CN112877274A - Extracellular vesicle magnetic imprinting material and preparation method and application thereof - Google Patents
Extracellular vesicle magnetic imprinting material and preparation method and application thereof Download PDFInfo
- Publication number
- CN112877274A CN112877274A CN202110123602.6A CN202110123602A CN112877274A CN 112877274 A CN112877274 A CN 112877274A CN 202110123602 A CN202110123602 A CN 202110123602A CN 112877274 A CN112877274 A CN 112877274A
- Authority
- CN
- China
- Prior art keywords
- magnetic
- magnetic particles
- extracellular
- extracellular vesicle
- imprinting material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000000463 material Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 239000006249 magnetic particle Substances 0.000 claims abstract description 134
- 229920000642 polymer Polymers 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 4
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 77
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 52
- 229910052681 coesite Inorganic materials 0.000 claims description 50
- 229910052906 cristobalite Inorganic materials 0.000 claims description 50
- 229910052682 stishovite Inorganic materials 0.000 claims description 50
- 229910052905 tridymite Inorganic materials 0.000 claims description 50
- 108091023037 Aptamer Proteins 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims description 12
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims description 9
- 150000001299 aldehydes Chemical class 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 238000006116 polymerization reaction Methods 0.000 claims description 7
- 238000006557 surface reaction Methods 0.000 claims description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 6
- 239000011324 bead Substances 0.000 claims description 6
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 229910000077 silane Inorganic materials 0.000 claims description 4
- 238000002444 silanisation Methods 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- 230000000379 polymerizing effect Effects 0.000 claims description 2
- 210000001124 body fluid Anatomy 0.000 abstract description 2
- 239000010839 body fluid Substances 0.000 abstract description 2
- 239000006143 cell culture medium Substances 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 239000011159 matrix material Substances 0.000 abstract 2
- 238000004140 cleaning Methods 0.000 abstract 1
- 125000000524 functional group Chemical group 0.000 abstract 1
- 238000004064 recycling Methods 0.000 abstract 1
- 238000005406 washing Methods 0.000 description 23
- 238000001035 drying Methods 0.000 description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 16
- 239000007864 aqueous solution Substances 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000002105 nanoparticle Substances 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 239000003480 eluent Substances 0.000 description 10
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000002604 ultrasonography Methods 0.000 description 8
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 210000002381 plasma Anatomy 0.000 description 7
- 238000003756 stirring Methods 0.000 description 7
- HPEPIADELDNCED-UHFFFAOYSA-N triethoxysilylmethanol Chemical compound CCO[Si](CO)(OCC)OCC HPEPIADELDNCED-UHFFFAOYSA-N 0.000 description 7
- 238000007885 magnetic separation Methods 0.000 description 6
- 238000001000 micrograph Methods 0.000 description 6
- 230000009870 specific binding Effects 0.000 description 6
- 102100025222 CD63 antigen Human genes 0.000 description 5
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- NBXZNTLFQLUFES-UHFFFAOYSA-N triethoxy(propyl)silane Chemical compound CCC[Si](OCC)(OCC)OCC NBXZNTLFQLUFES-UHFFFAOYSA-N 0.000 description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 4
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000006884 silylation reaction Methods 0.000 description 4
- 239000001632 sodium acetate Substances 0.000 description 4
- 235000017281 sodium acetate Nutrition 0.000 description 4
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 102100037904 CD9 antigen Human genes 0.000 description 3
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- CPLASELWOOUNGW-UHFFFAOYSA-N benzyl(triethoxy)silane Chemical compound CCO[Si](OCC)(OCC)CC1=CC=CC=C1 CPLASELWOOUNGW-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000001878 scanning electron micrograph Methods 0.000 description 3
- 238000005199 ultracentrifugation Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102100027221 CD81 antigen Human genes 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 2
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 2
- 101000914479 Homo sapiens CD81 antigen Proteins 0.000 description 2
- 101000606465 Homo sapiens Inactive tyrosine-protein kinase 7 Proteins 0.000 description 2
- 102100039813 Inactive tyrosine-protein kinase 7 Human genes 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102100021768 Phosphoserine aminotransferase Human genes 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 238000005576 amination reaction Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 1
- 101710165845 CD81 protein Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000003012 bilayer membrane Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000002122 magnetic nanoparticle Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 150000001282 organosilanes Chemical class 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- BONNPLTURUUHRQ-UHFFFAOYSA-K trisodium;n'-(3-trimethoxysilylpropyl)ethane-1,2-diamine;triacetate Chemical compound [Na+].[Na+].[Na+].CC([O-])=O.CC([O-])=O.CC([O-])=O.CO[Si](OC)(OC)CCCNCCN BONNPLTURUUHRQ-UHFFFAOYSA-K 0.000 description 1
Images
Classifications
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G49/00—Compounds of iron
- C01G49/02—Oxides; Hydroxides
- C01G49/08—Ferroso-ferric oxide (Fe3O4)
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/60—Particles characterised by their size
- C01P2004/62—Submicrometer sized, i.e. from 0.1-1 micrometer
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/80—Particles consisting of a mixture of two or more inorganic phases
- C01P2004/82—Particles consisting of a mixture of two or more inorganic phases two phases having the same anion, e.g. both oxidic phases
- C01P2004/84—Particles consisting of a mixture of two or more inorganic phases two phases having the same anion, e.g. both oxidic phases one phase coated with the other
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses an extracellular vesicle magnetic imprinting material and a preparation method and application thereof, wherein the magnetic imprinting material comprises a magnetic particle matrix, and extracellular vesicle imprinting is arranged on the surface of the magnetic particle matrix; the preparation method of the magnetic imprinting material comprises the following steps: (1) preparing magnetic particles and modifying surface functional groups; (2) connecting the template extracellular vesicles to the surfaces of the magnetic particles; (3) growing a polymer recognition layer on the surface of the magnetic particles along the extracellular vesicles; (4) ultrasonically cleaning to remove the template vesicles to obtain a magnetic imprinting material with extracellular vesicle marks on the surface; the magnetic imprinting material can be applied to in vitro separation of extracellular vesicles. The magnetic imprinting material can efficiently capture and separate extracellular vesicles from cell culture media, plasma and other body fluids, has high capture efficiency, simple preparation process, high yield, low cost, convenient use, easy storage, no need of large-scale instruments for assistance, recycling and reutilization, and can further reduce the cost.
