CN117417497A - Superparamagnetism magnetic bead, preparation method and application thereof - Google Patents
Superparamagnetism magnetic bead, preparation method and application thereof Download PDFInfo
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- 239000011324 bead Substances 0.000 title claims abstract description 56
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000005291 magnetic effect Effects 0.000 title abstract description 41
- 238000006243 chemical reaction Methods 0.000 claims abstract description 64
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000002105 nanoparticle Substances 0.000 claims abstract description 20
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 239000003945 anionic surfactant Substances 0.000 claims abstract description 14
- VBMVTYDPPZVILR-UHFFFAOYSA-N iron(2+);oxygen(2-) Chemical group [O-2].[Fe+2] VBMVTYDPPZVILR-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 12
- 238000012986 modification Methods 0.000 claims abstract description 10
- 230000004048 modification Effects 0.000 claims abstract description 10
- 239000012535 impurity Substances 0.000 claims abstract description 9
- 238000004132 cross linking Methods 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 19
- 230000008569 process Effects 0.000 claims description 12
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 10
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 229910052779 Neodymium Inorganic materials 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- QEFYFXOXNSNQGX-UHFFFAOYSA-N neodymium atom Chemical compound [Nd] QEFYFXOXNSNQGX-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 6
- 239000006249 magnetic particle Substances 0.000 claims description 6
- 238000007885 magnetic separation Methods 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 4
- 239000003054 catalyst Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 229940077388 benzenesulfonate Drugs 0.000 claims description 2
- -1 sodium alkyl benzene Chemical class 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 11
- 238000001179 sorption measurement Methods 0.000 abstract description 7
- 230000004044 response Effects 0.000 abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 40
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000001000 micrograph Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 230000005294 ferromagnetic effect Effects 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 229920002845 Poly(methacrylic acid) Polymers 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 239000006148 magnetic separator Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical compound O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 239000003517 fume Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000010419 fine particle Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000005408 paramagnetism Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F292/00—Macromolecular compounds obtained by polymerising monomers on to inorganic materials
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F2/00—Processes of polymerisation
- C08F2/44—Polymerisation in the presence of compounding ingredients, e.g. plasticisers, dyestuffs, fillers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention relates to the technical field of biological magnetic beads, in particular to a superparamagnetic magnetic bead, a preparation method and application thereof. The preparation method of the superparamagnetic beads comprises the following steps: 1) Mixing the iron oxide core nano particles, an anionic surfactant and water, and then carrying out surface modification treatment to obtain a reaction solution I; 2) Mixing the reaction solution I with methacrylic acid and then carrying out a crosslinking reaction to obtain a reaction solution II; 3) Reaction solutions II and K 2 S 2 O 8 Mixing and then carrying out polymerization reaction to obtain a reaction solution III; 4) Sequentially magnetically separating, removing impurities and cleaning the reaction solution IIIAnd eluting to obtain the superparamagnetic beads. The product has fast magnetic response, is suitable for large-scale automatic nucleic acid extraction, has high trace DNA/RNA capturing efficiency and low non-specific impurity adsorption rate, has no inhibition effect on downstream reaction, and is compatible with an alcohol extraction system and an alcohol-free extraction system.
Description
Technical Field
The invention relates to the technical field of biological magnetic beads, in particular to a superparamagnetic magnetic bead, a preparation method and application thereof.
Background
The magnetic beads refer to superparamagnetic microspheres with a fine particle size. The functionalized nano magnetic beads have super-strong paramagnetism, can be rapidly aggregated in a magnetic field, and can be uniformly dispersed after leaving the magnetic field. Secondly, the particles have proper and uniform particle size, and the magnetic response and the good dispersibility are ensured to be strong enough. And the kit has abundant surface active groups so as to be coupled with biochemical substances and realize separation from a sample to be detected under the action of an external magnetic field. Generally, the application of functionalized nano-magnetic beads in the fields of biology, medicine and food requires that several conditions be satisfied: (1) has high magnetic responsiveness; (2) the suspension stability is good; (3) the particle size is small and uniform; (4) can be specifically combined with biological macromolecules such as protein, nucleic acid and the like, and has good biocompatibility.
The magnetic bead product is a key material of an extraction link of nucleic acid detection. Compared with the column extraction method, the magnetic bead method nucleic acid extraction reagent is easier to adapt to automatic equipment, and can meet the requirements of large-scale nucleic acid detection on high efficiency and high reliability. On the basis of ensuring the accuracy and stability of nucleic acid detection, it is particularly important to further control the quality and cost of the raw materials for nucleic acid detection. Therefore, the invention provides a superparamagnetic magnetic bead through continuous research, development, optimization and repeated test verification so as to improve the defects.
