WO2022265330A1 - Procédé de préparation de nanoparticules magnétiques à surface modifiée et nanoparticules magnétiques à surface modifiée ainsi préparées - Google Patents

Procédé de préparation de nanoparticules magnétiques à surface modifiée et nanoparticules magnétiques à surface modifiée ainsi préparées Download PDF

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WO2022265330A1
WO2022265330A1 PCT/KR2022/008331 KR2022008331W WO2022265330A1 WO 2022265330 A1 WO2022265330 A1 WO 2022265330A1 KR 2022008331 W KR2022008331 W KR 2022008331W WO 2022265330 A1 WO2022265330 A1 WO 2022265330A1
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magnetic nanoparticles
modified magnetic
modified
magnetic
polyacrylic acid
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PCT/KR2022/008331
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English (en)
Korean (ko)
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박기범
한연수
김남연
배영민
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전남대학교 산학협력단
주식회사 인바이러스테크
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Publication of WO2022265330A1 publication Critical patent/WO2022265330A1/fr

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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • H—ELECTRICITY
    • 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
    • H—ELECTRICITY
    • 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
    • H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
    • H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
    • H—ELECTRICITY
    • 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
    • H01F1/01—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials
    • H01F1/03—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity
    • H01F1/032—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials
    • H01F1/04—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials metals or alloys
    • H01F1/06—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties of inorganic materials characterised by their coercivity of hard-magnetic materials metals or alloys in the form of particles, e.g. powder

