CN112563016A - Preparation method of magnetic microspheres for nucleic acid extraction, prepared product and application - Google Patents
Preparation method of magnetic microspheres for nucleic acid extraction, prepared product and application Download PDFInfo
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- CN112563016A CN112563016A CN202011537465.2A CN202011537465A CN112563016A CN 112563016 A CN112563016 A CN 112563016A CN 202011537465 A CN202011537465 A CN 202011537465A CN 112563016 A CN112563016 A CN 112563016A
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- 238000000605 extraction Methods 0.000 title claims abstract description 29
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 25
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 25
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title description 6
- 239000006249 magnetic particle Substances 0.000 claims abstract description 65
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000000034 method Methods 0.000 claims abstract description 45
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 44
- 239000010703 silicon Substances 0.000 claims abstract description 44
- 238000007306 functionalization reaction Methods 0.000 claims abstract description 26
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- 241001678559 COVID-19 virus Species 0.000 claims description 8
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- 238000002123 RNA extraction Methods 0.000 claims description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 239000003002 pH adjusting agent Substances 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 238000000658 coextraction Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 11
- 238000010438 heat treatment Methods 0.000 description 34
- 238000003756 stirring Methods 0.000 description 27
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- 235000011114 ammonium hydroxide Nutrition 0.000 description 19
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- 239000000243 solution Substances 0.000 description 16
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 12
- 229920002477 rna polymer Polymers 0.000 description 11
- 239000001509 sodium citrate Substances 0.000 description 11
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
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- 230000015572 biosynthetic process Effects 0.000 description 9
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- 238000001514 detection method Methods 0.000 description 9
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 9
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 9
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
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- 239000000126 substance Substances 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000002270 dispersing agent Substances 0.000 description 3
- 239000011859 microparticle Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
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- -1 RNA Chemical class 0.000 description 2
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- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
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- 206010036790 Productive cough Diseases 0.000 description 1
- 229910008051 Si-OH Inorganic materials 0.000 description 1
- 229910006358 Si—OH Inorganic materials 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- QCWXUUIWCKQGHC-UHFFFAOYSA-N Zirconium Chemical compound [Zr] QCWXUUIWCKQGHC-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
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- 238000000975 co-precipitation Methods 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002681 effect on RNA Effects 0.000 description 1
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- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 239000004530 micro-emulsion Substances 0.000 description 1
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- 229910052759 nickel Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
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- 238000000197 pyrolysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 238000012360 testing method Methods 0.000 description 1
- UQMOLLPKNHFRAC-UHFFFAOYSA-N tetrabutyl silicate Chemical compound CCCCO[Si](OCCCC)(OCCCC)OCCCC UQMOLLPKNHFRAC-UHFFFAOYSA-N 0.000 description 1
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 description 1
- ZQZCOBSUOFHDEE-UHFFFAOYSA-N tetrapropyl silicate Chemical compound CCCO[Si](OCCC)(OCCC)OCCC ZQZCOBSUOFHDEE-UHFFFAOYSA-N 0.000 description 1
- 239000010936 titanium Substances 0.000 description 1
- 229910052719 titanium Inorganic materials 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- JCVQKRGIASEUKR-UHFFFAOYSA-N triethoxy(phenyl)silane Chemical compound CCO[Si](OCC)(OCC)C1=CC=CC=C1 JCVQKRGIASEUKR-UHFFFAOYSA-N 0.000 description 1
- ZNOCGWVLWPVKAO-UHFFFAOYSA-N trimethoxy(phenyl)silane Chemical compound CO[Si](OC)(OC)C1=CC=CC=C1 ZNOCGWVLWPVKAO-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 229910052726 zirconium Inorganic materials 0.000 description 1
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- 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
- H01F41/02—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 for manufacturing cores, coils, or magnets
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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- H01F1/0018—Diamagnetic or paramagnetic materials, i.e. materials with low susceptibility and no hysteresis
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- H01—ELECTRIC ELEMENTS
- 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
- H01F1/42—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of organic or organo-metallic materials, e.g. graphene
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Abstract
The invention relates to a method for preparing silicon hydroxyl magnetic microspheres, which comprises the following steps: synthesizing magnetic particles; performing hydrophilic layer modification on the magnetic particles; and performing functionalization treatment on the modified magnetic particles, wherein the functionalization treatment comprises: dispersing the modified magnetic particles in an anhydrous lower alcohol solution; adding a monomer for endowing the magnetic particles with silicon hydroxyl functionalization to react, wherein no water is added in the step of functionalization treatment and no aqueous reagent except a pH regulator is used. The invention improves the extraction effect of nucleic acid, especially RNA. The invention also relates to the prepared silicon hydroxyl magnetic microsphere and the application thereof in nucleic acid extraction.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to preparation of magnetic microspheres for nucleic acid extraction.
