CN116550303A - Preparation method of spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction - Google Patents
Preparation method of spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction Download PDFInfo
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- CN116550303A CN116550303A CN202310306993.4A CN202310306993A CN116550303A CN 116550303 A CN116550303 A CN 116550303A CN 202310306993 A CN202310306993 A CN 202310306993A CN 116550303 A CN116550303 A CN 116550303A
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- 229920002125 Sokalan® Polymers 0.000 title claims abstract description 98
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 96
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 96
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 96
- 239000004584 polyacrylic acid Substances 0.000 title claims abstract description 95
- 238000000605 extraction Methods 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 116
- 239000002073 nanorod Substances 0.000 claims abstract description 114
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 57
- 238000000034 method Methods 0.000 claims abstract description 22
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 10
- 239000011248 coating agent Substances 0.000 claims abstract description 7
- 238000000576 coating method Methods 0.000 claims abstract description 7
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 41
- IEECXTSVVFWGSE-UHFFFAOYSA-M iron(3+);oxygen(2-);hydroxide Chemical compound [OH-].[O-2].[Fe+3] IEECXTSVVFWGSE-UHFFFAOYSA-M 0.000 claims description 33
- 229960004887 ferric hydroxide Drugs 0.000 claims description 32
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 30
- 238000003756 stirring Methods 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 27
- CUPCBVUMRUSXIU-UHFFFAOYSA-N [Fe].OOO Chemical compound [Fe].OOO CUPCBVUMRUSXIU-UHFFFAOYSA-N 0.000 claims description 26
- 238000001354 calcination Methods 0.000 claims description 26
- 229910021519 iron(III) oxide-hydroxide Inorganic materials 0.000 claims description 26
- 239000000243 solution Substances 0.000 claims description 26
- 238000005406 washing Methods 0.000 claims description 25
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 24
- 238000010992 reflux Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 18
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 claims description 14
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 13
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 13
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 13
- 239000001257 hydrogen Substances 0.000 claims description 13
- 229910052739 hydrogen Inorganic materials 0.000 claims description 13
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 10
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 10
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 239000002086 nanomaterial Substances 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 6
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000006722 reduction reaction Methods 0.000 claims description 5
- 238000007789 sealing Methods 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 241000711573 Coronaviridae Species 0.000 claims description 2
- GWESVXSMPKAFAS-UHFFFAOYSA-N Isopropylcyclohexane Natural products CC(C)C1CCCCC1 GWESVXSMPKAFAS-UHFFFAOYSA-N 0.000 claims description 2
- 235000014413 iron hydroxide Nutrition 0.000 claims 1
- NCNCGGDMXMBVIA-UHFFFAOYSA-L iron(ii) hydroxide Chemical compound [OH-].[OH-].[Fe+2] NCNCGGDMXMBVIA-UHFFFAOYSA-L 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 25
- 238000009987 spinning Methods 0.000 abstract description 22
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 abstract description 13
- 230000004048 modification Effects 0.000 abstract description 12
- 238000012986 modification Methods 0.000 abstract description 12
- 238000012546 transfer Methods 0.000 abstract description 8
- 230000002195 synergetic effect Effects 0.000 abstract description 5
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 abstract 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 abstract 1
- 239000011324 bead Substances 0.000 description 29
- 239000006185 dispersion Substances 0.000 description 23
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 16
- 241001112090 Pseudovirus Species 0.000 description 14
- 239000012876 carrier material Substances 0.000 description 13
- 239000000463 material Substances 0.000 description 11
- 238000000926 separation method Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- 238000007885 magnetic separation Methods 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 125000000524 functional group Chemical group 0.000 description 6
- 229910002588 FeOOH Inorganic materials 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- -1 0.5 g) Chemical compound 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000009792 diffusion process Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 235000012239 silicon dioxide Nutrition 0.000 description 4
- 108010067770 Endopeptidase K Proteins 0.000 description 3
- 238000002123 RNA extraction Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 230000009089 cytolysis Effects 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000012807 PCR reagent Substances 0.000 description 2
- 229910004298 SiO 2 Inorganic materials 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000001132 ultrasonic dispersion Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000007886 magnetic bead extraction Methods 0.000 description 1
- 239000011553 magnetic fluid Substances 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
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- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
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Abstract
The invention discloses a preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction. The preparation method comprises the following steps: preparing a spinning magnetic iron oxide nanorod carrier core, coating mesoporous silica outside the spinning magnetic iron oxide nanorod carrier core, performing amino modification on the mesoporous silica, and finally using an acrylic acid modification layer for modification to obtain the spinning polyacrylic acid magnetic mesoporous carrier. According to the invention, through the synergistic effect of the spin magnetic iron oxide nanorod carrier core, the mesoporous silica structural layer and the polyacrylic acid modification layer, the promotion of the nucleic acid adsorption sites and the promotion of the mass transfer rate in the nucleic acid adsorption process are realized, and the nucleic acid extraction efficiency is finally improved.
Description
Technical field:
the invention relates to the technical field of nano materials and bioengineering, in particular to a preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction.
The background technology is as follows:
the separation and purification of nucleic acid is extremely important in molecular biology research and clinical molecular diagnosis, and whether high-quality nucleic acid molecules can be extracted from precious clinical samples obtained in molecular diagnosis is a key for providing accurate analysis and diagnosis results later. The traditional nucleic acid extraction method based on liquid phase separation requires a large sample amount, has long separation time, is easy to pollute and has high degradation rate, and the residual risk of the PCR inhibitor is obviously increased by the residual organic solvent. Compared with the liquid phase extraction method, the solid phase extraction method reduces the use of organic solvents, has relatively simple and convenient operation and short separation time, is not easy to pollute and degrade in the experimental process, and has relatively high concentration and purity of the extracted nucleic acid. The magnetic micro-nano material (hereinafter referred to as magnetic beads) separation and purification technology is the most widely used nucleic acid extraction method at present, and the automation, high flux and rapid extraction of nucleic acid are realized comprehensively. The magnetic bead method utilizes the selective adsorption of nano magnetic bead microsphere with specific functional group (hydroxyl, carboxyl or amino) on the surface to realize the separation and purification of nucleic acid, in the detection system, impurities such as protein and the like can not be adsorbed with the magnetic beads, and the magnetic beads can be aggregated and dispersed under a magnetic field to realize the separation of nucleic acid and impurities. The magnetic adsorption material is the core of magnetic bead separation and purification technology, because of the silicon dioxide (SiO 2 ) Has good capability of binding nucleic acid, siO 2 SiO formed by coating magnetic fluid 2 @Fe 3 O 4 Magnetic beads are the most commonly used nucleic acid magnetic separation nanomaterial. However, siO is currently used in the nucleic acid extraction kit on the market 2 @Fe 3 O 4 SiO on the surface of magnetic bead 2 The coating is a compact and nonporous structure with adsorption capacitySmall size, slow diffusion mass transfer speed, etc. Along with the continuous improvement of the requirements of the biological medicine industry and clinical diagnosis on indexes such as the sensitivity, the extraction efficiency, the extraction rate and the like of nucleic acid extraction, the research and development of novel magnetic separation nano materials for nucleic acid extraction, which are high in efficiency and sensitivity and can improve the mass transfer rate in the extraction process, has important significance.
The invention comprises the following steps:
the invention solves the problems existing in the prior art, and provides a preparation method of a spin-type polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction.
The invention aims to provide a preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction, which comprises the following steps:
s1: sealing the ferric chloride aqueous solution, standing in an oven to obtain a ferric hydroxide nano rod, and dispersing the ferric hydroxide nano rod in water to obtain a ferric hydroxide nano rod storage solution;
s2: diluting the ferric hydroxide nano rod storage solution obtained in the step S1 to the concentration of 0.0005-0.0010g/mL to obtain ferric hydroxide nano rod liquid, adding urea, cetyl Trimethyl Ammonium Bromide (CTAB), isopropyl alcohol and cyclohexane into the ferric hydroxide nano rod liquid, fully and uniformly mixing, controlling the mass ratio of the ferric hydroxide nano rod to the urea and the cetyl trimethyl ammonium bromide in the ferric hydroxide nano rod liquid to be 1:25-35:45-55, the volume ratio of the isopropyl alcohol, the cyclohexane and the ferric hydroxide nano rod liquid to be 0.08-0.12:1.4-1.6:1, adding Tetraethoxysilane (TEOS), the volume ratio of the tetraethoxysilane to the ferric hydroxide nano rod liquid to be 0.02-0.06:1, stirring and refluxing for reaction, controlling the coating of mesoporous silica layers with different thicknesses, cooling, washing and drying by water to obtain the ferric hydroxide nano rod coated with mesoporous silica (FeH@mSiO 2 )；
S3: the mesoporous silica coated iron oxyhydroxide nano rod (FeOOH@mSiO) obtained in the step S2 2 ) Calcining in air, and then calcining in hydrogen for reduction reaction to obtain black mesoporous silica coated ferroferric oxide nano rod (Fe 3 O 4 @mSiO 2 )；
S4: the black ferroferric oxide nanorod coated with mesoporous silica obtained in the step S3 (Fe 3 O 4 @mSiO 2 ) Dispersing into absolute ethyl alcohol, adding aminopropyl triethoxysilane (APTES), stirring and refluxing for reaction, wherein the mass ratio of black ferroferric oxide nanorod coated with mesoporous silica to aminopropyl triethoxysilane is 1:5-1:10, magnetically separating the reacted product, washing and drying to obtain amino-modified ferroferric oxide nanorod (Fe 3 O 4 @mSiO 2 -NH 2 )；
S5: the amino-modified mesoporous silica obtained in step S4 was treated with ferroferric oxide nanorods (Fe 3 O 4 @mSiO 2 -NH 2 ) Dispersing in water, adding polyacrylic acid (PAA), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS), stirring at room temperature, wherein the mass ratio of the ferroferric oxide nanorod of amino modified mesoporous silica to the polyacrylic acid is 4.5-5.5:1, the molar ratio of the polyacrylic acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide is 1:1.4-1.6, and performing magnetic separation on the product, washing and vacuum drying to obtain the magnetic mesoporous nanomaterial loaded with the polyacrylic acid, namely the spin polyacrylic acid magnetic mesoporous carrier.
