeping the magnetic strength of the magnetic bead unchanged, thereby increasing the loading of the magnetic bead on nucleic acid extraction.
Drawings
FIG. 1 is a flow chart of the preparation of the magnetic beads for nucleic acid extraction with high loading according to the present invention.
FIG. 2 is an IR characterization chart of silicon hydroxyl magnetic beads before and after treatment in example 1 of the present invention.
FIG. 3 is a TEM and SEM representation of silicon hydroxyl magnetic beads before and after treatment in example 1 of the present invention.
FIG. 4 is a diagram showing the hysteresis loop of silicon hydroxyl magnetic beads before and after the treatment of example 1.
FIG. 5 shows measurement of unit load of 162bp nucleic acid extraction by using silicon hydroxyl magnetic beads before and after treatment in example 1 of the present invention.
FIG. 6 shows the measurement of unit load of 1017bp nucleic acid extraction by using silicon hydroxyl magnetic beads before and after the treatment in example 1 of the present invention.
FIG. 7 is a TEM and SEM representation of amino magnetic beads before and after treatment in example 2 of the present invention.
FIG. 8 shows the recovery rate of nucleic acids with different fragment sizes from amino magnetic beads before and after the treatment in example 2 of the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1 preparation of silicon-hydroxyl magnetic beads for high-load nucleic acid extraction
1. Process for preparing magnetic core by high-temperature thermal decomposition method
3.0g of ferric chloride was added to the beaker(FeCl 3 ) 7.2g anhydrous sodium acetate (NaOAc), 900mg trisodium citrate, 30mL Ethylene Glycol (EG) and 90mL diethylene glycol (DEG) were added and stirred at 150r/min for 8h to completely disperse all solids to form a reddish brown liquid precursor. Then pouring the lead liquid into a 250mL glass lining, transferring the lining into a high-pressure reaction kettle, heating to 200 ℃, reacting for 12 hours, cooling to room temperature, magnetically absorbing to remove supernatant, and washing magnetic cores with ethanol and deionized water for three times, wherein the dosage of the washing liquid is 500mL each time. Finally, the resulting magnetic nuclei were dispersed in 200mL of water to form a magnetic nucleus suspension.
2. Process for coating magnetic core by using tetraethyl silicate (TEOS)
And (3) adding 1.2g of the magnetic core dry weight prepared in the step (1) into a 1L three-neck flask, adding 240mL of water, 600mL of ethanol, 15mL of ammonia water, stirring at 150r/min, and performing ultrasonic-assisted dispersion at 40kHz for 0.5h. Then 8mL TEOS was slowly added dropwise to the flask via a dropping funnel, and after completion of the addition, the reaction was continued for 10 hours with ultrasonic stirring. And (4) magnetically absorbing to remove supernatant, and washing the magnetic beads with ethanol and deionized water for three times, wherein the using amount of the washing solution is 500mL each time. Finally, the beads were dispersed in 200mL of water to form a suspension.
3. Process for treating silicon hydroxyl magnetic beads by using treatment liquid
Weighing 220mg sodium carbonate (Na) 2 CO 3 ) Dissolving the mixture in 300mL of water, adding 300mg of the magnetic beads prepared in the step 2 into the solution, and carrying out ultrasonic treatment for 3 hours at 40kHz in a three-necked flask under the stirring of 150 r/min. And (4) magnetically absorbing to remove supernatant, and washing the magnetic beads with deionized water for three times, wherein the dosage of the washing solution is 500mL each time. Finally preparing 40mg/mL magnetic bead dispersion liquid for extracting high-load nucleic acid.
4. Characterization of silica-based magnetic beads for high-loading nucleic acid extraction
The magnetic beads before and after activation treatment were characterized by IR (infrared spectroscopy), TEM, SEM and hysteresis loop, with the IR characterization results shown in fig. 2, the TEM and SEM characterization results shown in fig. 3, and the hysteresis loop characterization results shown in fig. 4.