Description
Technical Field
The invention relates to a blotting material, a preparation method and application thereof, and more particularly relates to an extracellular vesicle magnetic blotting material, and a preparation method and application thereof.
Background
Extracellular Vesicles (EVs) are encapsulated by a phospholipid bilayer membrane, have a diameter of 30-200nm, and are secreted by various cells including tumor cells, endothelial cells, immune cells, platelets and the like. In recent years, more and more studies have shown that extracellular vesicles contain abundant DNA, RNA, and proteins as a means for remote communication between cells in vivo. Especially for the extracellular vesicles secreted by tumor cells, which contain various cancer-specific proteins and nucleotides (mRNA, microRNA, and DNA fragments), they can be used as circulating biomarkers for cancer diagnosis, prediction, and therapy monitoring. However, there is currently no standard efficient method to obtain high purity, high yield extracellular vesicles. The existing acquisition methods comprise an ultracentrifugation method, an ultrafiltration and size exclusion chromatography method, a polyethylene glycol precipitation method and an immunomagnetic bead separation method, wherein the ultracentrifugation method is the most common extracellular vesicle separation method at present, can process a larger amount of samples and obtain relatively pure extracellular vesicles, but has longer time consumption, higher requirement on instruments and lower recovery rate, so that the application range of the ultracentrifugation method is limited; ultrafiltration and size exclusion chromatography process sample volumes too large to be suitable for the collection of extracellular vesicles in small volume samples (serum, plasma); the extracellular vesicle obtained by the treatment of the polyethylene glycol precipitation method has a plurality of impurities and is not single in component; although the immunomagnetic bead separation method can separate relatively pure extracellular vesicles, the antibody is expensive and difficult to store, which greatly limits the wide application of the immunomagnetic bead separation method.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a preparation method of an extracellular vesicle magnetic imprinting material, which can efficiently capture and separate extracellular vesicles, has simple preparation process, easy storage and low cost; the invention also aims to provide application of the extracellular vesicle magnetic imprinting material.
The technical scheme is as follows: the extracellular vesicle magnetic imprinting material comprises magnetic particles, wherein extracellular vesicle imprints are arranged on the surfaces of the magnetic particles.
Wherein the magnetic particles are Fe3O4@SiO2The particle size of the magnetic particles is 450-600 nm.
The preparation method of the extracellular vesicle magnetic imprinting material comprises the following steps:
(1) preparing magnetic particles and carrying out surface functionalization;
(2) connecting the template extracellular vesicles to the surfaces of the magnetic particles;
(3) growing a polymer recognition layer on the surface of the magnetic particles along extracellular vesicles through siloxane polymerization reaction by utilizing molecular imprinting reaction;
(4) and removing the template extracellular vesicles on the surfaces of the magnetic particles by ultrasonic cleaning to obtain the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting.
When the magnetic particles are subjected to surface functionalization in the step 1, the magnetic particles are dispersed in water to be a solution of 1-2 mg/mL, and a functionalized silane reagent with the volume fraction of 1-5% is added to obtain a mixed solution, so that the mixed solution reacts at room temperature, wherein the functionalized silane reagent is a silane reagent containing amino groups or a silylation reagent containing carboxyl groups.
Wherein, the step 2 is to connect the template extracellular vesicles to the surface of the magnetic particles through covalent bonds, and comprises the following steps:
(21) activating amino on the surface of the magnetic beads or carboxyl on the surface of the magnetic particles to obtain activated magnetic particles;
(22) and (3) ultracentrifuging and purifying to obtain template extracellular vesicles, and reacting the extracellular vesicles with the activated magnetic particles in a phosphate buffer solution to obtain a magnetic particle compound with the extracellular vesicles connected to the surface. Wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is 0.5-5 mug: 1 mg.
Wherein, the step 2 of connecting the template extracellular vesicles to the surfaces of the magnetic particles through non-covalent bonds comprises the following steps:
(21) activating amino groups on the surfaces of the magnetic particles by using glutaraldehyde to obtain aldehyde-based magnetic particles;
(22) adding an aminated aptamer or an antibody into the aldehyde magnetic particles to react at room temperature to obtain aptamer or antibody modified magnetic particles;
(23) mixing ultracentrifugally purified extracellular vesicles with magnetic particles modified by aptamers or antibodies in a buffer solution, and obtaining a magnetic particle compound with extracellular vesicles connected to the surface through antigen-antibody combination or aptamer-protein specific combination; wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is 0.5-5 mug: 1mg, the antibody is CD63 antibody, CD81 antibody or CD9 antibody, and the aptamer is an aptamer corresponding to CD63, CD81, CD9, EpCAM, PTK7, PSA, LZH8 and Lib.
Wherein, step 3 comprises the following steps:
(31) dispersing the magnetic particle compound into a buffer solution, adding tetraethoxysilane, and reacting at room temperature, wherein the mass ratio of the magnetic particle compound to the tetraethoxysilane is 1 mg: 1-10 μ g;
(32) adding a silanization reagent into the reaction product, and polymerizing and growing on the surface of the magnetic particle compound along the surface of the extracellular vesicle through hydrolysis reaction to form a recognition layer, wherein the hydrolysis reaction temperature is 10-25 ℃, and the mass ratio of the magnetic particle compound to the silanization reagent is 1 mg: 1-10 mu g of silylation agent, 0.5-10% of volume fraction of silylation agent and 8-72 h of time required for forming the identification layer, wherein the silylation agent is at least one of 3-aminopropyl triethoxysilane, hydroxymethyl triethoxysilane, propyl triethoxysilane or benzyl triethoxysilane.
Wherein the power of removing the template extracellular vesicles by ultrasonic cleaning in the step 4 is 25-45 Hz, and the ultrasonic time is 2-10 min.
The magnetic imprinting material of the extracellular vesicles is applied to in-vitro separation of the extracellular vesicles.