Disclosure of Invention
In order to solve the problems in the prior art, a superparamagnetic magnetic bead, a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of superparamagnetic beads, which comprises the following steps:
1) Mixing the iron oxide core nano particles, an anionic surfactant and water, and then carrying out surface modification treatment to obtain a reaction solution I;
2) Mixing the reaction solution I in the step 1) with methacrylic acid and then carrying out a crosslinking reaction to obtain a reaction solution II
3) Mixing the reaction liquid II and K in the step 2) 2 S 2 O 8 Mixing and then carrying out polymerization reaction to obtain a reaction solution III;
4) And (3) sequentially carrying out magnetic separation, impurity removal, cleaning and elution on the reaction solution III in the step (3) to obtain the superparamagnetic beads.
Preferably, the mass volume ratio of the iron oxide magnetic nano particles, the anionic surfactant and the water in the step 1) is 0.8-1.2 g:1-1.5 g: 220-270 mL, wherein the anionic surfactant is one or more of sodium dodecyl sulfate and sodium alkyl benzene sulfonate.
Preferably, the surface modification treatment in the step 1) is performed at a temperature of 65 to 75 ℃ for 25 to 35 minutes.
Preferably, the volume ratio of the methacrylic acid to the reaction liquid I in the step 2) is 0.8-1.2:220-270.
Preferably, K in said step 2) 2 S 2 O 8 The mass volume ratio of the catalyst to the reaction solution II is 1.5-2.5 g:220-270 mL.
Preferably, the temperature of the crosslinking reaction in the step 2) is 65-75 ℃, and the reaction time is 40-55 min.
Preferably, the polymerization reaction temperature in the step 3) is 68-74 ℃ and the reaction time is 100-145 min.
Preferably, the instrument used in the magnetic separation process in the step 4) is a powerful neodymium permanent magnet, the instrument used in the impurity removal process is a magnetic particle concentrator, and the solvent used in the elution process is deionized water or ddH 2 O。
The invention also provides the superparamagnetism magnetic bead prepared by the preparation method.
The invention also provides superparamagnetism magnetic beads prepared by the preparation method and application of the superparamagnetism magnetic beads in preparation of nucleic acid detection reagents.
Compared with the prior art, the invention has the following beneficial effects:
the invention modifies the surface of the iron oxide core nanoparticle by the anionic surfactant, reduces the surface energy, reduces the interaction among the nanoparticles, and stabilizes the nanoparticle to prevent agglomeration.
The superparamagnetism magnetic bead has fast magnetic response, is suitable for large-scale automatic nucleic acid extraction, has good nucleic acid binding capacity, high trace DNA/RNA capturing efficiency and low non-specific impurity adsorption rate, has no inhibition effect on downstream reaction, and is compatible with an alcohol extraction system and an alcohol-free extraction system.
Drawings
FIG. 1 is a scanning electron microscope image of the superparamagnetic beads of example 2 at a particle size of 20 nm; wherein, the left image is a scanning electron microscope image when 10000 times, and the right image is a scanning electron microscope image when 350000 times;
FIG. 2 is a scanning electron microscope image of the superparamagnetic beads of example 2 at a particle size of 10 nm; wherein, the left side of the figure is a scanning electron microscope image at 15000 times, and the right side of the figure is a scanning electron microscope image at 350000 times.
Detailed Description
The invention provides a preparation method of superparamagnetic beads, which comprises the following steps:
1) Mixing the iron oxide core nano particles, an anionic surfactant and water, and then carrying out surface modification treatment to obtain a reaction solution I;
2) Mixing the reaction solution I in the step 1) with methacrylic acid and then carrying out a crosslinking reaction to obtain a reaction solution II
3) Mixing the reaction liquid II and K in the step 2) 2 S 2 O 8 Mixing and then carrying out polymerization reaction to obtain a reaction solution III;
4) And (3) sequentially carrying out magnetic separation, impurity removal, cleaning and elution on the reaction solution III in the step (3) to obtain the superparamagnetic beads.