Background
Nucleic acids are a class of biological macromolecules formed by connecting nucleotides or deoxynucleotides through phosphodiester bonds, and include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). As a carrier of genetic information, nucleic acids are important as an object of research in the field of molecular biology. The quality of the nucleic acid sample, particularly the extraction effect thereof, has an important influence on the subsequent analysis process. The traditional nucleic acid extraction method comprises an alkaline lysis method, a boiling method, a column separation method and the like.
The magnetic microsphere method is a new extraction method developed in recent years, and the method is used for preparing superparamagnetic nano magnetic microspheres with some functional groups (such as hydroxyl and carboxyl) introduced on the surfaces. The magnetic microsphere can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface. Under the action of an external magnetic field, nucleic acid can be separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like. Compared with the traditional method, the method has the characteristics of no need of participation of highly toxic reagents, avoidance of multi-step high-rotation-speed centrifugation, and simplicity and feasibility. The silicon hydroxyl magnetic microspheres are common magnetic microspheres for nucleic acid extraction, and have good effect in DNA extraction; however, the RNA extraction has the disadvantages of extremely weak signal and even no extraction.
Therefore, in the field of nucleic acid extraction, there is a strong demand for improving the RNA extraction effect of silica hydroxyl microspheres.
Disclosure of Invention
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
As used herein, the terms "having," "including," or "including" are used in a non-exclusive manner. Thus, these terms may refer both to the case where no additional features are present in the entity described in this context, in addition to the features introduced by these terms, and to the case where one or more additional features are present. As an example, the expressions "a has B", "a comprises B" and "a comprises B" may refer both to the case where no further elements other than B are present in a (i.e. the case where it consists solely of B), and to the case where one or more further elements other than B are present in entity a, such as elements C, C and D or even other elements.
The term "about" in the context of a particular value or ratio of the invention refers to +/-10% of the value or ratio, or +/-5% of a given value or ratio in one embodiment.
As mentioned above, the currently prepared silicon hydroxyl magnetic microspheres have the problems of weak signals and even no extraction when used for RNA extraction, which greatly restricts the application of the silicon hydroxyl magnetic microspheres in nucleic acid extraction.
In order to solve the above problems, the present inventors have studied a process for preparing silicon hydroxyl magnetic microspheres. The present inventors found that the reason for the above problems is: in the functionalization treatment step in the preparation of the magnetic microspheres, the magnetic particles are often added into hydrous ethanol, or water or a hydrous reagent (such as a dispersing agent and the like) is added after the anhydrous ethanol is added, and the use of the water aims to promote the hydrolysis speed and accelerate the silicon hydroxylation; however, surprisingly, the introduction of water in this step or the resulting magnetic microspheres makes it difficult to achieve RNA extraction. Accordingly, the present inventors have adjusted the functionalization process steps, and have completed the present invention.
Accordingly, in a first aspect, the present invention provides a method for preparing a silica-hydroxyl magnetic microsphere, comprising:
synthesizing magnetic particles;
performing hydrophilic layer modification on the magnetic particles; and
functionalizing the modified magnetic particles, the functionalizing comprising:
-dispersing the modified magnetic particles in an anhydrous lower alcohol solution;
-adding a monomer that confers a silicon hydroxyl functionalization on said magnetic particles,
wherein no water is added and no aqueous reagent other than a pH adjusting agent is used in the step of the functionalization treatment.