Definitions

  • the present invention relates to a method for preparing magnetic nanoparticles by surface-modifying the surface of magnetic particles for extracting nucleic acids with polyacrylic acid, and surface-modified magnetic nanoparticles prepared therefrom.
  • nucleic acid separation, nucleic acid binding, nucleic acid washing, and nucleic acid recovery steps can be automated using specific buffer conditions and strong magnets that have a modified surface with nucleic acids or have affinity for nucleic acids. Therefore, it is known as a next-generation nucleic acid extraction technique.
  • the surfactant was not easily removed during the washing process because it was a method of forming and dispersing charges on the surface of the beads using a surfactant.
  • the iron core was treated with a basic solution without using toxic volatile substances such as ammonia or organic solvents such as ethanol. Then, a method was developed to obtain magnetic nanoparticles uniformly coated with carboxyl groups by mixing the surface-modified magnetic particles with a water-soluble acrylic polymer. With the secured magnetic nanoparticles, a magnetic nanoparticle-based nucleic acid extraction kit was developed.
  • the present invention relates to a method for preparing magnetic nanoparticles by surface-modifying the surface of magnetic particles for extracting nucleic acids with polyacrylic acid, and surface-modified magnetic nanoparticles prepared therefrom.
  • a composition for gene extraction including surface-modified magnetic nanoparticles containing the same.
  • magnetic nanoparticles are materials that exhibit magnetic properties and are materials in an ultra-fine area. Magnetic nanoparticles can be applied in various ways because of their structural and magnetic properties, such as MRI contrast agents, biodiagnostic devices, anti-counterfeiting inks, and device control devices inside speakers. In real life, where there are many devices that use electricity, they can be controlled by magnetism.
  • Types include iron oxide (Fe 2 O 3 , Fe 3 O 4 ), ferrite (a form in which one Fe in Fe 3 O 4 is replaced with another magnetism-related atom, CoFe 2 O 4 , MnFe 2 O 4 , etc.), alloys (magnetic atoms Oxidation problems caused by it, alloys with precious metals to increase conductivity and stability, FePt, CoPt, etc.).
  • these magnetic particles are not used in powder form, but are dispersed in liquid and used in various fields. When a magnetic field is applied when a solution (ferrofluid) is made, only the part with the magnetic field becomes sharp along the magnetic force line. It's because it shows a phenomenon that rises.
  • the use of a magnetic field in this way is very low in terms of cost and has the advantage of being able to be freely adjusted by changing the strength and direction of the magnetic field.
  • the magnetic particles since the magnetic particles must be well dispersed in the ferrofluid without being entangled with each other, it is necessary to use an appropriate surfactant or attach a hydrocarbon ring to the surface of the magnetic particles to ensure good dispersion, and the co-precipitation method is mainly used as a manufacturing method. .
  • surface modification refers to imparting physical, chemical, and biological characteristics to the surface of a material that were not originally present in the material. That is, it refers to changing the characteristics of only the surface within a range of several tens of nm to several ⁇ m from the surface of the material through an appropriate process.
  • polynucleotide refers to a polymer of nucleotides in which nucleotide units are formed in a long chain shape by covalent bonds, and refers to DNA or RNA strands of a certain length or longer.
  • oligopeptide is a nucleic acid or base sequence that includes both RNA and DNA, and includes cDNA, genomic DNA, and synthetic DNA.
  • the RNA includes mRNA, rRNA, and synthetic RNA.
  • oligonucleotide, nucleic acid, and base are used interchangeably in this specification.
  • nucleic acid or “gene” is a polymer composed of units called nucleotides and is a kind of biomolecule. Nucleic acids or genes can be roughly divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genetic information of nucleic acids is contained in the base sequence of nucleic acids, and is expressed in the form of proteins through biosynthesis. In addition, since the expressed proteins or enzymes play an important role in the growth, maintenance, and division of cells, different organisms may have different combinations of DNA and RNA sequences. In addition, even in the same living organism, gene expression patterns may vary depending on external environmental conditions, nutritional status, pathogen infection, developmental stage, tissue differentiation, and growth status.
  • the expression pattern of the gene that changes is expressed as RNA expression level, gene expression level, gene expression pattern, RNA transcription level, RNA transcription amount change, transcriptome profile, etc.
  • the first thing to be basically performed is to purify RNA or DNA from the sample.
  • purified DNA is used for functional genomics research, and the sequence of the purified DNA is secured through a specific site of DNA, for example, COI barcode, ITS barcode, ribosomal RNA sequence, etc., and 1:1 with open databases such as Genbank. Through comparison, it can be used for species identification.
