CN114459877A - DNA tetrahedral composite magnetic nano material for enriching exosome and preparation - Google Patents
DNA tetrahedral composite magnetic nano material for enriching exosome and preparation Download PDFInfo
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Abstract
The invention relates to the technical field of composite magnetic nano materials and exosome enrichment, and provides a DNA tetrahedral composite magnetic nano material for enriching exosomes and preparation thereof, wherein a polymer with active reaction groups is modified by a precipitation polymerization method on the surface of a prepared ferroferric oxide magnetic core; then, the DNA tetrahedron is stably immobilized on the surface of the magnetic sphere through covalent bonds, and finally, metal ions are immobilized by utilizing the chelation of the phosphate group of the DNA tetrahedron. The material can realize separation and enrichment of exosome through specific interaction of metal ions and exosome phospholipid bilayers. According to the invention, phosphate groups in the DNA tetrahedron are used as action sites for immobilizing metal ions for the first time, and the special rigid skeleton and spatial characteristics of the DNA tetrahedron are utilized, so that the enrichment efficiency of exosomes can be effectively improved. The operation process of using the material to enrich exosome is convenient and fast, and the sample loss caused by centrifugal operation is avoided.
Description
Technical Field
The invention relates to the technical field of composite magnetic nano materials and exosome enrichment, in particular to a DNA tetrahedral composite magnetic nano material for enriching exosomes and a preparation method thereof.
Background
Exosomes are extracellular vesicles released by the multivesicular bodies of most cells after fusion with the cytoplasmic membrane. Exosomes have two main functions: they transport proteins, lipids and nucleic acids from cell to cell, and transmit important biological signals during normal physiopathological processes. In recent years, it has been found that exosome-stabilized lipid bilayer membrane structures can protect their contents compared to markers in serum and urine, and thus can provide biomarkers of higher stability and specificity. The recognition and separation of exosomes are the prerequisite of exosome research, however, as exosomes are small in size, interfering substances in body fluid are too many, and the difficulty of separation and enrichment is greatly increased. Ultracentrifugation is a commonly used method for separating exosomes, but is long in time consumption, low in separation purity and low in efficiency; some commercial kits have a complex process, although they have high sensitivity for the isolation of exosomes.
The DNA tetrahedron is a DNA three-dimensional structure with a tetrahedron shape formed by self-assembling and combining 4 DNA single strands by precise DNA sequence design and applying the principle of base complementary pairing. The DNA tetrahedron has excellent biocompatibility and abundant functional modification sites, and is simple to prepare, high in yield, and adjustable in size and dynamic property. The framework structure of the DNA tetrahedron has certain rigidity. In our previous patent application (202110071495.7), we use the spatial separation of DNA tetrahedrons to effectively control antibody distribution, thereby achieving efficient enrichment and quantification of specific proteins in complex matrices.
At present, most of the enrichment materials used for exosomes are metal oxides (such as titanium dioxide, zirconium dioxide, and the like) or magnetic bead-based metal ion affinity materials, and such materials have a good effect in enriching molecules such as phosphorylated peptides or phosphorylated proteins. However, exosomes are a kind of biological vesicles with large volume (diameter 40-200 nm), and the above materials can only provide a few action sites when interacting with exosomes, similar to point-to-point interaction between two tangent spheres, so that the effect of enriching exosomes is not good.
Disclosure of Invention
The invention aims to overcome at least one of the defects of the prior art, and provides a DNA tetrahedral composite magnetic nano material for enriching exosomes, a preparation method and application thereof, which can effectively improve the enrichment efficiency of exosomes.
The invention adopts the following technical scheme:
in one aspect, the invention provides a DNA tetrahedral composite magnetic nanomaterial for enriching exosomes, comprising, from inside to outside: the polymer layer is coated on the surface of the magnetic core by a precipitation polymerization method, the DNA tetrahedron is fixedly carried on the surface of the polymer layer, and the metal ions are fixedly carried on the DNA tetrahedron; the metal ions are immobilized by chelation with the phosphate groups of the DNA tetrahedron.