In the preparation method of the superparamagnetic beads, the reaction of the ferromagnetic core nano particles and the anionic surfactant is mainly the process of physical adsorption and chemical adsorption, and is not the traditional chemical reaction. Physical adsorption: the anionic surfactant binds to the surface of the oxidized ferromagnetic core nanoparticle by electrostatic action and van der waals forces. This is a reversible process that does not involve the formation or cleavage of chemical bonds. Chemical adsorption: in some cases, the anionic surfactant may form a chemical bond with the surface of the oxidized ferromagnetic core nanoparticle. This typically involves the reaction of hydroxyl groups on the iron oxide surface with certain functional groups of the surfactant (such as sulfate or carboxylate) to form stable chemical bonds. The purpose of this surface modification treatment is to improve the dispersibility of the magnetic nanoparticles, preventing their aggregation in water, and thus improving their superparamagnetism. At the same time, the introduction of surfactants may also provide additional chemical functionality for further chemical modification or binding of biomolecules.
In the invention, the mass volume ratio of the iron oxide magnetic nano particles, the anionic surfactant and the water in the step 1) is 0.8-1.2 g:1 to 1.5g: 220-270 mL, the mass volume ratio is preferably 1.0-1.2 g:1 to 1.2g: 220-250 mL, more preferably 1g:1.15g:250mL.
In the present invention, the anionic surfactant is preferably one or more of sodium dodecyl sulfate and sodium alkylbenzenesulfonate, and more preferably sodium dodecyl sulfate.
In the present invention, the temperature of the surface modification treatment in the step 1) is 65 to 75 ℃, preferably 70 to 75 ℃, and more preferably 70 ℃; the surface modification treatment in step 1) is performed for 25 to 35 minutes, preferably 28 to 32 minutes, and more preferably 30 minutes.
In the present invention, the volume ratio of methacrylic acid to the reaction solution I in the step 2) is 0.8 to 1.2g:220 to 270, the volume ratio is preferably 0.9 to 1:220 to 250, and the volume ratio is more preferably 0.96:250.
in the present invention, K in the step 2) 2 S 2 O 8 The mass volume ratio of the catalyst to the reaction liquid II is 1.5-2.5 g:220-270 mL, and the mass volume ratio is preferably 1.8-2.2 g: 220-250 mL, more preferably 1.98g:250mL.
In the present invention, the crosslinking reaction temperature in the step 2) is 65 to 75 ℃, the temperature is preferably 70 to 75 ℃, and the temperature is more preferably 70 ℃; the reaction time in the step 2) is 40 to 55min, preferably 42 to 50min, and more preferably 45min.
In the present invention, the polymerization reaction temperature in the step 3) is 68 to 74 ℃, preferably 68 to 72 ℃, and more preferably 70 ℃; the reaction time of the polymerization reaction in the step 3) is 100 to 145min, preferably 110 to 140min, and more preferably 120min.
In the invention, the instrument used in the magnetic separation process in the step 4) is preferably a powerful neodymium permanent magnet, the instrument used in the impurity removal process is preferably a magnetic particle concentrator, and the solvent used in the elution process is preferably deionized water or ddH 2 O。
The present invention will be further described with reference to the drawings and examples, which are only for describing the technical scheme of the present invention, and are not limited to the present invention. The reagents used in the examples of the present invention are all commercially available products.
The invention is based on classical methacrylic acid (methyacrylic acid, MAA) polymerization to form carboxyl groups on the surface of ferromagnetic oxide core nanoparticles (MNPs).
Chemicals, apparatus and devices used in the various embodiments of the invention
The chemicals are shown in table 1 below:
TABLE 1
Apparatus and device:
fume hood (Fume hood)
Heating magnetic stirrer (e.g. IKAMAAG REO)
Strong neodymium permanent magnet (Strong neodymium permanent magnet, ndFeB N45 40X 20 mm)
Sterile plastic bottle
Example 1
A preparation method of superparamagnetism magnetic beads comprises the following steps:
1. 0.8g of commercially available iron oxide core nanoparticles (MNPs) having a particle diameter of 10nm were weighed into 30mL of water to prepare an iron oxide core nanoparticle solution.
2. 1.0g of Sodium Dodecyl Sulfate (SDS) was weighed and placed in 190mL of purified and degassed water, then placed in a 250mL flask together with the iron oxide core nanoparticle solution, magnetically stirred for 5min to uniformly mix, and then heated at a constant temperature of 65 ℃ for 25min to prepare 220mL of reaction solution I.