The preparation principle is as follows: the invention prepares the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting by using magnetic particles as a carrier, organosilane as a construction module and extracellular vesicles as a template, and utilizes the characteristic that a silicone polymerization hydrolysis can continuously grow an organosilicon layer during preparation to ensure that a silicone monomer mixture continuously grows a layer of silicon dioxide recognition layer on the surface of the magnetic particles along the extracellular vesicles of the template, removes the template extracellular vesicle template through ultrasonic cleaning, and leaves a cavity with specificity recognition and capture of the extracellular vesicles on the surface of the magnetic particles And (4) collecting and analyzing.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: 1. the magnetic imprinting material of the extracellular vesicles, provided by the invention, can efficiently capture and separate the extracellular vesicles from cell culture media, plasma and other body fluids, and has high capture efficiency; 2. the preparation process is simple, the yield is high, and the cost is low; 3. the magnetic imprinting material for the extracellular vesicles, provided by the invention, can be applied to the in vitro separation of the extracellular vesicles, is convenient to use and easy to store, and does not need the assistance of a large instrument; 4. can be recycled and reused, and further reduces the cost.
Drawings
FIG. 1 is a schematic diagram of a process for preparing an extracellular vesicle magnetic imprinting material;
FIG. 2 is Fe3O4@SiO2Scanning electron microscope images of nanoparticles;
FIG. 3 is EVs-Fe3O4@SiO2Scanning electron microscope images of the compound;
FIG. 4 is a scanning electron micrograph of magnetic particles grown with an extracellular vesicle recognition layer;
FIG. 5 is a scanning electron microscope image of a magnetic imprinting material with extracellular vesicle imprinting on the surface;
FIG. 6 is a scanning electron micrograph of a magnetic imprinting material with extracellular vesicles captured;
FIG. 7 shows the capture efficiency of extracellular vesicle magnetic colloids after 5 cycles.
Detailed Description
The process for preparing the extracellular vesicle magnetic imprinting material is shown in fig. 1, wherein a template extracellular vesicle is connected to the surface of a magnetic particle through an amino group, a polymer recognition layer grows on the surface of the magnetic particle along the extracellular vesicle through siloxane polymerization, and finally the template extracellular vesicle on the surface of the magnetic particle is removed through ultrasonic cleaning to obtain the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting, and the specific process is as follows:
(1) preparation of Fe3O4@SiO2Preparation and surface functionalization of magnetic particles:
dissolving 2.70g of ferric chloride hexahydrate and 7.20g of sodium acetate in 100mL of ethylene glycol, uniformly stirring, and reacting in an oven at 240 ℃ for 12 hours to form Fe3O4A nanoparticle; centrifugal washing of Fe3O4Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of tetraethoxysilane and 0.10g of Fe3O4Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring for reaction for 12 hours, washing with water, and drying to obtain Fe3O4@SiO2The scanning electron microscope of the magnetic particles is shown in FIG. 2, the left side is a larger-range image, the right side is a partial enlarged image, and the particle size is 550 nm;
the prepared Fe3O4@SiO2The magnetic particles are uniformly dispersed in the aqueous solution to obtain Fe with the concentration of 1mg/mL3O4@SiO2An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyltriethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe3O4@SiO2Magnetic particles;
(2) amination of Fe3O4@SiO2Magnetic particle covalent attachment to extracellular vesicles
30mg of aminated Fe3O4@SiO2Uniformly dispersing the magnetic particles in a glutaraldehyde solution containing 30mL of 1% by volume fraction, reacting for 0.5 hour at room temperature, and washing twice to obtain Fe with surface aldehyde group3O4@SiO2Magnetic particles;
taking 25 mu g of ultracentrifuge purified extracellular vesicles, and mixing the extracellular vesicles with 10mg of surface activated Fe3O4@SiO2The magnetic particles are put into 5mL phosphoric acid buffer solution and react for 2 to 4 hours at room temperature to obtain the extracellular vesicles with the surface connected withMagnetic particles EVs-Fe of3O4@SiO2Obtained EVs-Fe3O4@SiO2The scanning electron micrograph of the compound is shown in FIG. 3, the left side is a larger-range sketch, and the right side is a partial enlarged view;
(3) at EVs-Fe3O4@SiO2Growing a polymer recognition layer on the composite
24mg of EVs-Fe3O4@SiO2Dispersing into 5mL phosphoric acid buffer solution, adding 32 μ L ethyl orthosilicate, reacting at room temperature for 2 hours, then adding 24 μ L3-aminopropyltriethoxysilane APTES, and continuing to react at 10 deg.C for 72 hours; after the reaction is finished, removing the supernatant through magnetic separation, washing the supernatant with deionized water for three times to obtain the magnetic particles with the extracellular vesicle recognition layer, wherein a scanning electron microscope image of the magnetic particles is shown in fig. 4: the left side is a larger range diagram, and the right side is a partial enlarged diagram;
(4) template extracellular vesicles with removal of magnetic particles growing extracellular vesicle recognition layer
Placing the magnetic particles with the extracellular vesicle recognition layer in an eluent, performing water bath ultrasound for 30min to remove the template extracellular vesicles, wherein the eluent is an aqueous solution containing 0.1mol/L HCl + 0.01% Txiton X-100, and the ultrasound power is 45 HZ; and (3) continuing to perform ultrasonic treatment on the product for 20min, washing the product by using deionized water, and drying the product to obtain the magnetic imprinting material with extracellular vesicle marks on the surface, wherein a scanning electron microscope image of the magnetic imprinting material is shown in fig. 5: the left side is a larger scale drawing and the right side is a partial enlarged drawing.
Example 2
As shown in fig. 1, a carboxylated magnetic nanoparticle is used to connect a template extracellular vesicle to the surface of a magnetic particle, a molecular imprinting reaction is used to grow a polymer recognition layer on the surface of the magnetic particle along the extracellular vesicle through siloxane polymerization, and finally the template extracellular vesicle on the surface of the magnetic particle is removed by ultrasonic cleaning, so that the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting is obtained through the following specific processes:
(1) preparation of Fe3O4@SiO2Magnetic propertyPreparation of the particles and surface carboxylation:
dissolving 2.70g of ferric chloride hexahydrate and 7.20g of sodium acetate in 100mL of ethylene glycol, uniformly stirring, and reacting in an oven at 240 ℃ for 12 hours to form Fe3O4A nanoparticle; centrifugal washing of Fe3O4Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of tetraethoxysilane and 0.10g of Fe3O4Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring for reaction for 12 hours, washing with water, and drying to obtain Fe3O4@SiO2Magnetic particles;
the prepared Fe3O4@SiO2The magnetic particles are uniformly dispersed in the aqueous solution to obtain Fe with the concentration of 1mg/mL3O4@SiO2An aqueous solution of magnetic particles; adding 30 mu L of 35 volume percent N- (trimethoxysilylpropyl) ethylenediamine triacetic acid sodium salt into 30mL of the solution, reacting for 30min at room temperature, washing with water, and drying to obtain carboxylated Fe3O4@SiO2Magnetic particles;
(2) carboxylated Fe3O4@SiO2The magnetic particles are covalently linked to the extracellular vesicles by NHS/EDC activated carboxylated magnetic beads
Mixing Fe3O4@SiO2The magnetic particles were homogeneously dispersed in 2- (N-morpholino) ethanesulfonic acid buffer solution at pH 6 to give a concentration of 1mg/mL Fe3O4@SiO210mL of magnetic particle solution is added with 100mgNHS and 200mgEDC respectively to be activated for 0.5 hour at the middle room temperature and washed twice to obtain surface activated Fe3O4@SiO2Magnetic particles.