  • purified DNA is used as a key basic material for various molecular biology basic research such as genetic manipulation.
  • Purified RNA can be synthesized into total DNA through reverse transcription, and the DNA synthesized in this way is also referred to as a cDNA library.
  • cDNA may be prepared by the 1st RT-PCR method using only specific primers, and a cDNA library may be constructed using oligo dT between 10 and 20 mer or random sequences between 5 and 20 mer.
  • PCR is performed with the synthesized cDNA library, it is said to be a two-step RT-PCR process.
  • the DNA synthesized through this process can exponentially amplify DNA of a specific base sequence through a polymerase chain reaction, which is a unique characteristic of taq polymerase, using taq polymerase and sequence-specific DNA oligos.
  • quantitative real-time PCR and gene expression relative quantification methods which track the amount of amplified DNA in real time to estimate the expression level of a gene, have been developed and are widely used worldwide.
  • amplified DNA can be compared with infected samples or non-infected samples to diagnose genetic diseases or infectious diseases such as bacteria and viruses.
  • DNA polymerase which is a key element of PCR
  • reverse transcriptase which synthesizes RNA into cDNA
  • inhibitors which are factors that inhibit enzymes. Since these inhibitors inhibit the activity of enzymes in a concentration-dependent manner, the inhibitors must be maintained at low concentrations in all processes for a successful PCR experiment.
  • step (a) synthesizing polyacrylic acid by adding acrylic acid monomer, ultrapure water, and potassium persulfate; (b) obtaining basic-treated magnetic nanoparticles by dispersing the magnetic material in a basic atmosphere; and (c) coating the surface by introducing the magnetic nanoparticles obtained in step (b) into a polyacrylic acid reactor.
  • polyacrylic acid includes 3 to 6.5 parts by weight of acrylic acid monomer, 93 to 96.5 parts by weight of ultrapure water, and 0.05 to 0.5 parts by weight of potassium persulfate.
  • ultrapure water has a resistivity of 16 to 20 M ⁇ .cm at 25°C.
  • the ultrapure water has a resistivity of 17 to 18 M ⁇ .cm at 25°C.
  • step (a) is synthesized at 85 to 95° C. at 350-450 rpm.
  • step (c) is stirred at 350-450 rpm at 65-75°C for 2-4 hours followed by stirring at 350-450 rpm at 18-28°C for 2-4 hours.
  • step (c) is surface modification with carboxyl groups to extract genes.
  • the magnetic material is one or more selected from the group consisting of Fe 2 O 3 , Fe 3 O 4 , CoFe 2 O 4 , MnFe 2 O 4 , FePt, and CoPt.
  • the basic atmosphere is one or more selected from the group consisting of KOH, NaOH, ammonia, or amine-based so as to have a pH of 5-7.
  • the magnetic nanoparticles have a particle diameter of 80 to 200 nm.
  • composition for gene extraction including surface-modified magnetic nanoparticles including the above-described surface-modified magnetic nanoparticles is provided.
  • a gene extraction kit including surface-modified magnetic nanoparticles including the above-described surface-modified magnetic nanoparticles is provided.
  • the method for producing surface-modified magnetic nanoparticles according to the present invention and the surface-modified magnetic nanoparticles produced therefrom can extract oligopeptides at a low price and with simple synthesis equipment without emission of environmental pollutants.
  • 1 is a schematic view of manufacturing and treating polyacrylic acid for activating and modifying the surface of magnetic particles.
  • Figure 2 shows the comparison results of nucleic acid extraction tests between magnetic nanoparticles synthesized through the present invention and commercially available products.
  • FIG. 3 is a schematic diagram of magnetic nanoparticles whose surface is modified through the present invention.
  • Lane 5 shows the result of confirming the presence or absence of virus infection through RT-PCR after extracting nucleic acids from cultured cells in which viruses were proliferated using the magnetic nanoparticles synthesized through the present invention.
  • Lanes 1-4 are Dengue virus
  • lanes 5-7 are Japanese encephalitis virus
  • lane 8 is ZIKA virus.
  • Lane1 Dengue virus 1 (NCCP Sales No. 41501)
  • Lane2 Dengue virus 2 (NCCP Sales No. 43248)
  • Lane3 Dengue virus 3 (NCCP Sales No. 43256)
  • Lane4 Dengue virus 4 (NCCP Sales No.
  • Lane5 Japanese encephalitis virus 1 (NCCP sale number 41302)
  • Lane6 Japanese encephalitis virus 3 (NCCP sale number 41304)
  • Lane7 Japanese encephalitis virus 5 (NCCP sale number 43279)
  • Lane8 ZIKA virus (NCCP sale number 43245).
  • step (a) synthesizing polyacrylic acid by adding acrylic acid monomer, ultrapure water, and potassium persulfate; (b) obtaining basic-treated magnetic nanoparticles by dispersing the magnetic material in a basic atmosphere; and (c) coating the surface by introducing the magnetic nanoparticles obtained in step (b) into a polyacrylic acid reactor.
  • the synthesized magnetic nanoparticles were formed as a polyacrylic acid-magnetic nanoparticle complex in which several magnetic nanoparticles formed a complex. All processes could be scaled up and applied according to the above ratio.