3. Adding 0.8mL of methacrylic acid (MAA) into the reaction solution I, reacting for 5min, adding sodium hydroxide into the reaction solution I to reduce the pH value of the mixed solution to 3, and carrying out equilibrium reaction for 40min at the constant temperature of 65 ℃ to obtain a reaction solution II with an equilibrium state of 220 mL;
4. 1.5g of K is weighed out 2 S 2 O 8 Adding the mixture into the reaction solution II, continuously stirring for 15min, and then, maintaining the polymerization reaction at 70 ℃ for 100min to prepare 220mL of reaction solution III;
5. separating the iron oxide MNPs coated by the existing carboxyl in the reaction solution III by utilizing strong neodymium permanent magnet for 15min, cooling to room temperature, and removing free MAA and PMAA on the surface of the iron oxide MNPs coated by the existing carboxyl by utilizing a magnetic particle concentrator, wherein the process lasts for 28min until the reaction residual components and byproducts are removed.
6. After the ferric oxide MNPs with the carboxyl coating are dispersed in deionized water in an ultrasonic cleaner, magnetic beads are collected by a magnetic separator and ddH is utilized 2 O the collected beads were eluted 5 times until the detergent was completely removed.
7. And dispersing the eluted magnetic beads in 250mL of deionized water for 4min to obtain the superparamagnetic magnetic beads.
The preparation method of the superparamagnetic beads of the embodiment is not limited by nitrogen, and all steps can be performed in inert N 2 Under gaseous or atmospheric oxygen conditions.
Example 2
A preparation method of superparamagnetism magnetic beads comprises the following steps:
1.1 g of commercially available iron oxide core nanoparticles (MNPs) having a particle diameter of 10nm were weighed into 45mL of water to prepare an iron oxide core nanoparticle solution.
2. 1.15g of Sodium Dodecyl Sulfate (SDS) was weighed and placed in 200mL of purified and degassed water, and then placed in a 250mL flask together with the iron oxide core nanoparticle solution, and after magnetic stirring for 5min, the mixture was heated at a constant temperature of 70 ℃ for 30min to prepare 245mL of reaction solution I.
3. Adding 0.96mL of methacrylic acid (MAA) into the reaction solution I, reacting for 5min, adding sodium hydroxide into the reaction solution I to reduce the pH value of the mixed solution to 3, and carrying out equilibrium reaction for 45min at a constant temperature of 70 ℃ to obtain a reaction solution II in an equilibrium state of 245 mL;
4. 1.98g of K was weighed out 2 S 2 O 8 Adding the mixture into the reaction solution II, continuously stirring for 15min, and then, maintaining the polymerization reaction at 70 ℃ for 120min to prepare 245mL of reaction solution III;
5. separating the iron oxide MNPs coated by the existing carboxyl in the reaction solution III by utilizing strong neodymium permanent magnet for 15min, cooling to room temperature, and removing free MAA and PMAA on the surface of the iron oxide MNPs coated by the existing carboxyl by utilizing a magnetic particle concentrator, wherein the process lasts for 30min until the reaction residual components and byproducts are removed.
6. After the ferric oxide MNPs with the carboxyl coating are dispersed in deionized water in an ultrasonic cleaner, magnetic beads are collected by a magnetic separator and ddH is utilized 2 O the collected beads were eluted 5 times until the detergent was completely removed.
8. And dispersing the eluted magnetic beads in 250mL of deionized water for 5min to obtain the superparamagnetic magnetic beads.
The preparation method of the superparamagnetic beads of the embodiment is not limited by nitrogen, and all steps can be performed in inert N 2 Under gaseous or atmospheric oxygen conditions.
Example 3
A preparation method of superparamagnetism magnetic beads comprises the following steps:
1. 1.2g of commercially available iron oxide core nanoparticles (MNPs) having a particle diameter of 10nm were weighed into 50mL of water to prepare an iron oxide core nanoparticle solution.
2. 1.5g of Sodium Dodecyl Sulfate (SDS) was weighed and placed in 220mL of purified and degassed water, then placed in a 300mL flask together with the iron oxide core nanoparticle solution, magnetically stirred for 5min to uniformly mix, and then heated at a constant temperature of 75 ℃ for 35min to prepare 270mL of reaction solution I.