Taking 25 mu g of ultracentrifuge purified extracellular vesicles, and mixing the extracellular vesicles with 10mg of surface activated Fe3O4@SiO2The magnetic particles are placed in 5mL phosphoric acid buffer solution and react for 4 hours at room temperature to obtain the magnetic particles EVs-Fe with the surface connected with extracellular vesicles3O4@SiO2；
(3) At EVs-Fe3O4@SiO2Composite materialOn which a polymer recognition layer is grown
24mg of EVs-Fe3O4@SiO2Dispersing into 5mL phosphoric acid buffer solution, adding 32 μ L ethyl orthosilicate, reacting at room temperature for 2 hours, then adding 3-aminopropyltriethoxysilane APTES and 12 μ L hydroxymethyl triethoxysilane HMTEOS, and continuing to react at 10 deg.C for 50 hours; after the reaction is finished, removing supernatant through magnetic separation, and washing the supernatant for three times by using deionized water to obtain magnetic particles growing with an extracellular vesicle recognition layer;
(4) template extracellular vesicles with removal of magnetic particles growing extracellular vesicle recognition layer
Placing the magnetic particles with the extracellular vesicle recognition layer in an eluent, performing water bath ultrasound for 30min to remove the template extracellular vesicles, wherein the eluent is an aqueous solution containing 0.1mol/L HCl + 0.01% Txiton X-100, and the ultrasound power is 45 HZ; and continuing to perform ultrasonic treatment on the product for 20min, washing the product by using deionized water, and drying the product to obtain the magnetic imprinting material with extracellular vesicle marks on the surface.
Example 3
As shown in fig. 1, the process for preparing the extracellular vesicle magnetic imprinting material is that by utilizing aptamer-vesicle specific binding, a template extracellular vesicle is connected to the surface of a magnetic particle, a polymer recognition layer grows on the surface of the magnetic particle along the extracellular vesicle through siloxane polymerization, and finally the template extracellular vesicle on the surface of the magnetic particle is removed through ultrasonic cleaning, so as to obtain the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting, and the specific process is as follows:
(1) preparation of Fe3O4@SiO2Preparation and surface functionalization of magnetic particles:
dissolving 2.70g of ferric chloride hexahydrate and 7.20g of sodium acetate in 100mL of ethylene glycol, uniformly stirring, and reacting in an oven at 240 ℃ for 12 hours to form Fe3O4A nanoparticle; centrifugal washing of Fe3O4Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of tetraethoxysilane and 0.10g of Fe3O4The nanoparticles were added to a mixture of 80mL ethanol, 20mL water and 1mL ammonia, and stirredReacting for 12h, washing and drying to obtain Fe3O4@SiO2Magnetic particles;
the prepared Fe3O4@SiO2The magnetic particles are uniformly dispersed in the aqueous solution to obtain Fe with the concentration of 1mg/mL3O4@SiO2An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyltriethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe3O4@SiO2Magnetic particles;
(2) magnetic particles are linked to extracellular vesicles by aptamer-target specific binding
30mg of aminated Fe3O4@SiO2Uniformly dispersing the magnetic particles in a glutaraldehyde solution containing 30mL of 1% by volume fraction, reacting for 0.5 hour at room temperature, and washing twice to obtain surface-activated Fe3O4@SiO2Magnetic particles, i.e. aldehydized magnetic particles;
adding 100 mu L of amination CD63 aptamer with the concentration of 10 mu M into 20mL of aldehyde group magnetic particle solution with the concentration of 1mg/mL, and connecting the amino group CD63 aptamer on the surface of the magnetic particle through aldehyde-amine condensation reaction;
Fe3O4@SiO2-aptamer for attaching EVs to the surface of magnetic particles by specific binding to extracellular vesicles, yielding EVs-Fe3O4@SiO2A complex;
the sequence of the aptamer was 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3'.
(3) At EVs-Fe3O4@SiO2Growing a polymer recognition layer on the composite
24mg of EVs-Fe3O4@SiO2Dispersing into 5mL phosphoric acid buffer solution, adding 32 mu L ethyl orthosilicate, reacting for 2 hours at room temperature, then adding 12 mu L3-aminopropyltriethoxysilane APTES, 6 mu L hydroxymethyl triethoxysilane HMTEOS and 12 mu L propyltriethoxysilane PTES, and continuing to react for 40 hours at 10 ℃; after the reaction, the supernatant was removed by magnetic separation and usedWashing with ionized water for three times to obtain magnetic particles growing with extracellular vesicle recognition layers;
(4) template extracellular vesicles with removal of magnetic particles growing extracellular vesicle recognition layer
Placing the magnetic particles with the extracellular vesicle recognition layer in an eluent, performing water bath ultrasound for 30min to remove the template extracellular vesicles, wherein the eluent is an aqueous solution containing 0.1mol/L HCl + 0.01% Txiton X-100, and the ultrasound power is 45 HZ; and continuing to perform ultrasonic treatment on the product for 20min, washing the product by using deionized water, and drying the product to obtain the magnetic imprinting material with extracellular vesicle marks on the surface.
Example 4
This example is different from example 3 in that: the aptamer used in step 3 was an EpCAM protein aptamer with an aptamer sequence of 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3'.
Example 5
This example is different from example 3 in that: the aptamer used in step 3 was a CA125 protein aptamer with the aptamer sequence 5'-TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-3'.
Example 6
This example is different from example 3 in that: the aptamer used in the step 3 is a HER2 protein aptamer, and the sequence of the aptamer is 5'-GGG CCG TCG AAC ACG AGC ATG GTG CGT GGA CCT AGG ATG ACC TGA GTA CTG TCC-3'.