The magnetic bead method utilizes the selective adsorption of nano magnetic bead microsphere with specific functional group (hydroxyl, carboxyl or amino) on the surface to realize the separation and purification of nucleic acid, in the detection system, the impurities such as protein and the like can not be adsorbed with the magnetic beads, and the magnetic beads can be aggregated and dispersed under the magnetic field to realize the separation of nucleic acid and impurities. The magnetic adsorption material is the core of magnetic bead separation and purification technology, because of the silicon dioxide (SiO 2 ) Has good capability of binding nucleic acid, siO 2 Coating magnetic fluidSiO formed 2 @Fe 3 O 4 Magnetic beads are the most commonly used nucleic acid magnetic separation nanomaterial. However, siO is currently used in the nucleic acid extraction kit on the market 2 @Fe 3 O 4 SiO on the surface of magnetic bead 2 The coating is a compact and nonporous structure, and has the defects of small adsorption capacity, low diffusion mass transfer speed and the like. In addition, in the step of nucleic acid adsorption, the existing automatic magnetic bead extraction instrument has the advantages that the combination and adsorption between the magnetic beads and the nucleic acid are promoted through a beating mode, and the dispersibility of the magnetic bead material has great influence on the extraction effect.
Aiming at the process, the invention provides the spinning polyacrylic acid magnetic mesoporous carrier for extracting the nucleic acid, and the spinning polyacrylic acid magnetic mesoporous carrier improves the nucleic acid extraction efficiency through triple structural characteristics.
Firstly, the ferric oxide nano rod core of the spin polyacrylic acid magnetic mesoporous carrier provides a structural basis for realizing micro spin of the material, and the binding efficiency of the polyacrylic acid magnetic mesoporous carrier and nucleic acid is greatly improved through the micro spin. The microcosmic shape of the spin polyacrylic acid magnetic mesoporous carrier is an elliptic rod-shaped structure, and the shape of the magnetic mesoporous carrier is similar to that of a stirrer. When the spinning polyacrylic acid magnetic mesoporous carrier dispersion liquid is placed on a magnetic stirrer, magnetic stirring is started, and the spinning polyacrylic acid magnetic mesoporous carrier starts microcosmic spinning to drive the dispersion liquid to stir. Based on the spin mechanism of the spin polyacrylic acid magnetic mesoporous carrier, in the nucleic acid adsorption step, the combination and adsorption between the magnetic beads and the nucleic acid are not required to be promoted through a beating mode, and only the magnetic field conversion driving force (magnetic stirrer principle) is provided at the bottom of the automatic extractor, so that the spin polyacrylic acid magnetic mesoporous carrier can be self-stirred, fully mixed and adsorbed with the nucleic acid, the diffusion mass transfer rate in the nucleic acid adsorption process is effectively improved, and the virus nucleic acid extraction efficiency is further improved.
Secondly, the mesoporous silica structure layer in the spin-type polyacrylic acid magnetic mesoporous carrier improves the specific surface area of the magnetic carrier.
Third, the polyacrylic acid modification layer in the spin polyacrylic acid magnetic mesoporous carrier provides carboxyl functional groups capable of specifically adsorbing nucleic acid, polyacrylic acid is coated on the surface of the mesoporous silica structural layer, and the mesoporous silica structural layer further increases modification sites of the carboxyl functional groups by improving the specific surface area of the magnetic carrier, namely greatly increases adsorption sites of the nucleic acid, and finally further improves the adsorption capacity of the nucleic acid.
The triple structural characteristics of the spin-type polyacrylic acid magnetic mesoporous carrier provided by the invention have a synergistic effect, and through the synergistic effect of the spin-type magnetic ferric oxide nanorod carrier core, the mesoporous silicon dioxide structural layer and the polyacrylic acid modification layer, the promotion of a nucleic acid adsorption site and the promotion of the mass transfer rate in the nucleic acid adsorption process are realized, and the nucleic acid extraction efficiency is finally improved.
Preferably, in the step S2, the mass ratio of the ferric oxide nano rod to the urea to the hexadecyl trimethyl ammonium bromide in the ferric oxide nano rod liquid is 1:30:50, and the volume ratio of the isopropanol to the cyclohexane to the ferric oxide nano rod liquid is 0.1:1.5:1.
Preferably, the specific condition of the stirring reflux reaction in the step S2 is stirring reflux at 70 ℃ for 15-18 hours.
Preferably, the specific steps of calcining in air in step S3 and then calcining in hydrogen for reduction reaction are as follows: calcining at 500 deg.C for 2.5-3.5 hr, and calcining at 300 deg.C in hydrogen for 3.5-4.5 hr.
Preferably, the mass volume ratio of the black ferroferric oxide nano rod coated with the mesoporous silica to the absolute ethyl alcohol in the step S4 is 0.0005-0.0015:1.
Preferably, the specific condition of the stirring reflux reaction in step S4 is that the stirring reflux reaction is carried out at 80 ℃ for 10-14 hours.
Preferably, the mass ratio of the ferroferric oxide nanorod of the amino modified mesoporous silica to the polyacrylic acid in the step S5 is 5:1, and the molar ratio of the polyacrylic acid, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and the N-hydroxysulfosuccinimide is 1:1.5:1.5.
The invention also protects the spin polyacrylic acid magnetic mesoporous carrier prepared by the preparation method.
The invention also protects the application of the spin polyacrylic acid magnetic mesoporous carrier in nucleic acid extraction.
Preferably, the nucleic acid is a novel coronavirus nucleic acid.
Compared with the prior art, the invention has the following advantages:
the spin polyacrylic acid magnetic mesoporous carrier provided by the invention improves the nucleic acid extraction efficiency through the triple structural characteristics. Firstly, the ferric oxide nano rod core of the spin polyacrylic acid magnetic mesoporous carrier provides a structural basis for realizing micro spin of the material, and the binding efficiency of the polyacrylic acid magnetic mesoporous carrier and nucleic acid is greatly improved through the micro spin. The microcosmic shape of the spin polyacrylic acid magnetic mesoporous carrier is an elliptic rod-shaped structure, and the shape of the magnetic mesoporous carrier is similar to that of a stirrer. When the spinning polyacrylic acid magnetic mesoporous carrier dispersion liquid is placed on a magnetic stirrer, magnetic stirring is started, and the spinning polyacrylic acid magnetic mesoporous carrier starts microcosmic spinning to drive the dispersion liquid to stir. Based on the spin mechanism of the spin polyacrylic acid magnetic mesoporous carrier, in the nucleic acid adsorption step, the combination and adsorption between the magnetic beads and the nucleic acid are not required to be promoted through a beating mode, and only the magnetic field conversion driving force (magnetic stirrer principle) is provided at the bottom of the automatic extractor, so that the spin polyacrylic acid magnetic mesoporous carrier can be self-stirred, fully mixed and adsorbed with the nucleic acid, the diffusion mass transfer rate in the nucleic acid adsorption process is effectively improved, and the virus nucleic acid extraction efficiency is further improved. Second, the mesoporous silica structure layer in the spin-type polyacrylic acid magnetic mesoporous carrier can improve the specific surface area of the magnetic carrier. Third, the polyacrylic acid modification layer in the spin polyacrylic acid magnetic mesoporous carrier provides carboxyl functional groups capable of specifically adsorbing nucleic acid, polyacrylic acid is coated on the surface of the mesoporous silica structural layer, and the mesoporous silica structural layer further increases modification sites of the carboxyl functional groups by improving the specific surface area of the magnetic carrier, namely greatly increases adsorption sites of the nucleic acid, and finally further improves the adsorption capacity of the nucleic acid. The triple structural characteristics of the spin-type polyacrylic acid magnetic mesoporous carrier provided by the invention have a synergistic effect, and through the synergistic effect of the spin-type magnetic ferric oxide nanorod carrier core, the mesoporous silicon dioxide structural layer and the polyacrylic acid modification layer, the promotion of a nucleic acid adsorption site and the promotion of the mass transfer rate in the nucleic acid adsorption process are realized, and the nucleic acid extraction efficiency is finally improved.
Description of the drawings:
FIG. 1 is a iron oxyhydroxide nanorod prepared in example 1.
Fig. 2 is a mesoporous silica coated iron oxyhydroxide nanorod prepared in example 1.
FIG. 3 is a spin-on polyacrylic acid magnetic mesoporous support prepared in example 1.
The specific embodiment is as follows:
the following examples are further illustrative of the invention and are not intended to be limiting thereof.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention. Unless otherwise indicated, the experimental materials and reagents herein are all commercially available products conventional in the art.
Example 1
A preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction comprises the following steps:
s1, sealing 1mM ferric chloride aqueous solution, and standing in a baking oven at 90 ℃ for 12 hours to obtain the ferric hydroxide nano-rod, namely the spin magnetic rod-shaped carrier core, wherein the size of the ferric hydroxide nano-rod is 400 multiplied by 50nm as shown in figure 1. And (3) after cooling, centrifuging, washing twice, and dispersing in water to obtain the ferric hydroxide nano rod storage solution for standby.
S2, diluting the iron oxyhydroxide nanorod storage solution (containing 10mg of iron oxyhydroxide nanorod) obtained in the step S1 by adding water until the volume of the iron oxyhydroxide nanorod storage solution is 15mL, adding urea (0.3 g), cetyltrimethylammonium bromide (CTAB, 0.5 g), isopropanol (1 mL) and cyclohexane (15 mL), performing ultrasonic dispersion, fully mixing, and then adding tetraethyl glycol600. Mu.L of oxysilane (TEOS) was refluxed with stirring at 70℃for 16 hours, and the thickness of the coated mesoporous silica layer was controlled to be about 150nm. Cooling, centrifuging, washing with water, and drying to obtain mesoporous silica coated iron oxyhydroxide nanorod (FeOOH@mSiO) 2 ) As shown in FIG. 2, the thickness of the mesoporous silica layer is about 150nm, the mesoporous silica layer is uniformly coated on the outer layer of the iron oxyhydroxide nanorods, and the size of the iron oxyhydroxide nanorods coated with the mesoporous silica layer is about 700×350nm.
S3, 200mg of iron oxyhydroxide nanorod coated with mesoporous silica (FeOOH@mSiO) 2 ) Calcining in air at 500 deg.c for 3 hr, and calcining and reducing in hydrogen at 300 deg.c for 4 hr to obtain black ferroferric oxide nanometer rod (Fe) with coated mesoporous silica 3 O 4 @mSiO 2 )。
S4, 100mg of black ferroferric oxide nanorod coated with mesoporous silica (Fe 3 O 4 @mSiO 2 ) Dispersing into 100mL absolute ethanol, adding aminopropyl triethoxysilane (APTES), stirring at 80deg.C, refluxing for 12 hr, fe 3 O 4 @mSiO 2 And APETS feeding mass ratio is 1:5, and the number of amino groups modified on the surface of the nanorod is regulated. Magnetic separation, three times of alcohol washing, one time of water washing, drying to obtain amino modified mesoporous silica ferroferric oxide nano rod (Fe 3 O 4 @mSiO 2 -NH 2 )。
S5, ferroferric oxide nanorods (Fe) of 100mg of amino-modified mesoporous silica 3 O 4 @mSiO 2 -NH 2 ) Dispersing into 50mL of water, adding polyacrylic acid (PAA), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS), and stirring at room temperature for 12 hours. Fe (Fe) 3 O 4 @mSiO 2 -NH 2 And PAA with a mass ratio of 5:1, PAA to EDC to NHS with a molar ratio of 1:1.5:1.5, magnetically separating the product, washing with water for three times, washing with ethanol for three times, and vacuum drying to obtain the polyacrylic acid-loaded magnetic mesoporous nanomaterial (Fe 3 O 4 @mSiO 2 PAA), i.e. a spin-on polyacrylic acid magnetic mesoporous support. As shown in FIG. 3, the size of the spin-type polyacrylic acid magnetic mesoporous carrierAbout 800×400nm, the thickness of the mesoporous silica-polyacrylic acid outer layer structure slightly increases after the polyacrylic acid is loaded.
Example 2
The same as in example 1, except that: and S2, diluting the ferric hydroxide nano rod storage solution obtained in the step S1 to the concentration of 0.0005g/mL to obtain ferric hydroxide nano rod solution, wherein the mass ratio of the ferric hydroxide nano rod to urea and hexadecyl trimethyl ammonium bromide in the ferric hydroxide nano rod solution is 1:25:45, the volume ratio of isopropanol, cyclohexane and the ferric hydroxide nano rod solution is 0.08:1.4:1, the volume ratio of tetraethoxysilane to the ferric hydroxide nano rod solution is 0.02:1, and the specific conditions of stirring reflux reaction are that stirring reflux is carried out for 18 hours at 70 ℃.
The specific steps of calcining in air in the step S3 and then calcining in hydrogen for reduction reaction are as follows: calcining at 500 ℃ for 3.5 hours in air, and then calcining and reducing in hydrogen at 300 ℃ for 4.5 hours.
In the step S4, the mass volume ratio of the black ferroferric oxide nano rod coated with the mesoporous silica to the absolute ethyl alcohol is 0.0005:1, the mass ratio of the black ferroferric oxide nano rod coated with the mesoporous silica to the aminopropyl triethoxysilane is 1:5, and the specific condition of stirring reflux reaction is stirring reflux reaction for 14 hours at 80 ℃.
The mass ratio of the ferroferric oxide nano rod of the amino modified mesoporous silica to the polyacrylic acid in the step S5 is 4.5:1, and the molar ratio of the polyacrylic acid to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysulfosuccinimide is 1:1.4:1.4.
Example 3
The same as in example 1, except that: and S2, diluting the ferric hydroxide nano rod storage solution obtained in the step S1 to the concentration of 0.0010g/mL to obtain ferric hydroxide nano rod solution, wherein the mass ratio of the ferric hydroxide nano rod to urea to cetyl trimethyl ammonium bromide in the ferric hydroxide nano rod solution is 1:35:55, the volume ratio of isopropanol to cyclohexane to the ferric hydroxide nano rod solution is 0.12:1.6:1, the volume ratio of tetraethoxysilane to the ferric hydroxide nano rod solution is 0.06:1, and the specific conditions of stirring reflux reaction are that the stirring reflux is carried out for 15 hours at 70 ℃.
The specific steps of calcining in air in the step S3 and then calcining in hydrogen for reduction reaction are as follows: calcining at 500 ℃ for 2.5 hours in air, and then calcining and reducing in hydrogen at 300 ℃ for 3.5 hours.
In the step S4, the mass volume ratio of the black ferroferric oxide nano rod coated with the mesoporous silica to the absolute ethyl alcohol is 0.0015:1, the mass ratio of the black ferroferric oxide nano rod coated with the mesoporous silica to the aminopropyl triethoxysilane is 1:10, and the specific condition of stirring reflux reaction is that stirring reflux reaction is carried out for 10 hours at 80 ℃.
The mass ratio of the ferroferric oxide nano rod of the amino modified mesoporous silica to the polyacrylic acid in the step S5 is 5.5:1, and the molar ratio of the polyacrylic acid to the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to the N-hydroxysulfosuccinimide is 1:1.6:1.6.
Comparative example 1
A preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction comprises the following steps:
s1, sealing 1mM ferric chloride aqueous solution, and standing for 12 hours in a 90 ℃ oven to obtain the ferric hydroxide nanorod (figure 1), namely the spin magnetic rod-shaped carrier core. After cooling, centrifuging, washing twice, and drying for standby. S2, calcining 200mg of the iron oxide hydroxide nano rod in air at 500 ℃ for 3 hours, and then calcining and reducing in hydrogen at 300 ℃ for 4 hours to obtain the black ferroferric oxide nano rod.
S3, dispersing 100mg of ferroferric oxide nanorods into 100mL of absolute ethyl alcohol, adding aminopropyl triethoxysilane (APTES), stirring and refluxing at 80 ℃ for 12 hours, wherein the mass ratio of the ferroferric oxide nanorods to the APETS is 1:5, and regulating and controlling the number of amino modified on the surface of the nanorods. And (3) performing magnetic separation, alcohol washing for three times, water washing for one time, and drying to obtain the amino modified ferroferric oxide nanorod.
S4, dispersing 100mg of the amino modified ferroferric oxide nanorods obtained in the step S3 into 50mL of water, and adding polyacrylic acid (PAA), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinatePercipimide (NHS), stirred at room temperature for 12 hours. Fe (Fe) 3 O 4 @mSiO 2 -NH 2 And the mass ratio of PAA to EDC to NHS is 5:1.5:1.5, the product is magnetically separated, washed three times with water, washed three times with ethanol, and dried in vacuum to obtain the magnetic ferroferric oxide nanorod material loaded with polyacrylic acid.
Comparative example 2
A preparation method of a spin polyacrylic acid magnetic mesoporous carrier for nucleic acid extraction comprises the following steps:
s1, sealing 1mM ferric chloride aqueous solution, and standing for 12 hours in a 90 ℃ oven to obtain the ferric hydroxide nanorod (figure 1), namely the spin magnetic rod-shaped carrier core. And (3) after cooling, centrifuging, washing twice, and dispersing in water to obtain the ferric hydroxide nano rod storage solution for standby.
S2, diluting the iron oxyhydroxide nanorod storage solution (containing 10mg of iron oxyhydroxide nanorods) obtained in the step S1 by adding water until the volume of the iron oxyhydroxide nanorod storage solution is 15mL, adding urea (0.3 g), cetyltrimethylammonium bromide (CTAB, 0.5 g), isopropanol (1 mL) and cyclohexane (15 mL), performing ultrasonic dispersion, fully mixing, adding Tetraethoxysilane (TEOS) 600 mu L, and stirring and refluxing at 70 ℃ for 16 hours. Cooling, centrifuging, washing with water, and drying to obtain mesoporous silica coated iron oxyhydroxide nanorod (FeOOH@mSiO) 2 ) As shown in fig. 2.
S3, 200mg of iron oxyhydroxide nanorod coated with mesoporous silica (FeOOH@mSiO) 2 ) Calcining in air at 500 deg.c for 3 hr, and calcining and reducing in hydrogen at 300 deg.c for 4 hr to obtain black ferroferric oxide nanometer rod (Fe) with coated mesoporous silica 3 O 4 @mSiO 2 )。
S4, 100mg of black ferroferric oxide nanorod coated with mesoporous silica (Fe 3 O 4 @mSiO 2 ) Dispersing into 100mL absolute ethanol, adding aminopropyl triethoxysilane (APTES), stirring at 80deg.C, refluxing for 12 hr, fe 3 O 4 @mSiO 2 And APETS feeding mass ratio is 1:5, and the number of amino groups modified on the surface of the nanorod is regulated. Magnetic separation, alcohol washing for three times, water washing for one time, drying,obtaining amino modified mesoporous silica coated ferroferric oxide nano rod (Fe) 3 O 4 @mSiO 2 -NH 2 )。
Application example 1: application of spin polyacrylic acid magnetic mesoporous carrier in extraction of novel crown 2019-nCov pseudovirus RNA (automatic extraction)
The spin-on polyacrylic acid magnetic mesoporous support material prepared in example 1 was dispersed in water at a concentration of 40mg/mL. Taking 96 deep hole plate, adding 600 μl of washing solution into columns 4 and 10, adding 100 μl of eluent into columns 6 and 12, sequentially adding 20 μl of proteinase K and 20 μl of aqueous dispersion of the above magnetic mesoporous carrier material into columns 1 and 7, and adding 200 μl of new crown 2019-nCov pseudovirus sample (concentration 5×10) 4 cobies/mL) and 500 μl of lysis conjugate. The sample nucleic acid is rapidly extracted by an automatic nucleic acid extractor, the mixing time of an operation program is set to be 4, 1 and 1min according to four steps of combination, washing, elution and magnetic discarding, the magnetic attraction times are 1, 1 and 0, and the temperature is 70 ℃, the room temperature and the room temperature are 70 ℃. And after the program operation is finished, adding the eluent into a fluorescent quantitative PCR reagent for quantitative amplification, and comparing the nucleic acid extraction effect of the spinning polyacrylic acid magnetic mesoporous carrier material and the original magnetic bead dispersion liquid (commercially available magnetic beads) of the kit according to the result.
The magnetic bead method for extracting the novel crown nucleic acid is described with reference to the specification of a commercial nucleic acid extraction or purification reagent product (Guangdong ear mechanical equipment 20170583; guangzhou da gene Co., ltd.).
The fluorescence PCR method nucleic acid detection procedure was described with reference to the extraction procedure described in the instructions of the commercial nucleic acid detection kit product (national mechanical standard 20203400749; guangzhou da genes Co., ltd.).
Table 1 shows the results of nucleic acid extraction (automatic extraction) of novel crown 2019-nCov pseudovirus, which shows that: the spinning polyacrylic acid magnetic mesoporous carrier material dispersion liquid can be normally used in a novel crown 2019-nCov pseudovirus RNA extraction experiment, and the nucleic acid extraction performance of the spinning polyacrylic acid magnetic mesoporous carrier material dispersion liquid is equivalent to that of a stock dispersion liquid containing magnetic beads in a nucleic acid extraction kit.
TABLE 1 New crown 2019-nCov pseudovirus nucleic acid extraction results (automatic extraction)
Meanwhile, the polyacrylic acid-loaded magnetic ferroferric oxide nanorod material prepared in comparative example 1 and the amino-modified mesoporous silica-coated ferroferric oxide nanorod prepared in comparative example 2 were respectively dispersed in water at a concentration of 40mg/mL, and nucleic acid extraction was performed according to the above extraction method.
The nucleic acid extraction effects of the spin-type polyacrylic acid magnetic mesoporous support material prepared in comparative example 1 and the polyacrylic acid-loaded magnetic ferroferric oxide nanorod material prepared in comparative example 1, and the amino-modified and mesoporous silica-coated ferroferric oxide nanorod prepared in comparative example 2 are shown in table 2.
Table 2 shows the results of nucleic acid extraction (automatic extraction) of novel crown 2019-nCov pseudovirus, showing that: the spinning polyacrylic acid magnetic mesoporous carrier material dispersion (i.e. example 1) and the polyacrylic acid-loaded magnetic ferroferric oxide nanorod dispersion (i.e. comparative example 1) can be normally used in a novel crown 2019-nCov pseudovirus RNA extraction experiment, but the extraction effect of the spinning polyacrylic acid magnetic mesoporous carrier material dispersion is better than that of the polyacrylic acid-loaded magnetic ferroferric oxide nanorod dispersion, the amino-modified and mesoporous silica-coated ferroferric oxide nanorod dispersion cannot be normally used in the novel crown 2019-nCov pseudovirus RNA extraction experiment, and the nucleic acid extraction performance of the polyacrylic acid-loaded magnetic ferroferric oxide nanorod dispersion (comparative example 2) is far worse than that of the spinning polyacrylic acid magnetic mesoporous carrier material dispersion.
TABLE 2 New crown 2019-nCov pseudovirus nucleic acid extraction results (automatic extraction)
Application example 2: application of spin polyacrylic acid magnetic mesoporous carrier in extraction of novel crown 2019-nCov pseudovirus RNA (manual extraction)
The spin-on polyacrylic acid magnetic mesoporous carrier material prepared in the embodiment 1 of the invention is dispersed in water, and the concentration is 40mg/mL. Taking 96 deep hole plate, adding 600 μl of washing solution into columns 4 and 10, adding 100 μl of eluent into columns 6 and 12, sequentially adding 20 μl of proteinase K and 20 μl of aqueous dispersion of the above magnetic mesoporous carrier material into columns 1 and 7, and adding 200 μl of new crown 2019-nCov pseudovirus sample (concentration 5×10) 4 cobies/mL) and 500 μl of lysis conjugate. The method mainly comprises three steps of combination, washing and elution, wherein the adsorption, washing and elution time is respectively 4, 1 and 1min, a magnetic stirrer (350 r.p.m) is placed below a 96-well plate in each operation process, the spin polyacrylic acid magnetic mesoporous carrier is self-stirred and fully mixed with the nucleic acid, and the magnetic separation is carried out after each step is finished, and then the next operation is carried out. After the extraction is finished, the eluent is added into a fluorescent quantitative PCR reagent for quantitative amplification, and the nucleic acid extraction effect of the spinning polyacrylic acid magnetic mesoporous carrier material and the original magnetic bead dispersion liquid (commercially available magnetic beads) of the kit is compared according to the result.
The nucleic acid extraction-related reagents used in this application example, including washing solution, eluting solution, proteinase K, lysis conjugate solution and raw magnetic bead dispersion, were all obtained from commercial nucleic acid extraction or purification reagent products (Guangzhou Daan Gene Co., ltd., guangdong ear mechanical equipment 20170583).
The fluorescence PCR method nucleic acid detection procedure was described with reference to the extraction procedure described in the instructions of the commercial nucleic acid detection kit product (national mechanical standard 20203400749; guangzhou da genes Co., ltd.).
Table 3 shows the results of the nucleic acid extraction (manual extraction) of the novel crown 2019-nCov pseudovirus, which shows that: the spinning polyacrylic acid magnetic mesoporous carrier material dispersion liquid can be normally used in a manual extraction experiment of novel crown 2019-nCov pseudovirus RNA, and when magnetic field conversion driving force is provided at normal temperature, the spinning polyacrylic acid magnetic mesoporous carrier material is self-stirred through micro-spinning, so that the extraction efficiency of virus nucleic acid is improved, and the nucleic acid extraction performance is better than that of the original dispersion liquid containing magnetic beads in a nucleic acid extraction kit. In addition, the manual extraction effect of the spinning polyacrylic acid magnetic mesoporous carrier material dispersion liquid is better than the automatic extraction effect of the original dispersion liquid containing magnetic beads in the nucleic acid extraction kit.
TABLE 3 New crown 2019-nCov pseudovirus nucleic acid extraction results (Manual extraction)
The method for extracting and purifying the viral nucleic acid based on the spin polyacrylic acid magnetic mesoporous carrier has no less separation efficiency and no less nucleic acid yield than the commercial magnetic bead products.
The above embodiments are only described to assist in understanding the technical solution of the present invention and its core idea, and it should be noted that it will be obvious to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.
Claims (10)
1. The preparation method of the spin polyacrylic acid magnetic mesoporous carrier for extracting the nucleic acid is characterized by comprising the following steps of:
s1: sealing the ferric chloride aqueous solution, standing in an oven to obtain a ferric hydroxide nano rod, and dispersing the ferric hydroxide nano rod in water to obtain a ferric hydroxide nano rod storage solution;
s2: diluting the iron oxyhydroxide nano rod storage solution obtained in the step S1 to the concentration of 0.0005-0.0010g/mL to obtain iron oxyhydroxide nano rod liquid, adding urea, cetyltrimethylammonium bromide, isopropyl alcohol and cyclohexane into the iron oxyhydroxide nano rod liquid, fully and uniformly mixing, controlling the mass ratio of the iron oxyhydroxide nano rod to the urea and the cetyltrimethylammonium bromide in the iron oxyhydroxide nano rod liquid to be 1:25-35:45-55, the volume ratio of the isopropyl alcohol, the cyclohexane to the iron oxyhydroxide nano rod liquid to be 0.08-0.12:1.4-1.6:1, adding tetraethoxysilane, the volume ratio of the tetraethoxysilane to the iron oxyhydroxide nano rod liquid to be 0.02-0.06:1, stirring and refluxing for reaction, controlling the coating of mesoporous silica layers with different thicknesses, cooling, washing and drying to obtain the iron oxyhydroxide nano rod coated with mesoporous silica;
s3: calcining the iron hydroxide nano rod coated with the mesoporous silica obtained in the step S2 in air, and then calcining and reducing in hydrogen to obtain a black ferroferric oxide nano rod coated with the mesoporous silica;
s4: dispersing the black ferroferric oxide nanorod coated with mesoporous silica obtained in the step S3 into absolute ethyl alcohol, adding aminopropyl triethoxysilane, stirring and refluxing for reaction, wherein the mass ratio of the black ferroferric oxide nanorod coated with mesoporous silica to the aminopropyl triethoxysilane is 1:5-1:10, magnetically separating the reacted product, washing and drying to obtain the ferroferric oxide nanorod of amino-modified mesoporous silica;
s5: dispersing the ferroferric oxide nanorod of the amino modified mesoporous silica obtained in the step S4 into water, adding polyacrylic acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, stirring at room temperature, wherein the mass ratio of the ferroferric oxide nanorod of the amino modified mesoporous silica to the polyacrylic acid is 4.5-5.5:1, the molar ratio of the polyacrylic acid, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide is 1:1.4-1.6, magnetically separating the product, and washing and vacuum drying to obtain the magnetic mesoporous nanomaterial loaded with polyacrylic acid, namely the spin polyacrylic acid magnetic mesoporous carrier.
2. The preparation method according to claim 1, wherein in the step S2, the mass ratio of the iron oxyhydroxide nanorods to urea and hexadecyl trimethyl ammonium bromide in the iron oxyhydroxide nanorod liquid is 1:30:50, and the volume ratio of isopropanol, cyclohexane and the iron oxyhydroxide nanorod liquid is 0.1:1.5:1.
3. The process according to claim 1 or 2, wherein the stirring reflux reaction in step S2 is carried out under stirring reflux at 70 ℃ for 15 to 18 hours.
4. The preparation method according to claim 1, wherein the specific steps of calcining in air and then calcining in hydrogen for reduction reaction in step S3 are as follows: calcining at 500 deg.C for 2.5-3.5 hr, and calcining at 300 deg.C in hydrogen for 3.5-4.5 hr.
5. The preparation method according to claim 1, wherein the mass-to-volume ratio of the black mesoporous silica coated ferroferric oxide nanorods to the absolute ethyl alcohol in the step S4 is 0.0005-0.0015:1.
6. The process according to claim 1 or 5, wherein the stirring reflux reaction in step S4 is carried out at 80℃for 10 to 14 hours.
7. The preparation method according to claim 1, wherein the mass ratio of the ferroferric oxide nanorod of the amino modified mesoporous silica to the polyacrylic acid in the step S5 is 5:1, and the molar ratio of the polyacrylic acid, the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and the N-hydroxysulfosuccinimide is 1:1.5:1.5.
8. The spin-on polyacrylic acid magnetic mesoporous carrier prepared by the preparation method of any one of claims 1 to 7.
9. The use of the spin-on polyacrylic acid magnetic mesoporous carrier according to claim 8 in nucleic acid extraction.
10. The use of claim 9, wherein the nucleic acid is a novel coronavirus nucleic acid.
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