3. Adding 1.2mL of methacrylic acid (MAA) into the reaction solution I, reacting for 5min, adding sodium hydroxide into the reaction solution I to reduce the pH value of the mixed solution to 3, and carrying out equilibrium reaction for 55min at a constant temperature of 70 ℃ to obtain reaction solution II in an equilibrium state of 270mL;
4. weigh 2.5g of K 2 S 2 O 8 Adding the mixture into the reaction solution II, continuously stirring for 15min, and then keeping the polymerization reaction at 70 ℃ for 145min to prepare 270mL of reaction solution III;
5. separating the iron oxide MNPs coated by the existing carboxyl in the reaction solution III for 20min by utilizing a strong neodymium permanent magnet, cooling to room temperature, and removing free MAA and PMAA on the surface of the iron oxide MNPs coated by the existing carboxyl by utilizing a magnetic particle concentrator, wherein the process lasts for 40min until the reaction residual components and byproducts are removed.
6. After the ferric oxide MNPs with the carboxyl coating are dispersed in deionized water in an ultrasonic cleaner, magnetic beads are collected by a magnetic separator and ddH is utilized 2 O the collected beads were eluted 5 times until the detergent was completely removed.
9. And dispersing the eluted magnetic beads in 300mL of deionized water for 6min to obtain the superparamagnetic magnetic beads.
The preparation method of the superparamagnetic beads of the embodiment is not limited by nitrogen, and all steps can be performed in inert N 2 Under gaseous or atmospheric oxygen conditions.
Experimental example 1
The superparamagnetic beads provided in example 1 of the present invention were subjected to performance tests, and the results are shown in Table 2.
TABLE 2
As can be seen from table 2 and fig. 1-2, the superparamagnetic beads of the present invention have uniform particle sizes and small batch-to-batch differences, and are very suitable for clinical detection application requirements; the surface of the magnetic beads is provided with rich holes, the specific surface area is large, the nucleic acid adsorption capacity is better, and the magnetic beads are very suitable for capturing trace nucleic acids from samples, and the scanning electron microscope images of the superparamagnetic magnetic beads with the particle diameters of 10nm/20nm are shown in figures 1-2.
The magnetic beads provided by the magnetic bead kit can be applied to molecular biology experiments such as virus nucleic acid extraction, genome DNA extraction, detection and the like, and specific operation steps and parameters are adjusted according to the using instructions of the used nucleic acid extraction kit.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the superparamagnetic beads is characterized by comprising the following steps:
1) Mixing the iron oxide core nano particles, an anionic surfactant and water, and then carrying out surface modification treatment to obtain a reaction solution I;
2) Mixing the reaction solution I in the step 1) with methacrylic acid and then carrying out a crosslinking reaction to obtain a reaction solution II;
3) Mixing the reaction liquid II and K in the step 2) 2 S 2 O 8 Mixing and then carrying out polymerization reaction to obtain a reaction solution III;
4) And (3) sequentially carrying out magnetic separation, impurity removal, cleaning and elution on the reaction solution III in the step (3) to obtain the superparamagnetic beads.
2. The preparation method according to claim 1, wherein the mass-to-volume ratio of the iron oxide magnetic nanoparticles, the anionic surfactant and the water in the step 1) is 0.8-1.2 g:1-1.5 g: 220-270 mL; the anionic surfactant is one or more of sodium dodecyl sulfate and sodium alkyl benzene sulfonate.
3. The method according to claim 1, wherein the surface modification treatment in step 1) is carried out at a temperature of 65 to 75 ℃ for 25 to 35 minutes.
4. The method according to claim 1, wherein the volume ratio of methacrylic acid to the reaction solution I in the step 2) is 0.8-1.2:220-270.
5. The method according to claim 1, wherein K in the step 2) 2 S 2 O 8 The mass volume ratio of the catalyst to the reaction solution II is 1.5-2.5 g:220-270 mL.
6. The method according to claim 1, wherein the crosslinking reaction in step 2) is carried out at a temperature of 65 to 75℃for a period of 40 to 55 minutes.
7. The process according to claim 1, wherein the polymerization reaction in step 3) is carried out at a temperature of 68 to 74℃for a period of 100 to 145 minutes.
8. The method according to claim 1, wherein the apparatus used in the magnetic separation in the step 4) is a powerful neodymium permanent magnet, the apparatus used in the impurity removal is a magnetic particle concentrator, and the solvent used in the elution is deionized water or ddH 2 O。
9. A superparamagnetic bead prepared by the method of any one of claims 1 to 8.
10. The superparamagnetic beads prepared by the preparation method of any one of claims 1 to 8 and the application of the superparamagnetic beads in the preparation of nucleic acid detection reagents.
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