Example 7
This example is different from example 3 in that: the aptamer used in step 3 was PTK7 protein aptamer with an aptamer sequence of 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3'.
Example 8
This example is different from example 3 in that: the aptamer used in step 3 was a PSA protein aptamer with the sequence 5'-AAT TAA AGC TCG CCA TCA AAT AGC-3'.
Example 9
This example is different from example 3 in that: the aptamer used in the step 3 is LZH8 protein aptamer, and the sequence of the aptamer is 5'-ATC CAG AGT GAC GCA GCA TAT TAG TAC GGC TTA ACC CPC ATG GTG GAC ACG GTG GCT TAG T-3'.
Example 10
This example is different from example 3 in that: the aptamer used in step 3 was Lib protein aptamer with the sequence 5'-CAC CCC ACC TAA AAA AAA AAA CAC TAA TGC TA-3'.
Example 11
As shown in fig. 1, a template extracellular vesicle is connected to the surface of a magnetic particle by utilizing antigen-antibody specific binding, a polymer recognition layer is grown on the surface of the magnetic particle along the extracellular vesicle by utilizing a molecular imprinting reaction through siloxane polymerization, and finally the template extracellular vesicle on the surface of the magnetic particle is removed by ultrasonic cleaning to obtain the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting, which comprises the following specific processes:
(1) preparation of Fe3O4@SiO2Preparation and surface functionalization of magnetic particles:
dissolving 2.70g of ferric chloride hexahydrate and 7.20g of sodium acetate in 100mL of ethylene glycol, uniformly stirring, and reacting in an oven at 240 ℃ for 12 hours to form Fe3O4A nanoparticle; centrifugal washing of Fe3O4Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of tetraethoxysilane and 0.10g of Fe3O4Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring for reaction for 12 hours, washing with water, and drying to obtain Fe3O4@SiO2Magnetic particles;
the prepared Fe3O4@SiO2The magnetic particles are uniformly dispersed in the aqueous solution to obtain Fe with the concentration of 1mg/mL3O4@SiO2An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyltriethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe3O4@SiO2Magnetic particles；
(2) Magnetic particles are linked to extracellular vesicles by antigen-antibody specific binding
30mg of aminated Fe3O4@SiO2Uniformly dispersing the magnetic particles in a glutaraldehyde solution containing 30mL of 1% by volume fraction, reacting for 0.5 hour at room temperature, and washing twice to obtain surface-activated Fe3O4@SiO2Magnetic particles, i.e. aldehydized magnetic particles;
adding 100 mu L of CD63 antibody with the concentration of 1mg/mL into 10mL of aldehyde-based magnetic particles with the concentration of 1mg/mL, and connecting the aldehyde-based magnetic particles on the surfaces of the magnetic particles through an aldehyde-amine condensation reaction;
connecting EVs to the surface of the magnetic particle through antigen-antibody specific binding to obtain EVs-Fe3O4@SiO2A complex;
(3) at EVs-Fe3O4@SiO2Growing a polymer recognition layer on the composite
24mg of EVs-Fe3O4@SiO2Dispersing into 5mL phosphoric acid buffer solution, adding 32 μ L ethyl orthosilicate, reacting at room temperature for 2 hours, then adding 12 μ L3-Aminopropyltriethoxysilane (APTES), 18 μ L hydroxymethyl triethoxysilane (HMTEOS), 12 μ L Propyltriethoxysilane (PTES) and 12 μ L Benzyltriethoxysilane (BTES), and continuing the reaction at 10 deg.C for 20 hours; after the reaction is finished, removing supernatant through magnetic separation, and washing the supernatant for three times by using deionized water to obtain magnetic particles growing with an extracellular vesicle recognition layer;
(4) template extracellular vesicles with removal of magnetic particles growing extracellular vesicle recognition layer
Placing the magnetic particles with the extracellular vesicle recognition layer in an eluent, performing water bath ultrasound for 30min to remove the template extracellular vesicles, wherein the eluent is an aqueous solution containing 0.1mol/L HCl + 0.01% Txiton X-100, and the ultrasound power is 45 HZ; and continuing to perform ultrasonic treatment on the product for 20min, washing the product by using deionized water, and drying the product to obtain the magnetic imprinting material with extracellular vesicle marks on the surface.
Example 12
This example is different from example 11 in that: the antibody used in step 3 was a CD9 protein antibody.
Example 13
This example is different from example 11 in that: the antibody used in step 3 was a CD81 protein antibody.
Example 14
The capture capacity of the extracellular vesicle magnetic imprinting material is verified by the following method:
diluting healthy human plasma by 1000 times by using PBS, uniformly mixing 1mL of the plasma with 2mg of extracellular vesicle magnetic imprinting material, oscillating and reacting for 20min at room temperature, removing supernatant through magnetic separation after the reaction is finished, washing twice by using phosphoric acid buffer solution, finally placing the precipitate in phosphoric acid buffer solution containing 2.5% of glutaraldehyde, incubating overnight at 4 ℃ for fixation, and then performing gradient dehydration and drying by using 30%, 50%, 70%, 80%, 90%, 95% and 100% of ethanol. The scanning electron microscope image of the final product is shown in fig. 6, and the mark on the surface of the magnetic imprinting material disappears and many extracellular vesicle particles appear, which indicates that the extracellular vesicle magnetic imprinting material can rapidly capture and collect extracellular vesicles from blood plasma.
Example 15
The ability of the extracellular vesicle magnetic imprinting material for repeatedly capturing extracellular vesicles is verified, and the method comprises the following steps:
diluting healthy human plasma by 1000 times by using PBS, uniformly mixing 5mL of the plasma with 10mg of extracellular vesicle magnetic imprinting material, oscillating and reacting at room temperature for 20min, and collecting precipitates by magnetic separation after the reaction is finished. And measuring the concentration of extracellular vesicles in the solution before and after capture by using an enzyme-linked immunosorbent assay, and calculating capture efficiency. Then, the obtained precipitate is washed by eluent to remove extracellular vesicles, and then the precipitate is washed for three times by ethanol and water respectively and can be used for the next round of capture, wherein the eluent is aqueous solution containing 0.1mol/L HCl + 0.01% Txiton X-100. The whole process is carried out for 5 times, and the experimental result shows that the magnetic imprinting material still maintains high capture efficiency after being repeatedly used for 5 times, and the calculated capture efficiency of 5 times of circulation is shown in figure 7.