  • the surface-modified magnetic particles were mixed with a water-soluble polymeric acrylic polymer to finally obtain magnetic nanoparticles uniformly coated with carboxyl groups.
  • the mixture was washed three times with 500 ml of distilled water while stirring vigorously. After drying, the particles were weighed and dispensed into bottles.
  • bead stocks were prepared and used by diluting to 50 mg/ml (see FIG. 3).
  • the synthesized magnetic nanoparticles were formed as a polyacrylic acid-magnetic nanoparticle complex in which several magnetic nanoparticles formed a complex. All processes could be scaled up and applied according to the above ratio.
  • the surface-modified magnetic particles were mixed with a water-soluble polymeric acrylic polymer to finally obtain magnetic nanoparticles uniformly coated with carboxyl groups.
  • Magnetic nanoparticles were prepared according to Preparation Example 1 or 2 according to the present invention.
  • a commercially available product Bionner
  • Electrophoresis was performed by preparing the concentration of each sample at 5ul and 10ul.
  • Aedes albopictus were homogenized with 1ml of HB buffer (20M EDTA, 10mM Tris-HCl, 25.8mM Sodium citrate), and then centrifuged at 14000rpm for 10 minutes to remove the residue. 100ul was put into a 1.5ml tube. 100 ul of LNB buffer and 100 ul of CP buffer were sequentially mixed with 100 ul of the supernatant, followed by centrifugation at 14000 rpm for 10 minutes to obtain 250 ul of the supernatant from which the residue was removed. 150ul of isopropanol was added and mixed, and then 50ul of magnetic particles were added and mixed.
  • HB buffer 20M EDTA, 10mM Tris-HCl, 25.8mM Sodium citrate
  • Bioneer-COOH TS-1012-1, AccuBeadTM COOH Magnetic Beads, Size 1-5 ⁇ m
  • IVT-MNP4 magnetic nanoparticles according to Preparation Example 1 of the present invention.
  • the mixture mixed with magnetic beads was attached to the wall of the tube with a magnet to collect the magnetic particles on the wall of the tube, and the remaining solution was removed by suction with a micropipette. At this time, care was taken not to mix the magnetic beads.
  • the magnet was separated, and 600ul of WA buffer was added and mixed by vortex to completely wash the beads. Again, a magnet was attached to the wall of the tube to collect magnetic particles, and the remaining solution was sucked up and removed with a micropipette.
  • the magnet As a step for separating the magnet, the magnet was separated, and 500ul of wash buffer (70% ethanol) was added and mixed with a vortex to completely wash the beads. Again, a magnet was attached to the wall of the tube to collect magnetic particles, and the remaining solution was sucked up and removed with a micropipette. The magnet separation step described above was performed one more time.
  • wash buffer 70% ethanol
  • the lid was opened and dried at 60 ° C for 2 to 3 minutes. 50ul of RNase free water was added and the beads were completely mixed with water. After incubation at 60 ° C. for 1 minute, the magnet was collected on the wall using a magnet, and the remaining solution was transferred to a new tube to complete nucleic acid extraction. 10x DNA loading dye was added to the extracted nucleic acid, and electrophoresis was performed under the condition of 1% TBE gel, 100v, 30 minutes.
  • lane 1 was 5ul electrophoresis after nucleic acid extraction with magnetic beads synthesized by this method
  • lane 2 was 5ul electrophoresis after nucleic acid extraction with Bioneer magnetic beads
  • lane 3 was 10ul electrophoresis after nucleic acid extraction with magnetic beads synthesized by this method
  • lane 4 was 10ul electrophoresis after nucleic acid extraction with Bioneer magnetic beads.
  • nucleic acids could be extracted at low cost and with simple synthesis equipment without emission of environmental pollutants.
  • the surface-modified magnetic nanoparticles according to the present invention are expected to be widely used in bio, medicine, and health fields because oligopeptides can be extracted at a low price and with simple synthesis equipment without emission of environmental pollutants.

Abstract

La présente invention concerne un procédé de préparation de nanoparticules magnétiques à surface modifiée et des nanoparticules magnétiques à surface modifiée ainsi préparées, le procédé comprenant les étapes consistant : (a) à synthétiser de l'acide polyacrylique par addition d'un monomère d'acide acrylique, d'eau ultrapure et de persulfate de potassium ; (b) à disperser un agent magnétique dans une atmosphère basique pour obtenir des nanoparticules magnétiques essentiellement traitées ; et (c) à ajouter les nanoparticules magnétiques obtenues à l'étape (b) dans un réservoir de réaction d'acide polyacrylique pour revêtir leurs surfaces.
PCT/KR2022/008331 2021-06-15 2022-06-14 Procédé de préparation de nanoparticules magnétiques à surface modifiée et nanoparticules magnétiques à surface modifiée ainsi préparées WO2022265330A1 (fr)

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CN101612541A (zh) * 2009-07-17 2009-12-30 江南大学 聚丙烯酸包覆四氧化三铁磁性纳米粒子的制备及其应用
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