As can be seen from FIG. 2, before and after the treatment in step 3, the relative ratios of signals of Fe-O bonds and Si-O bonds change, and the signal ratio of Si-O bonds decreases, indicating that the Si coating layer changes and the treated liquid degrades during the treatment of the magnetic beads.
As can be seen from FIG. 3, the diameter R of the magnetic core prepared in step 1 is about 200nm, and the thickness D of the clad is about 50nm; the surface of the magnetic beads became rough and wrinkled from a smooth surface by the treatment with the treatment solution, indicating that the specific surface area of the magnetic beads increased.
As can be seen from fig. 4, the magnetic field strength of the magnetic beads did not change significantly before and after the treatment.
5. Load test of silicon hydroxyl magnetic beads for high-load nucleic acid extraction
Extracting the nucleic acids with the lengths of 162bp and 1017bp respectively by using untreated magnetic beads (namely, the magnetic beads with the coating layers prepared in the step 2) and the treated magnetic beads, wherein the specific test process comprises the following steps: 50 mu L of diluted PCR (polymerase chain reaction) product system is taken, 100 mu g of magnetic beads are added, then 100 mu L of PEG (polyethylene glycol) binding solution (30 wt% PEG-8000 solution) is added, and after standing for 15min, the supernatant is removed by magnetic attraction. The magnetic beads were washed by adding 200. Mu.L of 80% ethanol solution, after removing ethanol by magnetic attraction, the nucleic acids on the magnetic beads were eluted by adding Tris-HCl eluent (pH =8.5, concentration 10 mM), and the eluted nucleic acids were quantitatively detected by using Qubit. The results showed that the loading of the magnetic beads before and after the treatment showed great differences, the unit loading of the magnetic beads after the treatment to the two nucleic acids was 26. Mu.g/mg and 84. Mu.g/mg, respectively, while the unit loading of the untreated magnetic beads was only 4. Mu.g/mg and 10. Mu.g/mg. The specific results are shown in fig. 5 and 6. (Note: the nucleic acid sample is a PCR product of plasmid DNA corresponding to the size of the fragment, the amount of nucleic acid is quantified by the Qibit assay, and the amount of magnetic beads is 100. Mu.g).
Example 2 preparation of silica-coated amino magnetic beads for nucleic acid extraction with high Loading
1. Preparation process of amino magnetic beads
Silicon hydroxyl magnetic beads were prepared by steps 1 and 2 of example 1. Then 300mg of silicon hydroxyl magnetic beads are added into a three-necked flask, 240mL of water, 600mL of ethanol and 15mL of ammonia water are added, and ultrasonic assisted dispersion is carried out at 40kHz for 0.5h under the stirring of 150 r/min. Then, 6mL of KH-540 ((3-aminopropyl) trimethoxysilane) as a silane coupling agent was slowly added dropwise from a dropping funnel, and after completion of the addition, the ultrasonic stirring reaction was continued for 10 hours. And (4) removing the supernatant by magnetic attraction, and washing the magnetic beads by using ethanol and deionized water for three times respectively, wherein the dosage of the washing solution is 500mL each time. Finally, 200mL 80% ethanol solution is used for dispersing the magnetic beads to form suspension.
2. Process for treating amino magnetic beads by using treatment liquid
1.0g of tetrabutylammonium fluoride (TABF) was dissolved in 300mL of water, 300mg of the amino magnetic beads prepared in step 1 were added to the solution, and the mixture was sonicated at 40kHz for 2.5 hours in a three-necked flask with stirring at 150 r/min. And (4) magnetically absorbing to remove supernatant, and washing the magnetic beads three times by using 80% ethanol, wherein the using amount of the washing solution is 500mL each time. Finally preparing 40mg/mL magnetic bead dispersion liquid for extracting high-load nucleic acid.
3. Correlation characterization of amino magnetic beads for high-load nucleic acid extraction
The magnetic beads before and after the activation treatment were characterized by TEM and SEM, and the results are shown in fig. 7. As a result, the surface of the magnetic beads was roughened and wrinkled from a smooth surface by the treatment with the treatment solution, and the specific surface area was significantly increased.
4. Recovery test of high-load amino magnetic beads for nucleic acid extraction
Nucleic acids with different fragment sizes of 162, 341, 535, 806, 1017 and 1612bp were extracted by using untreated amino magnetic beads and the treated amino magnetic beads, and the extraction process was as in step 5 of example 1. The results showed that the recovery rates of magnetic beads before and after the treatment were greatly different when nucleic acids of the same fragment size were adsorbed, and that the recovery rates of magnetic beads after the treatment were all more than 90%, whereas the recovery rate of untreated magnetic beads was only about 75% (specifically, the results are shown in FIG. 8). (Note: the nucleic acid sample is a PCR product of plasmid DNA corresponding to the size of the fragment, the amount of nucleic acid added is a 10-fold diluted concentration PCR product of 50L, the amount of nucleic acid is determined by Qibit measurement, and the amount of magnetic beads is 100. Mu.g).
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The preparation method of the magnetic bead is characterized in that the magnetic bead is prepared by activating the magnetic core with the silicon dioxide coating layer by using a treatment solution;
the treatment liquid is an aqueous solution of tetrabutylammonium fluoride;
the activation treatment comprises ultrasonic treatment under stirring of a treatment liquid containing magnetic cores with silicon dioxide coating layers; the ultrasonic time is 0.5-8h, and the ultrasonic frequency is 20-100kHz;
the magnetic bead comprises a magnetic core and a silicon dioxide coating layer coated on the surface layer of the magnetic core, wherein the magnetic core is made of a superparamagnetic material, the ratio R/D of the particle size R of the magnetic core to the thickness D of the silicon dioxide coating layer is 1-5, the thickness of the silicon dioxide coating layer is 10-100nm, the silicon dioxide coating layer is provided with amino groups, and the surface of the silicon dioxide coating layer is provided with a folded microstructure;
wherein R represents the diameter of the magnetic core.
2. The method according to claim 1, wherein the magnetic core has a spherical, cubic, rectangular parallelepiped, cylindrical, rod-like or amorphous shape.
3. The method according to claim 1, wherein the magnetic core is gamma-ferric oxide, ferroferric oxide, or ferrous oxide.
4. The method according to claim 1, wherein the silica coating layer has a thickness of 20nm, 25nm, 30nm, 40nm, or 50nm.
5. The method according to claim 1, wherein the magnetic beads have a saturation magnetization of 25 to 80emu/g;
and/or the amount of bound nucleic acid per mg of magnetic beads is 10-100. Mu.g.
6. A method for preparing a magnetic bead as defined in claim 1 wherein the concentration of tetrabutylammonium fluoride in the treatment solution is between 0.1 and 0.5wt%.
7. A method for preparing a magnetic bead as defined in any one of claims 1 to 6, further comprising a step of modifying the magnetic core having a silica coating layer before the activation treatment.
8. A magnetic bead obtained by the method according to any one of claims 1 to 7.
9. Use of the magnetic beads of claim 8 for extracting nucleic acids.
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| TWI771253B (en) * | 2021-12-27 | 2022-07-11 | 財團法人工業技術研究院 | Simple nucleic acid extraction kit for outdoor use and method for extracting nucleic acid of sample outdoors |
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| CN115612686B (en) * | 2022-12-20 | 2023-03-31 | 北京迈佳致和科技有限公司 | Effervescent tablet of nucleic acid extraction magnetic beads, synthesis method and application thereof |
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| Magnetic particles for the separation and purification of nucleic acids;Sonja Berensmeier等;《Appl Microbiol Biotechnol》;20061231;第73卷;第495-504页 * |
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