Sequence listing
<110> Nanjing university
<120> extracellular vesicle magnetic imprinting material, preparation method and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caccccacct cgctcccgtg acactaatgc ta 32
<210> 2
<211> 48
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cactacagag gttgcgtctg tcccacgttg tcatgggggg ttggcctg 48
<210> 3
<211> 40
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tatcaattac ttaccctagt ggtgtgatgt cgtatggatg 40
<210> 4
<211> 54
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gggccgtcga acacgagcat ggtgcgtgga cctaggatga cctgagtact gtcc 54
<210> 5
<211> 41
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atctaactgc tgcgccgccg ggaaaatact gtacggttag a 41
<210> 6
<211> 24
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aattaaagct cgccatcaaa tagc 24
<210> 7
<211> 60
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atccagagtg acgcagcata ttagtacggc ttaaccccat ggtggacacg gtggcttagt 60
<210> 8
<211> 32
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
caccccacct aaaaaaaaaa acactaatgc ta 32
Claims (9)
1. An extracellular vesicle magnetic imprinting material, which is characterized by comprising magnetic particles, wherein the surfaces of the magnetic particles are provided with extracellular vesicle imprints.
2. The extracellular vesicular magnetic imprinting material of claim 1, wherein the magnetic particles are Fe3O4@SiO2Magnetic particles, the particle size of the magnetic particles is 450-600 nm.
3. A preparation method of the extracellular vesicle magnetic imprinting material of claim 1, comprising the following steps:
(1) preparing magnetic particles and carrying out surface functionalization;
(2) connecting the template extracellular vesicles to the surfaces of the magnetic particles;
(3) growing a polymer recognition layer on the surface of the magnetic particles along the extracellular vesicles through siloxane polymerization reaction;
(4) and removing the template extracellular vesicles on the surfaces of the magnetic particles by ultrasonic cleaning to obtain the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting.
4. The method for preparing an extracellular vesicle magnetic imprinting material according to claim 3, wherein in the step 1, the magnetic particles are first dispersed in water to be a solution of 1-2 mg/mL when being subjected to surface functionalization, and then a functionalized silane reagent with a volume fraction of 1% -5% is added to obtain a mixed solution, and the mixed solution is reacted at room temperature.
5. The method for preparing the extracellular vesicle magnetic imprinting material according to claim 3, wherein the step 2 of attaching the template extracellular vesicle to the surface of the magnetic particle through a covalent bond comprises the following steps:
(21) activating amino groups on the surfaces of the magnetic beads or carboxyl groups on the surfaces of the magnetic particles to obtain activated magnetic particles;
(22) and (3) ultracentrifuging and purifying to obtain extracellular vesicles, reacting the extracellular vesicles with the activated magnetic particles in a buffer solution to obtain a magnetic particle compound with extracellular vesicles connected to the surface, wherein the mass ratio of the extracellular vesicles to the magnetic particles is 0.5-5 [ mu ] g: 1 mg.
6. The method for preparing the extracellular vesicle magnetic imprinting material according to claim 3, wherein the step 2 of attaching the template extracellular vesicle to the surface of the magnetic particle through non-covalent bond comprises the following steps:
(21) activating amino groups on the surfaces of the magnetic particles by using glutaraldehyde to obtain aldehyde-based magnetic particles;
(22) adding an aminated aptamer or an antibody into the aldehyde magnetic particles to react at room temperature to obtain aptamer or antibody modified magnetic particles;
(23) mixing ultracentrifugally purified extracellular vesicles with magnetic particles modified by aptamers or antibodies in a buffer solution, and obtaining a magnetic particle compound with extracellular vesicles connected to the surface through antigen-antibody combination or aptamer-protein specific combination; wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is 0.5-5 mug: 1 mg.
7. The method for preparing the extracellular vesicle magnetic imprinting material according to claim 3, wherein the step 3 comprises the following steps:
(31) dispersing the magnetic particle compound into a buffer solution, adding tetraethoxysilane, and reacting at room temperature, wherein the mass ratio of the magnetic particle compound to the tetraethoxysilane is 1 mg: 1-10 μ g;
(32) adding a silanization reagent into the reaction product, and polymerizing and growing on the surface of the magnetic particle compound along the surface of the extracellular vesicle through hydrolysis reaction to form a recognition layer, wherein the hydrolysis reaction temperature is 10-25 ℃, and the mass ratio of the magnetic particle compound to the silanization reagent is 1 mg: 1-10 μ g.
8. The method for preparing the extracellular vesicle magnetic imprinting material according to claim 3, wherein the power for removing the template extracellular vesicles by ultrasonic cleaning in the step 4 is 25-45 Hz, and the time for ultrasonic cleaning is 2-10 min.