There is further provided in any of the possible implementations described above, an implementation in which the magnetic core comprises a ferriferrous oxide magnetic core.
In any of the above possible implementation manners, there is further provided an implementation manner, where the magnetic core is a ferroferric oxide magnetic nanoparticle, and the particle size is 50 to 800 nm.
According to any possible implementation manner, a polymer is wrapped outside the ferroferric oxide magnetic core, the ferroferric oxide magnetic core is of a core-shell structure, and the thickness of the polymer layer is 50-500 nm.
Any one of the above possible implementation manners, further providing an implementation manner that the 4 DNA single strands of the DNA tetrahedron are synthesized by self-assembly, each DNA single strand comprising 10-130 deoxyribonucleotide monomers; the 3 'end or the 5' end of the DNA single chain is provided with a first active functional group, and the first active functional group is sulfydryl, carboxyl, aldehyde group, epoxy group or amino; and the first active functional group and the second active functional group of the polymer are subjected to chemical reaction so as to fix the DNA tetrahedron on the surface of the polymer.
In any of the possible implementations described above, there is further provided an implementation in which the DNA tetrahedron is formed by base complementary pairing of four DNA single strands each at a concentration of 1. mu. mol/L.
There is further provided in accordance with any of the possible implementations described above an implementation in which the metal ions include titanium ions, zirconium ions.
Any of the possible implementations described above, further providing an implementation in which the polymer layer is obtained by polymerizing the magnetic core with polyglycidyl methacrylate.
In another aspect, the present invention also provides a preparation method of the DNA tetrahedral composite magnetic nanomaterial for enriching exosomes, which comprises:
s1, preparing ferroferric oxide magnetic nuclei to obtain a product I, namely Fe3O4；
S2, modifying a polymer layer on the surface of the product I through in-situ polymerization reaction, wherein the polymer is poly glycidyl methacrylate to obtain a product II, Fe3O4@PGMA；
S3, modifying amino groups on the surface of the product II to obtain a product III, Fe3O4@PGMA@NH2；
S4, immobilizing DNA tetrahedron on the surface of the product III through aldehyde-amine condensation reaction to obtain a product IV: fe3O4@PGMA@DNA TETs；
S5, chelating titanium ions and DNA tetrahedral phosphate groups on the surface of the product IV to obtain a product V: fe3O4@PGMA@DNA TETs@Ti4+And obtaining the product V which is the composite magnetic nano material.
In any of the foregoing possible implementation manners, there is further provided an implementation manner, in step S1, preparing ferroferric oxide magnetic cores by a solvothermal method; in step S5, the metal ions are titanium ions or zirconium ions.
On the other hand, the invention also provides application of the DNA tetrahedral composite magnetic nano material for enriching the exosomes in exosome enrichment and detection.
The beneficial effects of the invention are as follows: the magnetic nano material is combined with a DNA tetrahedron, phosphate groups of the DNA tetrahedron are used for immobilizing metal ions, and the immobilized metal ions and an exosome phospholipid bilayer are specifically interacted, so that efficient and high-selectivity enrichment of exosomes is realized. The invention utilizes the special rigid framework and space characteristics of the DNA tetrahedron for the first time. In the process of enriching exosomes, the material can provide a three-dimensional interaction system, so that the enrichment efficiency of exosomes can be effectively improved. The time of the operation process of using the material to enrich the exosome is short, the consumption of the sample reagent is low, and the manufacturing cost is low. High-quality exosomes can be quickly separated from serum and cell supernatant, and the processing cost and time of samples are greatly reduced.
Drawings
FIG. 1 is a schematic diagram of a synthetic route of a DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to an embodiment of the present invention.
FIG. 2 shows the product II Fe obtained in the synthesis of example 13O4Transmission Electron microscopy of @ PGMA.