By adopting the preparation method, the effect of the prepared silicon hydroxyl magnetic microspheres is obviously improved when the prepared silicon hydroxyl magnetic microspheres are used for extracting nucleic acid, particularly RNA, the sensitivity is high, and the repeatability and the detection rate are improved.
In a specific embodiment, the silicon hydroxyl magnetic microspheres are used for nucleic acid extraction, such as DNA extraction, RNA extraction, or DNA/RNA co-extraction; especially for RNA extraction.
As used herein, "magnetic microparticle" is used interchangeably with "magnetic matrix material" and refers to a magnetic particle having a superparamagnetic, intact and uniform crystalline form. It will be appreciated from the context of the present invention that the magnetic particles of the present invention have not been functionally and/or hydrophilically modified. The magnetic fine particles (magnetic seed cores) may be, for example, Fe3O4Form, or AFe3O4Forms wherein A is two or more of zinc, manganese, titanium, nickel, cobalt, zirconium, chromium.
The magnetic particles can be synthesized by methods well known in the art, such as chemical coprecipitation, hydrothermal, solvothermal, microemulsion, dc arc plasma, or pyrolysis.
As used herein, "magnetic microspheres" are used interchangeably with "magnetic beads".
As used herein, "hydrophilic layer modification" refers to modification of magnetic microparticles with a hydrophilic substance. The hydrophilic layer modification is intended to improve the dispersibility of the particles.
In the present invention, the hydrophilic substance may be a citric acid (e.g., citrate), a polyethylene glycol, or a polyvinylpyrrolidone type hydrophilic substance.
In an exemplary embodiment, in the case of using sodium citrate as the hydrophilic substance, 0.01M sodium citrate may be added and maintained at 90 ℃ for 1h, thereby obtaining surface-modified magnetic particles.
It should be understood that the functionalization step of the invention is carried out in a manner that avoids as much as possible the introduction of water; in other words, no water is added in the functionalization step and no aqueous reagent other than the pH adjuster is used. Any water may be, for example: ultrapure water, distilled water, water left in or intentionally added to the reaction vessel, and the like; any aqueous agent may be, for example, a dispersant, an alcohol/water mixture, or the like.
In a specific embodiment, "lower alcohol" refers to an alcohol having a carbon chain length of 3 or less.
In exemplary embodiments, the lower alcohol may be selected from one or more of methanol, ethanol, n-propanol, and isopropanol.
In the functionalization treatment step, after the modified magnetic particles are dispersed in the lower alcohol solution, a certain concentration of magnetic fluid is formed. The magnetic fluid concentration is defined as the ratio of the mass of the modified magnetic particles used to the volume of the lower alcohol solution. For example, when 300mg of the modified magnetic particles were dispersed in 150mL of a lower alcohol, the magnetofluid concentration was 2 mg/mL.
In the present invention, the concentration of the magnetic fluid may be 1-15 mg/mL. For example, about 1mg/mL, about 2mg/mL, about 3mg/mL, about 4mg/mL, about 5mg/mL, about 6mg/mL, about 7mg/mL, about 8mg/mL, about 9mg/mL, about 10mg/mL, about 11mg/mL, about 12mg/mL, about 13mg/mL, about 14mg/mL, or about 15 mg/mL.
In a preferred embodiment, the magnetic fluid concentration of the present invention is 2-14 mg/mL.
By selecting the concentration of the magnetic fluid of 2-14mg/mL in the step of functional treatment, the invention further improves the effect of the prepared magnetic microspheres in nucleic acid extraction.
The present invention is not particularly limited as to the length of the substep of treatment with a lower alcohol, as long as the treated magnetic particles can be sufficiently dispersed.
As used herein, "silicon hydroxyl functionalized monomer" is used interchangeably with "donor of silicon hydroxyl" and refers to a substance that imparts silicon hydroxyl functionalization to the modified magnetic microparticles.
Specifically, the "silicon hydroxyl-functionalized monomer" may be selected from one or more of the group consisting of methyl orthosilicate, ethyl orthosilicate, propyl orthosilicate, butyl orthosilicate, alkyltrimethoxysilane, alkyltriethoxysilane, alkyltriethylsilane, dialkyldimethoxysilane, dialkyldiethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, aminopropyltrimethoxysilane, and epoxypropyltrimethoxysilane.
Before or while the "silicon hydroxyl-functionalized monomer" is added for the reaction, the method of the present invention may further comprise a step of adjusting the reaction solution to a suitable reaction condition. Suitable pH conditions may for example be 8-13; the adjustment of the pH can be effected, for example, by adding a weakly basic pH regulator, such as 1-2mL of aqueous ammonia. Suitable reaction temperatures may be, for example, from 50 to 90 ℃.
In a particular embodiment, the substep of carrying out the reaction by adding a silicon hydroxyl functionalized monomer may last, for example, for 1 to 6 hours.
Illustratively, the aqueous agent may be, for example, an aqueous alcohol (alcohol/water mixed solution), a dispersant, or the like.
The method of the present invention may further comprise additional steps. For example, after the functionalization process step, the method of the invention may comprise a magnetic separation, washing, and/or drying step.
In a second aspect, the present invention provides a silicon hydroxyl magnetic microsphere, which is prepared by the method of the present invention.
In a third aspect, the present invention provides the use of silica-hydroxyl magnetic microspheres for extracting nucleic acids in a sample.
In particular embodiments, the sample may be selected from the group consisting of blood, pharyngeal swabs, sputum, alveolar lavage, tissue, food, and environmental samples, among others.
In particular embodiments, the nucleic acid is DNA, RNA, or both DNA and RNA.
In an exemplary embodiment, the RNA is an RNA derived from a virus, such as the RNA of a novel coronavirus (SARS-CoV-2).
Drawings
Fig. 1 shows a preparation process route of the silicon hydroxyl magnetic microsphere.
Fig. 2 shows an SEM image of the magnetic particles prepared in example 1;
fig. 3 shows the VSM test results of the magnetic particles prepared in example 1.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Example 1
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
The obtained magnetic seed core was observed under SEM, and the result is shown in fig. 2; the SEM result shows that the magnetic seed core is basically spherical and has a good crystal structure and the grain diameter is about 10 nm. Meanwhile, the obtained magnetic seed core was measured by a magnetic experiment using VSM, and the result is shown in fig. 3; VSM results show that the magnetic seed nuclei are superparamagnetic.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the second step in 150mL of absolute ethanol solution, adding 1mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, and stopping the reaction after 2h to obtain the silicon hydroxyl magnetic microspheres.
Example 2
Using the silica-hydroxyl magnetic microspheres obtained in example 1, sample 1(SARS-CoV-2 high concentration sample 5X 10) was extracted using the Ausheng Auto Pure32A extractor3copies/mL) and sample 2(SARS-CoV-2 Low concentration sample 400copies/mL), wherein sample 1 is at high concentration (5X 10)3copies/mL) of a pseudoviral sample containing the SARS-CoV-2 gene, and sample 2 is a low concentration (400copies/mL) of a pseudoviral sample containing the SARS-CoV-2 gene.
The RNA extracted from sample 1 and sample 2 was reverse-transcribed using a Michelle SLAN96P amplification apparatus, and the results are shown in Table 1.
Channels 1 to 4 represent the results of detection of four different genes of SARS-CoV-2, respectively, as follows;
detection rate: if there is no Ct value or the Ct value is greater than 38, it is considered that no Ct value is detected, as follows.
As can be seen from table 1, the experimental data for high concentration sample 1 indicates that: the magnetic microspheres prepared in example 1 have good extraction effect and good repeatability.
According to the result of the low concentration sample 2, the detection rate of the magnetic microspheres prepared in example 1 is 100%, and the repeatability is good.
Comparative example 1
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the second step in 150mL of ethanol/water solution, adding 1mL of ammonia water after 30min, heating to 77 ℃, adding 5mL of TEOS after the temperature is kept constant, wherein the dilution ratio of TEOS to ethanol is 1: 1-1: 10, and stopping the reaction after 2 h. Obtaining the silicon hydroxyl magnetic microspheres.
Comparative example 2
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the second step in 120mL of absolute ethyl alcohol, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after keeping the temperature constant, adding 30mL of ultrapure water after reacting for 2h, continuing to react for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Comparative example 3
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the step two in 30mL of water, stirring for 30min for dispersion, then adding 120mL of absolute ethyl alcohol, and continuing stirring for 30 min. Adding 2mL of ammonia water, heating to 80 ℃, keeping the temperature constant, adding 5mL of TEOS, wherein the dilution ratio of TEOS to ethanol is 1: 1-1: 10, reacting for 2h, adding 30mL of ultrapure water, continuing to react for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Example 3
Samples 1 and 2 were tested according to the extraction method and the detection method of example 2 using the silicon hydroxyl magnetic microspheres prepared in comparative examples 1 to 3, respectively, and the results are shown in table 2 below.
TABLE 2
In the methods of comparative examples 1 to 3, water was added in the functionalization treatment step in different orders, respectively. Table 2 the results show: the magnetic microspheres synthesized by the three methods have almost no effect on RNA extraction, and only the detectable part of the channel 2 can be detected, while the rest channels can not be detected. It is shown that the silicon hydroxyl magnetic microspheres synthesized by adopting an alcohol/water reaction system in the functionalization step cannot be used for extracting RNA.
Example 4
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic particles prepared above were dispersed in 200mL of 0.01M aqueous sodium citrate solution and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing the magnetic particles obtained in the step two in 150mL of absolute ethyl alcohol to ensure that the concentration of the magnetic fluid is 2mg/mL, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, reacting for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Example 5
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing the magnetic particles obtained in the step two in 150mL of absolute ethyl alcohol to ensure that the concentration of the magnetic fluid is 14mg/mL, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, reacting for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Comparative example 4
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing the magnetic particles obtained in the step two in 150mL of absolute ethyl alcohol to ensure that the concentration of the magnetic fluid is 1mg/mL respectively, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, reacting for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Comparative example 5
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing the magnetic particles obtained in the step two in 150mL of absolute ethyl alcohol to ensure that the concentration of the magnetic fluid is 15mg/mL, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, reacting for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Example 6
Samples 1 and 2 were tested according to the extraction method and the detection method of example 2 using the silicon hydroxyl magnetic microspheres prepared in examples 4 to 5 and comparative examples 4 to 5, respectively, and the results are shown in table 3 below.
TABLE 3
As can be seen from table 3, the extraction effect of the magnetic microspheres obtained in example 4 and example 5 in sample 1 is not significantly different; in sample 2 extraction, the Ct value of example 4 in the first channel advances example 5 by about 1.5, and the Ct value effect of example 5 in the third channel advances example 4 by about 0.6; and the detection rate of the magnetic microspheres prepared by the two examples is 100%. When the concentration of the magnetic fluid is 1mg/mL (namely comparative example 4), the extraction cycle number of the sample 1 lags behind that of the sample 4 by 1.5-3 Ct; the extraction cycle number of the sample 2 lags behind 0.5-3.5 Ct, and the detection rate is low. When the concentration of the magnetic fluid is 15mg/mL (namely the comparative example 5), the extraction cycle number of the sample 1 lags behind the extraction cycle number of the sample 4 by 0-4 Ct; the number of extraction cycles of sample 2 lags by 0.8-2.5 Ct and is not detectable. The optimal concentration of the magnetofluid is 2-14 g/mL.
Example 7
The method comprises the following steps: synthesis of magnetic seed cores
And (3) putting 4.0g of ferrous sulfate heptahydrate and 7.0g of ferric chloride hexahydrate in 150mL of water, setting the stirring speed to 300rpm, stirring for 30min to fully mix the solution, heating to 40 ℃, adding 50mL of ammonia water, reacting for 30min, heating again to 90 ℃, and curing for 1h to obtain the superparamagnetic nano magnetic particles with the particle size of about 5-15 nm.
Step two: hydrophilic layer modification of magnetic particles
The magnetic seed core prepared above was dispersed in 200mL of 0.01M aqueous solution of sodium citrate and kept at 90 ℃ for 1h to obtain surface-modified magnetic particles.
Step three: functionalization process
Silicon hydroxyl modification: and (3) dispersing 1g of the magnetic particles obtained in the second step in 150mL of anhydrous isopropanol, adding 2mL of ammonia water after 30min, heating to 80 ℃, adding 5mL of TEOS after the temperature is kept constant, reacting for 2h, stopping heating, and stirring overnight. Obtaining the silicon hydroxyl magnetic microspheres.
Example 8
Using the silica-based magnetic microspheres prepared in example 7, samples 1 and 2 were tested according to the extraction method and the detection method of example 2, and the results are shown in table 4 below.
TABLE 4
As is clear from Table 4, the magnetic microspheres with Si-OH groups obtained in example 7, which used isopropanol as the anhydrous lower alcohol, also exhibited a superior effect in extracting nucleic acid.
Claims (10)
1. A method of preparing a silicon hydroxyl magnetic microsphere comprising:
synthesizing magnetic particles;
performing hydrophilic layer modification on the magnetic particles; and
functionalizing the modified magnetic particles, the functionalizing comprising:
-dispersing the modified magnetic particles in an anhydrous lower alcohol solution;
-adding a monomer that confers a silicon hydroxyl functionalization on said magnetic particles,
wherein no water is added and no aqueous reagent other than a pH adjusting agent is used in the step of the functionalization treatment.
2. The method of claim 1, wherein the lower alcohol is an alcohol having a carbon chain length of 1 to 3.
3. The method of claim 2, wherein the lower alcohol is selected from one or more of methanol, ethanol, n-propanol, and isopropanol.
4. The method of any one of claims 1 to 3, wherein the modified magnetic particles are dispersed in an anhydrous lower alcohol solution such that the magnetic fluid concentration is 1-15 mg/mL.
5. The method of any one of claims 1 to 3, wherein the modified magnetic particles are dispersed in an anhydrous lower alcohol solution such that the magnetic fluid concentration is 2-14 mg/mL.
6. The method of claim 1, wherein the silica-hydroxyl magnetic microspheres are used for nucleic acid extraction.
7. The method of claim 6, wherein the silica-hydroxyl magnetic microspheres are used for RNA extraction, or for RNA and DNA co-extraction; for example, for extracting nucleic acids from SARS-CoV-2.
8. Silicon hydroxyl magnetic microspheres produced by the method of any one of claims 1 to 7.
9. Use of the silica-hydroxyl magnetic microspheres prepared by the method of any one of claims 1-7 in nucleic acid extraction.
10. The use of claim 9, wherein the nucleic acid is RNA, or DNA and RNA; for example, RNA from SARS-CoV-2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022135420A1 (en) * | 2020-12-23 | 2022-06-30 | 四川安可瑞新材料技术有限公司 | Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof |
| WO2023125592A1 (en) * | 2021-12-28 | 2023-07-06 | 南京金斯瑞生物科技有限公司 | Magnetic bead, and preparation method therefor and use thereof in nucleic acid extraction |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030148101A1 (en) * | 2000-03-24 | 2003-08-07 | Philippe Sauer | Porous ferro-or ferrimagnetic glass particles for isolating molecules |
| JP2006249528A (en) * | 2005-03-11 | 2006-09-21 | Hitachi Metals Ltd | Metal particulate, method for producing the same, and magnetic bead |
| CN1935744A (en) * | 2006-09-15 | 2007-03-28 | 东南大学 | Method for preparing silicon dioxide embedded nano composite particles |
| CN102151527A (en) * | 2010-11-23 | 2011-08-17 | 苏州照康生物技术有限公司 | Preparation method of monodisperse silicon oxide magnetic microspheres used for DNA purification and protein separation |
| CN102240532A (en) * | 2011-05-20 | 2011-11-16 | 天津大学 | Method for preparing inorganic nano particle/silicon dioxide composite microspheres with core shell structure |
| CN105344310A (en) * | 2015-11-10 | 2016-02-24 | 洛阳吉恩特生物科技有限公司 | Preparation method for polydisperse DNA extraction magnetic beads and applications |
| CN110304662A (en) * | 2019-07-17 | 2019-10-08 | 东莞东阳光科研发有限公司 | Silicone hydroxyl magnetic bead and its preparation method and application |
| CN111330558A (en) * | 2020-03-10 | 2020-06-26 | 上海领骏生物科技有限公司 | Method for manufacturing magnetic microspheres for extracting and purifying trace nucleic acid |
-
2020
- 2020-12-23 CN CN202011537465.2A patent/CN112563016B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030148101A1 (en) * | 2000-03-24 | 2003-08-07 | Philippe Sauer | Porous ferro-or ferrimagnetic glass particles for isolating molecules |
| JP2006249528A (en) * | 2005-03-11 | 2006-09-21 | Hitachi Metals Ltd | Metal particulate, method for producing the same, and magnetic bead |
| CN1935744A (en) * | 2006-09-15 | 2007-03-28 | 东南大学 | Method for preparing silicon dioxide embedded nano composite particles |
| CN102151527A (en) * | 2010-11-23 | 2011-08-17 | 苏州照康生物技术有限公司 | Preparation method of monodisperse silicon oxide magnetic microspheres used for DNA purification and protein separation |
| CN102240532A (en) * | 2011-05-20 | 2011-11-16 | 天津大学 | Method for preparing inorganic nano particle/silicon dioxide composite microspheres with core shell structure |
| CN105344310A (en) * | 2015-11-10 | 2016-02-24 | 洛阳吉恩特生物科技有限公司 | Preparation method for polydisperse DNA extraction magnetic beads and applications |
| CN110304662A (en) * | 2019-07-17 | 2019-10-08 | 东莞东阳光科研发有限公司 | Silicone hydroxyl magnetic bead and its preparation method and application |
| CN111330558A (en) * | 2020-03-10 | 2020-06-26 | 上海领骏生物科技有限公司 | Method for manufacturing magnetic microspheres for extracting and purifying trace nucleic acid |
Non-Patent Citations (4)
| Title |
|---|
| BORDELON H，ET AL: "A Magnetic Bead-Based Method for Concentrating DNA from Human Urine for Downstream Detection", 《PLOS ONE》, vol. 8, no. 7, 8 July 2013 (2013-07-08), pages 68369, XP055420219, DOI: 10.1371/journal.pone.0068369 * |
| 李海洋等: "硅羟基磁珠的制备及全基因组DNA提取优化", 《生物技术通报》 * |
| 李海洋等: "硅羟基磁珠的制备及全基因组DNA提取优化", 《生物技术通报》, vol. 33, no. 6, 31 December 2017 (2017-12-31), pages 223 - 229 * |
| 黄庚锐;梁志玲;罗宜苑;王亮;: "Fe_3O_4@SiO_2磁性纳米粒子制备方法的综述", 化学工程与装备, no. 02, 15 February 2020 (2020-02-15), pages 211 - 212 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022135420A1 (en) * | 2020-12-23 | 2022-06-30 | 四川安可瑞新材料技术有限公司 | Preparation method for magnetic microspheres for nucleic acid extraction, prepared product and use thereof |
| WO2023125592A1 (en) * | 2021-12-28 | 2023-07-06 | 南京金斯瑞生物科技有限公司 | Magnetic bead, and preparation method therefor and use thereof in nucleic acid extraction |
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