9. Use of the extracellular vesicle magnetic imprinting material of claim 1 for in vitro separation of extracellular vesicles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110123602.6A CN112877274B (en) | 2021-01-29 | 2021-01-29 | Extracellular vesicle magnetic imprinting material and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110123602.6A CN112877274B (en) | 2021-01-29 | 2021-01-29 | Extracellular vesicle magnetic imprinting material and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112877274A true CN112877274A (en) | 2021-06-01 |
| CN112877274B CN112877274B (en) | 2024-02-09 |
Family
ID=76053489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110123602.6A Active CN112877274B (en) | 2021-01-29 | 2021-01-29 | Extracellular vesicle magnetic imprinting material and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112877274B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115772525A (en) * | 2022-11-29 | 2023-03-10 | 中国科学院基础医学与肿瘤研究所(筹) | Aptamer combination for detecting early ovarian cancer and application thereof |
| CN116387007A (en) * | 2023-04-14 | 2023-07-04 | 西华大学 | Preparation method of synthetic oil-based magnetorheological fluid with high dispersion stability |
Citations (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347721A (en) * | 2008-09-17 | 2009-01-21 | 南开大学 | Method for preparing protein magnetic blotting nanospheres |
| WO2013012924A2 (en) * | 2011-07-18 | 2013-01-24 | President And Fellows Of Harvard College | Engineered microbe-targeting molecules and uses thereof |
| WO2014144325A1 (en) * | 2013-03-15 | 2014-09-18 | President And Fellows Of Harvard College | Methods and compositions for improving detection and/or capture of a target entity |
| CN104356323A (en) * | 2014-10-13 | 2015-02-18 | 北京大学 | Magnetic molecularly imprinted nano-particle as well as preparation method and application thereof |
| WO2015068772A1 (en) * | 2013-11-06 | 2015-05-14 | Jsr株式会社 | Separation method, detection method, signal measurement method, method for determining disease, method for evaluating drug efficacy of disease treatment drug, kit, and liquid composition |
| CN105921129A (en) * | 2016-05-16 | 2016-09-07 | 南昌大学 | Preparation method of magnetic molecularly imprinted microspheres and application in separation and enrichment of cucurbitane triterpenoid |
| CN106861650A (en) * | 2017-01-20 | 2017-06-20 | 贵州理工学院 | A kind of preparation method of the magnetic mesoporous imprinted material of sandwich structure of energy separating gallium |
| CN107200812A (en) * | 2017-06-07 | 2017-09-26 | 中国农业大学 | A kind of preparation method of magnetic molecularly imprinted material |
| CN108318693A (en) * | 2017-12-15 | 2018-07-24 | 北京大学 | Abasic magnetic molecularly imprinted nano particle of endonuclease and its preparation method and application |
| WO2018220390A1 (en) * | 2017-06-01 | 2018-12-06 | Oxford University Innovation Limited | Syncytiotrophoblast extracellular vesicles as biomarker for gestational diabetes mellitus |
| US20190178879A1 (en) * | 2012-08-23 | 2019-06-13 | Stemcell Technologies Canada Inc. | Compositions and methods for rapid and reversible biomolecular labeling |
| KR20190093141A (en) * | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | Nanovesicles from Adult Stem Cells and its use for targeted therapy |
| CN111358957A (en) * | 2020-03-06 | 2020-07-03 | 西安组织工程与再生医学研究所 | Magnetic nanoparticles |
| CN111467483A (en) * | 2020-04-17 | 2020-07-31 | 南京鼓楼医院 | Preparation method and application of magnetic nano microcarrier wrapping tumor cell membrane |
| CN112048462A (en) * | 2019-06-05 | 2020-12-08 | 北京丰特云基科技发展有限公司 | Extracellular vesicle separation and enrichment method based on anionic polymer modified matrix |
| WO2021086139A1 (en) * | 2019-10-30 | 2021-05-06 | 연세대학교 원주산학협력단 | Method for promoting stem cell-derived exosome production by means of magnetic nanoparticle cluster |
| WO2021143576A1 (en) * | 2020-01-19 | 2021-07-22 | 北京热景生物技术股份有限公司 | Lectin-magnetic carrier conjugate complex for isolating glycosylated exosomes from clinical samples |
| CN113671185A (en) * | 2021-08-23 | 2021-11-19 | 安龄(上海)生物科技有限公司 | Exosome recognition device and method |
| CN114755401A (en) * | 2022-03-24 | 2022-07-15 | 北京理工大学 | Molecular imprinting material for identifying unknown or fuzzy target object and sandwich type sensor detection method based on molecular imprinting |
| CN114867866A (en) * | 2019-12-27 | 2022-08-05 | 东曹株式会社 | Method for detecting protein inside extracellular vesicle and extracellular vesicle membrane permeation treatment agent |
| CN115461445A (en) * | 2020-04-15 | 2022-12-09 | 合同会社予幸集团中央研究所 | Method for recovering extracellular vesicles |
| CN116376124A (en) * | 2023-03-01 | 2023-07-04 | 西北大学 | Method for rapidly separating and enriching exosomes based on chitosan with specific molecular weight |
-
2021
- 2021-01-29 CN CN202110123602.6A patent/CN112877274B/en active Active
Patent Citations (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347721A (en) * | 2008-09-17 | 2009-01-21 | 南开大学 | Method for preparing protein magnetic blotting nanospheres |
| WO2013012924A2 (en) * | 2011-07-18 | 2013-01-24 | President And Fellows Of Harvard College | Engineered microbe-targeting molecules and uses thereof |
| US20190178879A1 (en) * | 2012-08-23 | 2019-06-13 | Stemcell Technologies Canada Inc. | Compositions and methods for rapid and reversible biomolecular labeling |
| WO2014144325A1 (en) * | 2013-03-15 | 2014-09-18 | President And Fellows Of Harvard College | Methods and compositions for improving detection and/or capture of a target entity |
| WO2015068772A1 (en) * | 2013-11-06 | 2015-05-14 | Jsr株式会社 | Separation method, detection method, signal measurement method, method for determining disease, method for evaluating drug efficacy of disease treatment drug, kit, and liquid composition |
| CN105723221A (en) * | 2013-11-06 | 2016-06-29 | Jsr株式会社 | Separation method, detection method, signal measurement method, method for determining disease, method for evaluating drug efficacy of disease treatment drug, kit, and liquid composition |
| CN104356323A (en) * | 2014-10-13 | 2015-02-18 | 北京大学 | Magnetic molecularly imprinted nano-particle as well as preparation method and application thereof |
| CN105921129A (en) * | 2016-05-16 | 2016-09-07 | 南昌大学 | Preparation method of magnetic molecularly imprinted microspheres and application in separation and enrichment of cucurbitane triterpenoid |
| CN106861650A (en) * | 2017-01-20 | 2017-06-20 | 贵州理工学院 | A kind of preparation method of the magnetic mesoporous imprinted material of sandwich structure of energy separating gallium |