FIG. 3 shows the Fe product of the example synthesis3O4（a）、Fe3O4@PGMA（b）、Fe3O4@PGMA@NH2- (c) and Fe3O4@PGMA@NH2-@DNA TET@Ti4+(d) A magnetic characterization map.
FIG. 4 shows Fe3O4@PGMA@NH2-@DNA TET@Ti4+EDS profile of (a).
FIG. 5 shows a DNA tetrahedral composite and a conventional TiO according to an embodiment of the present invention2Effect of enriched exosomes versus graphs.
Detailed Description
Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered in isolation, and they may be combined with each other to achieve better technical effects. In the drawings of the embodiments described below, the same reference numerals appearing in the respective drawings denote the same features or components, and may be applied to different embodiments.
The embodiment of the invention provides a DNA tetrahedron composite magnetic nano material for enriching exosomes, which is characterized in that a ferroferric oxide magnetic core is firstly prepared, poly glycidyl methacrylate and amino groups are modified on the surface of the magnetic core through a precipitation polymerization method, four DNA tetrahedrons with the vertexes modified by aldehyde groups are synthesized through the self-assembly reaction of DNA single chains, then the DNA tetrahedron is stably and fixedly carried on the surface of a magnetic sphere modified by the amino groups, and finally metal ions are modified on the DNA tetrahedron.
In a specific embodiment, the ferroferric oxide magnetic nanoparticles have a particle size of 50-800 nm, as shown in fig. 3 (a), and have strong magnetism.
In a specific embodiment, the polymer is wrapped outside the ferroferric oxide magnetic core, and is in an obvious core-shell structure as shown in fig. 2, and the thickness of the polymer layer is 50-500 nm. Due to the encapsulating polymer, the magnetic properties are only slightly reduced as shown in fig. 3 (b).
Preferably, 4 DNA single-strands of the DNA tetrahedron are synthesized in a self-assembly mode, and each DNA single-strand comprises 10-130 deoxyribonucleotide monomers; the 3 'end or the 5' end of the DNA single chain is provided with a first active functional group, and the first active functional group is sulfydryl, carboxyl, aldehyde group, epoxy group or amino; and the first active functional group and the second active functional group of the polymer are subjected to chemical reaction so as to fix the DNA tetrahedron on the surface of the polymer. As shown in fig. 3 (c) and (d), the material retains good magnetic properties during the synthesis process, and the magnetic properties are hardly reduced as compared with those of (b).
In one embodiment, the DNA tetrahedron is formed by base-complementary pairing of four DNA single strands each at a concentration of 1. mu. mol/L.
In a specific embodiment, the metal ions include titanium ions, zirconium ions. It can be seen from fig. 4 that the material contains a large amount of Ti elements, indicating that the material successfully carries a large amount of Ti ions.
In a particular embodiment, the polymer layer is obtained by polymerization of the magnetic core with polyglycidyl methacrylate.
As shown in fig. 1, the preparation method of the composite magnetic nanomaterial based on DNA tetrahedron in the embodiment of the present invention includes the following steps:
s1, preparing ferroferric oxide magnetic cores by a solvothermal method to obtain a product I, namely Fe3O4；
S2, modifying the poly glycidyl methacrylate on the surface of the product I through in-situ polymerization reaction to obtain a product II, namely Fe3O4@PGMA；
S3, modifying amino groups on the surface of the product II to obtain a product III, Fe3O4@PGMA@NH2；
S4, immobilizing DNA tetrahedron on the surface of the product III through aldehyde-amine condensation reaction to obtain a product IV: fe3O4@PGMA@DNA TETs。
S5, chelating titanium ions and DNA tetrahedral phosphate groups on the surface of the product IV to obtain a product V: fe3O4@PGMA@DNA TETs@Ti4+And obtaining the product V which is the composite magnetic nano material.
In the following specific examples:
and (3) a product I: 200mL of ethylene glycol was measured and placed in a flask, and 3g of ferric chloride hexahydrate was added thereto, and the mixture was sonicated for 30 minutes. 8g of anhydrous sodium acetate was added thereto, and stirred for 1 hour. Transferring the mixed solution to a stainless steel autoclave, reacting for 12 hours in a 220 ℃ oven, washing with ethanol and pure water respectively under the assistance of an external magnetic field, and collecting magnetic core Fe3O4And dried in an oven at 50 ℃ overnight;
and (3) a product II: taking 200mg of Fe3O4 Dispersing in a mixed solution containing 50mL of ethanol and 20mL of water, adding 2mL of concentrated ammonia water into the mixed solution, and carrying out ultrasonic treatment for 1 hour until the mixed solution is uniformly dispersed. Stirring the mixed solution in an oil bath at 70 ℃ until the solution is uniform, dropwise adding 600 mu L of 3- (trimethoxysilyl) methacrylate, and stirring for 24 hours. After the reaction is finished, under the assistance of a magnet, washing the product for a plurality of times by ethanol and pure water, and drying to obtain Fe3O4@ MPS. Weighing 70 mg of Fe3O4@ MPS was ultrasonically dispersed in 60mL acetonitrile. 200. mu.L of glycidyl methacrylate, 200mg of N, N-methylene bisacrylamide and 10mg of azobisisobutyronitrile were added to the solution, and after ultrasonic dispersion, the mixture was placed in an oil bath pan and reacted at 95 ℃ for 2 hours. Washing and washing the product with ethanol and water, and drying for later use;
and (3) a product III: weighing 100mgFe3O4Ultrasonically dispersing @ PGMA in 90ml of propylenediamine solution, carrying out oil bath reaction at 90 ℃ for 2 hours, washing the product with ethanol and water, and drying the product in an oven for later use;
the DNA tetrahedron used is prepared by the following steps: each single-stranded DNA was prepared at a concentration of 150. mu. mol/L. mu.L of each single strand was added to 96. mu.L of TE buffer to give a final concentration of 1. mu. mol/L per single strand. After the mixture is kept at 95 ℃ for 20 minutes, the mixture reacts for 30 minutes at 4 ℃ to form a DNA tetrahedron through self-assembly;
and (3) a product IV: weighing Fe3O4@PGMA-NH220mg of composite material, adding 100 mu L of the prepared DNA tetrahedron, 500 mu L of TE buffer solution and 30 mu L of NaCl solution with the concentration of 50 mmol/L for reaction, reacting for 16 hours at 4 ℃, and storing a sample in a refrigerator at 4 ℃ for later use;
and (3) a product V: mixing Fe3O4@ PGMA @ DNA TETs were added to a 5mM titanium sulfate solution and incubated at 40 ℃ for 8 hours with shaking. Washing the obtained product with a buffer solution and storing at 4 ℃, wherein the product V is the composite magnetic nano material.
Comparative experiment
Taking the protein and peptide fragment for detecting exosome in mixed serum as an example, the commercial TiO is used2As a comparative material, the performance of the novel magnetic composite nano material of the invention for enriching exosomes in an actual sample is examined. And (3) sucking 200 mul of mixed serum, adding the mixed serum into 1mL of the novel magnetic composite nano material, and uniformly mixing and incubating the mixed solution for 30 minutes at the temperature of 4 ℃. After incubation, the supernatant was removed under the action of an external magnet. And (3) washing the material with a washing solution, carrying out enzymolysis reaction, and sucking 10 mu l of the reacted enzyme digestion solution for high-resolution mass spectrometry detection. As shown in fig. 5: among them, conventional TiO 2298 exosome-associated proteins and 1202 peptides are enriched and identified; the DNA tetrahedral composite nano material prepared by the invention enriches and identifies 534 exosome-associated proteins and 3655 peptide fragments, which are greatly superior to the traditional TiO2And (4) material enrichment mode.
According to the invention, the special rigid framework and space characteristics of the DNA tetrahedron are utilized for the first time, phosphate groups in the DNA tetrahedron are used as action sites for immobilizing metal ions, in the process of enriching exosomes, the material can provide a three-dimensional interaction system, specific metal ions (such as titanium Ions (IV), zirconium Ions (IV) and the like) are chelated through the phosphate groups in each single chain of the DNA tetrahedron, and then the exosomes are adsorbed by utilizing the unique three-dimensional framework structure of the DNA tetrahedron, so that the exosomes are embedded into the DNA tetrahedron array, the number of interaction sites between the metal ions and the exosome phospholipid bilayer is obviously increased, and the high-efficiency enrichment of the exosomes is realized.
The operation process of enriching exosomes by using the material is convenient and fast, and the sample loss caused by centrifugal operation is avoided.
While several embodiments of the present invention have been presented herein, it will be appreciated by those skilled in the art that changes may be made to the embodiments herein without departing from the spirit of the invention. The above examples are merely illustrative and should not be taken as limiting the scope of the invention.
Claims (10)
1. A DNA tetrahedral composite magnetic nano material for enriching exosomes is characterized in that the composite magnetic nano material sequentially comprises from inside to outside: the polymer layer is coated on the surface of the magnetic core by a precipitation polymerization method, the DNA tetrahedron is fixedly carried on the surface of the polymer layer, and the metal ions are fixedly carried on the DNA tetrahedron; the metal ions are immobilized by chelation with the phosphate groups of the DNA tetrahedron.
2. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 1, wherein the magnetic core is ferroferric oxide magnetic core.
3. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 1, wherein the magnetic core is ferroferric oxide magnetic nanoparticles, and the particle size is 50-800 nm.
4. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 2, wherein the polymer layer is formed by wrapping a polymer outside a ferroferric oxide magnetic core and has a core-shell structure, and the thickness of the polymer layer is 50-500 nm.
5. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 1, wherein 4 DNA single strands of the DNA tetrahedron are synthesized by means of self-assembly, and each DNA single strand comprises 10-130 deoxyribonucleotide monomers; the 3 'end or the 5' end of the DNA single chain is provided with a first active functional group, and the first active functional group is sulfydryl, carboxyl, aldehyde group, epoxy group or amino; and the first active functional group and the second active functional group of the polymer layer are subjected to chemical reaction so as to immobilize the DNA tetrahedron on the surface of the polymer layer.
6. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 1, wherein the metal ions comprise titanium ions and zirconium ions.
7. The DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 1, wherein the polymer layer is obtained by polymerization reaction of the magnetic core and polyglycidyl methacrylate.
8. A method for preparing a DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to any one of claims 1 to 7, wherein the method comprises the following steps:
s1, preparing ferroferric oxide magnetic core to obtain a product I, namely Fe3O4；
S2, modifying a polymer layer on the surface of the product I through in-situ polymerization reaction, wherein the polymer is poly glycidyl methacrylate to obtain a product II, Fe3O4@PGMA；
S3, modifying amino groups on the surface of the product II to obtain a product III, Fe3O4@PGMA@NH2；
S4, immobilizing DNA tetrahedron on the surface of the product III through aldehyde-amine condensation reaction to obtain a product IV: fe3O4@PGMA@DNA TETs；
S5, chelating metal ions and DNA tetrahedral phosphate groups on the surface of the product IV to obtain a product V: fe3O4@PGMA@DNA TETs@Ti4+And obtaining the product V which is the composite magnetic nano material.
9. The method for preparing a DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to claim 8, wherein in step S1, ferroferric oxide magnetic cores are prepared by a solvothermal method; in step S5, the metal ions are titanium ions or zirconium ions.
10. Use of the DNA tetrahedral composite magnetic nanomaterial for enriching exosomes according to any one of claims 1 to 7 in exosome enrichment and detection.
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