| WO2018220390A1 (en) * | 2017-06-01 | 2018-12-06 | Oxford University Innovation Limited | Syncytiotrophoblast extracellular vesicles as biomarker for gestational diabetes mellitus |
| CN107200812A (en) * | 2017-06-07 | 2017-09-26 | 中国农业大学 | A kind of preparation method of magnetic molecularly imprinted material |
| CN108318693A (en) * | 2017-12-15 | 2018-07-24 | 北京大学 | Abasic magnetic molecularly imprinted nano particle of endonuclease and its preparation method and application |
| KR20190093141A (en) * | 2018-01-31 | 2019-08-08 | 서울대학교산학협력단 | Nanovesicles from Adult Stem Cells and its use for targeted therapy |
| CN112048462A (en) * | 2019-06-05 | 2020-12-08 | 北京丰特云基科技发展有限公司 | Extracellular vesicle separation and enrichment method based on anionic polymer modified matrix |
| WO2021086139A1 (en) * | 2019-10-30 | 2021-05-06 | 연세대학교 원주산학협력단 | Method for promoting stem cell-derived exosome production by means of magnetic nanoparticle cluster |
| CN114867866A (en) * | 2019-12-27 | 2022-08-05 | 东曹株式会社 | Method for detecting protein inside extracellular vesicle and extracellular vesicle membrane permeation treatment agent |
| WO2021143576A1 (en) * | 2020-01-19 | 2021-07-22 | 北京热景生物技术股份有限公司 | Lectin-magnetic carrier conjugate complex for isolating glycosylated exosomes from clinical samples |
| CN111358957A (en) * | 2020-03-06 | 2020-07-03 | 西安组织工程与再生医学研究所 | Magnetic nanoparticles |
| CN115461445A (en) * | 2020-04-15 | 2022-12-09 | 合同会社予幸集团中央研究所 | Method for recovering extracellular vesicles |
| CN111467483A (en) * | 2020-04-17 | 2020-07-31 | 南京鼓楼医院 | Preparation method and application of magnetic nano microcarrier wrapping tumor cell membrane |
| CN113671185A (en) * | 2021-08-23 | 2021-11-19 | 安龄(上海)生物科技有限公司 | Exosome recognition device and method |
| CN114755401A (en) * | 2022-03-24 | 2022-07-15 | 北京理工大学 | Molecular imprinting material for identifying unknown or fuzzy target object and sandwich type sensor detection method based on molecular imprinting |
| CN116376124A (en) * | 2023-03-01 | 2023-07-04 | 西北大学 | Method for rapidly separating and enriching exosomes based on chitosan with specific molecular weight |
Non-Patent Citations (6)
| Title |
|---|
| RUIXI A GAO等: "Combination of surface imprinting and immobilized template techniques for preparation of core-shell molecularly imprinted polymers based on directly amino-modifi ed Fe3O4 nanoparticles for specific recognition of bovine hemoglobin", JOURNAL OF MATERIALS CHEMISTRY B, no. 2, pages 1733 * |
| 周静静;王雨霏;胡小玲;张秋禹;: "磁性蛋白印迹复合微球的研究进展", 高分子通报, no. 10, pages 43 - 52 * |
| 张朋杰: "外泌体分子印迹材料的研究和应用", 中国优秀硕士学位论文全文数据库工程科技Ⅰ辑, no. 1, pages 014 - 706 * |
| 费贵强;白浩;邵彦明;王海花;李璐璐;: "磁性表面分子印迹聚合物(Fe_3O_4@mSiO_2@MIP)的制备及四溴双酚A检测研究", 陕西科技大学学报, no. 02, pages 96 - 102 * |
| 赵晓丽;何锡文;李文友;: "基于磁球表面的抗原决定基印迹法用于选择性识别细胞色素c的研究", 分析科学学报, no. 05, pages 619 - 625 * |
| 邓燕芳;刘桉如;罗明辉;范杰平;: "分子印迹膜分离技术进展", 化工进展, no. 06, pages 2166 - 2176 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115772525A (en) * | 2022-11-29 | 2023-03-10 | 中国科学院基础医学与肿瘤研究所(筹) | Aptamer combination for detecting early ovarian cancer and application thereof |
| CN116387007A (en) * | 2023-04-14 | 2023-07-04 | 西华大学 | Preparation method of synthetic oil-based magnetorheological fluid with high dispersion stability |
| CN116387007B (en) * | 2023-04-14 | 2024-01-02 | 西华大学 | Preparation method of synthetic oil-based magnetorheological fluid with high dispersion stability |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112877274B (en) | 2024-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Review on strategies and technologies for exosome isolation and purification | |
| CN112877274B (en) | Extracellular vesicle magnetic imprinting material and preparation method and application thereof | |
| US7169618B2 (en) | Magnetic particles and methods of producing coated magnetic particles | |
| KR101729687B1 (en) | Method for manufacturing suprerparamagnetic nanocomposite and suprerparamagnetic nanocomposite manufactured by the method | |
| JPH10502494A (en) | Highly dispersible magnetic metal oxides, their production and use | |
| CN108355625B (en) | Glycoprotein surface imprinted polymer based on boron affinity of team and preparation method and application thereof | |
| Chang et al. | Biomimetic immunomagnetic gold hybrid nanoparticles coupled with inductively coupled plasma mass spectrometry for the detection of circulating tumor cells | |
| CN1702782A (en) | Water-based magnetic liquid and method for making same | |
| WO2018171318A1 (en) | Silicon dioxide nanowire array chip for gathering and detecting circulating tumor cells in whole blood and preparation method therefor | |
| CN113933501A (en) | Immunomagnetic bead and preparation method and application thereof | |
| CN110812497B (en) | Bionic Janus magnetic-mesoporous silica nanoparticle for specifically capturing CTCs, and preparation method and application thereof | |
| Zhang et al. | One-step and high-density protein immobilization on epoxysilane-modified silica nanoparticles | |
| Wang et al. | Advances in aptamer-based nanomaterials for separation and analysis of non-genetic biomarkers in biofluids | |
| Jin et al. | Novel superparamagnetic core-shell molecular imprinting microspheres towards high selective sensing | |
| CN114276992A (en) | Complete exosome separation and purification kit and detection analysis method | |
| CN113416535A (en) | Fluorescent carbon quantum dot @ silicon dioxide microsphere and preparation method and application thereof | |
| CN111100840A (en) | Magnetic nano-composite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof | |
| CN109576206A (en) | A method of excretion body is separated based on combination method | |
| CN112394167A (en) | Fluorescent nano magnetic bead for capturing and identifying CTCs (biological chemical centers), and preparation method and application thereof | |
| CN114577746B (en) | Gold-doped covalent organic framework material, preparation method and application thereof, covalent organic framework nanoenzyme biological probe, application thereof and kit | |
| CN108318693B (en) | Dealkalized endonuclease magnetic molecularly imprinted nano-particle as well as preparation method and application thereof | |
| CN112300995A (en) | Substrate material for capturing circulating tumor cells and preparation method and application thereof | |
| CN114736868A (en) | Temperature response functional compound and exosome homogeneous phase separation and purification method | |
| CN114487391A (en) | Bionic immune magnetic nanoparticle, preparation method and application thereof | |
| Han et al. | Functionalized magnetic Fe3O4-β-cyclodextran nanoparticles for efficient removal of bilirubin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |