CN112877274B - 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
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Abstract
The invention discloses an extracellular vesicle magnetic imprinting material, a preparation method and application thereof, wherein the magnetic imprinting material comprises a magnetic particle matrix, and the surface of the magnetic particle matrix is provided with extracellular vesicle imprinting; the preparation method of the magnetic imprinting material comprises the following steps: (1) preparing magnetic particles and performing surface functional group modification; (2) Connecting the extracellular vesicles of the template on the surface of the magnetic particles; (3) Growing a polymer recognition layer on the surface of the magnetic particle along the extracellular vesicles; (4) Removing the template vesicle by ultrasonic cleaning to obtain a magnetic imprinting material with extracellular vesicle marks on the surface; the magnetic imprinting material can be applied to in vitro isolation of extracellular vesicles. The magnetic imprinting material can efficiently capture and separate extracellular vesicles from a cell culture medium, blood plasma and other body fluids, has high capture efficiency, simple preparation process, large yield and low cost, is convenient to use and easy to store, does not need large-scale instrument assistance, can be recycled, and can further reduce cost.
Description
Technical Field
The invention relates to a imprinting material and a preparation method and application thereof, in particular to an extracellular vesicle magnetic imprinting material and a preparation method and application thereof.
Background
Extracellular Vesicles (EVs) are extracellular vesicles which are wrapped by phospholipid bilayer membranes and have diameters of 30-200nm, and can be secreted by various cells including tumor cells, endothelial cells, immune cells, platelets and the like. In recent years, more and more researches show that extracellular vesicles are taken as a means for in-vivo intercellular remote communication, and are rich in DNA, RNA, protein and other substances. In particular, extracellular vesicles secreted by tumor cells, which contain a variety of cancer-specific proteins and nucleotides (mRNA, micrornas, and DNA fragments), are useful as circulating biomarkers for cancer diagnosis, prognosis, and therapeutic monitoring. However, there is currently no standard and efficient method to obtain high purity, high yield extracellular vesicles. The existing acquisition methods include 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 commonly used extracellular vesicle separation method at present, can process a large amount of samples and obtain relatively pure extracellular vesicles, but has the defects of long time consumption, high instrument requirement and low recovery rate, so that the application range of the ultracentrifugation method is limited; ultrafiltration and size exclusion chromatography to handle excessive sample volumes are not suitable for collection of extracellular vesicles in small volumes of samples (serum, plasma); the extracellular vesicles obtained by the polyethylene glycol precipitation method have the advantages of non-single components and more impurities; although the immunomagnetic bead separation method can separate relatively pure extracellular vesicles, the antibody is expensive and difficult to preserve, which greatly limits the wide application of the immunoseparation method.
Disclosure of Invention
The invention aims to: the invention aims to provide a preparation method of an extracellular vesicle magnetic imprinting material, which can efficiently capture and separate extracellular vesicles, has the advantages of simple preparation process, easy storage and low cost; it is another object of the present invention to provide the use 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 imprinting is arranged on the surfaces of the magnetic particles.
Wherein the magnetic particles are Fe 3 O 4 @SiO 2 The particle size of the nano particles and 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 performing surface functionalization;
(2) Connecting the extracellular vesicles of the template on the surface of the magnetic particles;
(3) Using molecular imprinting reaction, and growing a polymer recognition layer on the surface of the magnetic particle along extracellular vesicles through siloxane polymerization reaction;
(4) And (3) 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 firstly dispersed into a solution with the volume fraction of 1-2 mg/mL in water, and then 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 an amino-containing silane reagent or a carboxyl-containing silanization reagent.
Wherein, in the step 2, the extracellular vesicles of the template are connected on the surface of the magnetic particles through covalent bonds, comprising 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) performing ultracentrifugation and purification to obtain a template extracellular vesicle, and reacting the extracellular vesicle with the activated magnetic particles in a phosphate buffer solution to obtain a magnetic particle compound with the extracellular vesicle connected to the surface. Wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is 0.5-5 mug: 1mg.
Wherein, in the step 2, the extracellular vesicles of the template are connected on the surface of the magnetic particles through non-covalent bonds, comprising the following steps:
(21) Activating amino groups on the surfaces of the magnetic particles by glutaraldehyde to obtain aldehyde-modified magnetic particles;
(22) Adding amino aptamer or antibody into the aldehyde magnetic particles to react at room temperature to obtain aptamer or antibody modified magnetic particles;
(23) Mixing the ultracentrifuge-purified extracellular vesicles with aptamer or antibody-modified magnetic particles in a buffer solution, and obtaining a magnetic particle compound with the extracellular vesicles connected to the surface through antigen-antibody binding or aptamer-protein specific binding; wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is 0.5-5 mug: 1mg of the antibody is a CD63 antibody, a CD81 antibody or a CD9 antibody, and the aptamer is an aptamer corresponding to CD63, CD81, CD9, epCAM, PTK7, PSA, LZH8 and Lib.
Wherein, step 3 includes 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 1mg: 1-10 mug;
(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 1mg: 1-10 mug, the volume fraction of the silanization reagent is 0.5-10%, and the time required for forming the recognition layer is 8-72 hours, wherein the silanization reagent is at least one of 3-aminopropyl triethoxysilane, hydroxymethyl triethoxysilane, propyl triethoxysilane or benzyl triethoxysilane.
Wherein, the power of removing the extracellular vesicles of the template by ultrasonic cleaning in the step 4 is 25-45 Hz, and the ultrasonic time is 2-10 min.
The extracellular vesicle magnetic imprinting material is applied to in vitro separation of extracellular vesicles.
The preparation principle is as follows: according to the method, the magnetic particles are used as carriers, the organosilane is used as a construction module, the extracellular vesicles are used as templates, the extracellular vesicle magnetic imprinting material with extracellular vesicle marks is prepared, the characteristic that the organosilicon layer can continuously grow through siloxane polymerization and hydrolysis is utilized during preparation, a silicon dioxide recognition layer is continuously grown on the surface of the magnetic particles along the extracellular vesicles of the templates, the template extracellular vesicles are removed through ultrasonic cleaning, cavities with specific recognition and extracellular vesicle capture functions are reserved on the surface of the magnetic particles, and the extracellular vesicle magnetic imprinting material provided by the invention has both magnetism and extracellular vesicle capture functions, can be rapidly separated by using a magnet, is beneficial to cleaning and removing redundant impurities, and can be used for rapidly capturing, collecting and analyzing the extracellular vesicles without additional equipment.
The beneficial effects are that: compared with the prior art, the invention has the remarkable advantages that: 1. the magnetic imprinting material for the extracellular vesicles can efficiently capture and separate the extracellular vesicles from a cell culture medium, 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 can be applied to in-vitro separation of the extracellular vesicles, is convenient to use and easy to store, and does not need large-scale instrument assistance; 4. can be recycled, and further reduces the cost.
Drawings
FIG. 1 is a schematic diagram of a preparation process of an extracellular vesicle magnetic imprinting material;
FIG. 2 is Fe 3 O 4 @SiO 2 Nanoparticle scanning electron microscopy images;
FIG. 3 is EVs-Fe 3 O 4 @SiO 2 A composite scanning electron microscope image;
FIG. 4 is a scanning electron microscope image 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 microscope image of a magnetic blotting material with extracellular vesicles captured;
FIG. 7 shows the capture efficiency of 5 cycles of extracellular vesicle magnetic colloid.
Detailed Description
The process for preparing the extracellular vesicle magnetic imprinting material is shown in figure 1, wherein a template extracellular vesicle is connected to the surface of a magnetic particle through amino groups, a polymer recognition layer grows on the surface of the magnetic particle along the extracellular vesicle through siloxane polymerization reaction, and finally the template extracellular vesicle on the surface of the magnetic particle is removed through ultrasonic cleaning, so that the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting is obtained, and the specific process is as follows:
(1) Preparation of Fe 3 O 4 @SiO 2 Preparation of magnetic particles and surface functionalization:
dissolving 2.70g of ferric trichloride 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 Fe 3 O 4 A nanoparticle; centrifugal washing of Fe 3 O 4 Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of ethyl orthosilicate and 0.10g of Fe 3 O 4 Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring and reacting for 12h, washing with water, and drying to obtain Fe 3 O 4 @SiO 2 The magnetic particles are shown in the figure 2, the left side is a larger range diagram, the right side is a partial enlarged diagram, and the particle size is 550nm;
fe prepared by the method 3 O 4 @SiO 2 The magnetic particles are uniformly dispersed in the water solution to obtain Fe with the concentration of 1mg/mL 3 O 4 @SiO 2 An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyl triethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe 3 O 4 @SiO 2 Magnetic particles;
(2) Aminated Fe 3 O 4 @SiO 2 Covalent attachment of magnetic particles to extracellular vesicles
30mg of aminated Fe 3 O 4 @SiO 2 The magnetic particles are evenly dispersed in glutaraldehyde solution with the volume fraction of 30mL being 1%, reacted for 0.5 hour at room temperature, washed twice to obtain Fe with surface hydroformylation 3 O 4 @SiO 2 Magnetic particles;
the ultracentrifuge purified extracellular vesicles were taken at 25. Mu.g, and the extracellular vesicles and 10mg of surface-activated Fe were mixed 3 O 4 @SiO 2 The magnetic particles are placed in 5mL of phosphoric acid buffer solution and react for 2 to 4 hours at room temperature, and the magnetic particles EVs-Fe with extracellular vesicles connected to the surfaces can be obtained 3 O 4 @SiO 2 The obtained EVs-Fe 3 O 4 @SiO 2 The scanning electron microscope image of the compound is shown in fig. 3, the left side is a larger range image, and the right side is a partial enlarged image;
(3) At EVs-Fe 3 O 4 @SiO 2 Growing a polymer recognition layer on the composite
24mgEVs-Fe 3 O 4 @SiO 2 Dispersing into 5mL of phosphoric acid buffer solution, adding 32 mu L of tetraethoxysilane, reacting for 2 hours at room temperature, then adding 24 mu L of 3-aminopropyl triethoxysilane APTES, and then continuing to react for 72 hours at 10 ℃; after the reaction was completed, the supernatant was removed by magnetic separation, and washed three times with deionized water to obtain magnetic particles having an extracellular vesicle recognition layer grown thereon, the scanning electron microscope image of which 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 from which magnetic particles having an extracellular vesicle recognition layer grown are removed
Placing the magnetic particles growing with the extracellular vesicle recognition layer in an eluent, carrying out water bath ultrasonic treatment for 30min to remove the extracellular vesicles of the template, wherein the eluent is an aqueous solution containing 0.1mol/L HCl+0.01% Txiton X-100, and the ultrasonic power is 45HZ; and (3) continuing ultrasonic treatment for 20min, washing with deionized water, and drying to obtain the magnetic imprinting material with extracellular vesicle imprinting on the surface, wherein a scanning electron microscope chart is shown in fig. 5: the left side is a larger range diagram, and the right side is a partial enlarged diagram.
Example 2
The preparation process of the extracellular vesicle magnetic imprinting material is shown in figure 1, the carboxylated magnetic nano-particles are utilized to connect the template extracellular vesicle on the surface of the magnetic particles, then molecular imprinting reaction is utilized to grow a polymer recognition layer on the surface of the magnetic particles along the extracellular vesicle through siloxane polymerization reaction, finally, the template extracellular vesicle on the surface of the magnetic particles is removed by ultrasonic cleaning, and the specific process of the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting is as follows:
(1) Preparation of Fe 3 O 4 @SiO 2 Preparation of magnetic particles and surface carboxylation:
dissolving 2.70g of ferric trichloride 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 Fe 3 O 4 A nanoparticle; centrifugal washing of Fe 3 O 4 Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of ethyl orthosilicate and 0.10g of Fe 3 O 4 Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring and reacting for 12h, washing with water, and drying to obtain Fe 3 O 4 @SiO 2 Magnetic particles;
fe prepared by the method 3 O 4 @SiO 2 The magnetic particles are uniformly dispersed in the water solution to obtain Fe with the concentration of 1mg/mL 3 O 4 @SiO 2 An aqueous solution of magnetic particles; adding 30 μl of 35% N- (trimethoxysilylpropyl) ethylenediamine triacetic acid sodium salt into 30mL of the above solution, reacting at room temperature for 30min, washing with water, and drying to obtainTo carboxylated Fe 3 O 4 @SiO 2 Magnetic particles;
(2) Carboxylated Fe 3 O 4 @SiO 2 Covalent attachment of magnetic particles to extracellular vesicles, i.e.activation of carboxylated magnetic beads by NHS/EDC
Fe is added to 3 O 4 @SiO 2 The magnetic particles were uniformly dispersed in 2- (N-morpholino) ethanesulfonic acid buffer solution at ph=6 to give Fe at a concentration of 1mg/mL 3 O 4 @SiO 2 10mL of magnetic particle solution is added with 100mgNHS and 200mgEDC respectively, activated for 0.5 hour at medium room temperature, washed twice to obtain surface activated Fe 3 O 4 @SiO 2 Magnetic particles.
The ultracentrifuge purified extracellular vesicles were taken at 25. Mu.g, and the extracellular vesicles and 10mg of surface-activated Fe were mixed 3 O 4 @SiO 2 The magnetic particles are placed in 5mL of phosphoric acid buffer solution and react for 4 hours at room temperature, and the magnetic particles EVs-Fe with extracellular vesicles connected to the surfaces can be obtained 3 O 4 @SiO 2 ；
(3) At EVs-Fe 3 O 4 @SiO 2 Growing a polymer recognition layer on the composite
24mgEVs-Fe 3 O 4 @SiO 2 Dispersing into 5mL of phosphoric acid buffer solution, adding 32 mu L of tetraethoxysilane, reacting for 2 hours at room temperature, then adding 12 mu L of 3-aminopropyl triethoxysilane APTES and 12 mu L of hydroxymethyl triethoxysilane HMTEOS, and then continuing to react for 50 hours at 10 ℃; after the reaction is finished, removing the supernatant through magnetic separation, and washing with deionized water for three times to obtain magnetic particles growing with an extracellular vesicle recognition layer;
(4) Template extracellular vesicles from which magnetic particles having an extracellular vesicle recognition layer grown are removed
Placing the magnetic particles growing with the extracellular vesicle recognition layer in an eluent, carrying out water bath ultrasonic treatment for 30min to remove the extracellular vesicles of the template, wherein the eluent is an aqueous solution containing 0.1mol/L HCl+0.01% Txiton X-100, and the ultrasonic power is 45HZ; and (3) continuing ultrasonic treatment of the product for 20min, washing with deionized water, and drying to obtain the magnetic imprinting material with extracellular vesicle imprinting on the surface.
Example 3
The process for preparing the extracellular vesicle magnetic imprinting material is shown in figure 1, the template extracellular vesicle is connected to the surface of the magnetic particle by utilizing the specific combination of an aptamer and a vesicle, a polymer recognition layer grows on the surface of the magnetic particle along the extracellular vesicle by siloxane polymerization reaction, 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, and the specific process is as follows:
(1) Preparation of Fe 3 O 4 @SiO 2 Preparation of magnetic particles and surface functionalization:
dissolving 2.70g of ferric trichloride 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 Fe 3 O 4 A nanoparticle; centrifugal washing of Fe 3 O 4 Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of ethyl orthosilicate and 0.10g of Fe 3 O 4 Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring and reacting for 12h, washing with water, and drying to obtain Fe 3 O 4 @SiO 2 Magnetic particles;
fe prepared by the method 3 O 4 @SiO 2 The magnetic particles are uniformly dispersed in the water solution to obtain Fe with the concentration of 1mg/mL 3 O 4 @SiO 2 An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyl triethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe 3 O 4 @SiO 2 Magnetic particles;
(2) Attachment of magnetic particles to extracellular vesicles by aptamer-target specific binding
30mg of aminated Fe 3 O 4 @SiO 2 The magnetic particles are evenly dispersed in glutaraldehyde solution with the volume fraction of 30mL being 1%, reacted for 0.5 hour at room temperature, washed twice to obtain surface activated Fe 3 O 4 @SiO 2 Magnetic particles, i.e. hydroformylation magnetic particles;
100 mu L of amination CD63 aptamer with the concentration of 10 mu M is added into 20mL of hydroformylation magnetic particle solution with the concentration of 1mg/mL, and is connected to the surfaces of magnetic particles through aldehyde-amine condensation reaction;
Fe 3 O 4 @SiO 2 aptamer binds specifically to extracellular vesicles, and attaches EVs to the surface of magnetic particles, resulting in EVs-Fe 3 O 4 @SiO 2 A complex;
the sequence of the aptamer was 5'-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-3'.
(3) At EVs-Fe 3 O 4 @SiO 2 Growing a polymer recognition layer on the composite
24mgEVs-Fe 3 O 4 @SiO 2 Dispersing into 5mL of phosphoric acid buffer solution, adding 32 mu L of tetraethoxysilane, reacting for 2 hours at room temperature, then adding 12 mu L of 3-aminopropyl triethoxysilane APTES, 6 mu L of hydroxymethyl triethoxysilane HMTEOS and 12 mu L of propyl triethoxysilane PTES, and then continuing to react for 40 hours at 10 ℃; after the reaction is finished, removing the supernatant through magnetic separation, and washing with deionized water for three times to obtain magnetic particles growing with an extracellular vesicle recognition layer;
(4) Template extracellular vesicles from which magnetic particles having an extracellular vesicle recognition layer grown are removed
Placing the magnetic particles growing with the extracellular vesicle recognition layer in an eluent, carrying out water bath ultrasonic treatment for 30min to remove the extracellular vesicles of the template, wherein the eluent is an aqueous solution containing 0.1mol/L HCl+0.01% Txiton X-100, and the ultrasonic power is 45HZ; and (3) continuing ultrasonic treatment of the product for 20min, washing with deionized water, and drying to obtain the magnetic imprinting material with extracellular vesicle imprinting on the surface.
Example 4
This embodiment differs from embodiment 3 in that: the aptamer used in step 3 was EpCAM protein aptamer and the sequence of the aptamer used was 5'-CAC TAC AGA GGT TGC GTC TGT CCC ACG TTG TCA TGG GGG GTT GGC CTG-3'.
Example 5
This embodiment differs from embodiment 3 in that: the aptamer used in the step 3 was a CA125 protein aptamer, and the sequence of the aptamer used was 5'-TAT CAA TTA CTT ACC CTA GTG GTG TGA TGT CGT ATG GAT G-3'.
Example 6
This embodiment differs from embodiment 3 in that: the aptamer used in step 3 was HER2 protein aptamer and the sequence of the aptamer used was 5'-GGG CCG TCG AAC ACG AGC ATG GTG CGT GGA CCT AGG ATG ACC TGA GTA CTG TCC-3'.
Example 7
This embodiment differs from embodiment 3 in that: the aptamer used in step 3 was a PTK7 protein aptamer, and the sequence of the aptamer used was 5'-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3'.
Example 8
This embodiment differs from embodiment 3 in that: the aptamer used in step 3 was PSA protein aptamer, and the sequence of the aptamer used was 5'-AAT TAA AGC TCG CCA TCA AAT AGC-3'.
Example 9
This embodiment differs from embodiment 3 in that: the aptamer used in step 3 was the LZH8 protein aptamer, and the sequence of the aptamer used was 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 embodiment differs from embodiment 3 in that: the aptamer used in step 3 was Lib protein aptamer, and the sequence of the aptamer used was 5'-CAC CCC ACC TAA AAA AAA AAA CAC TAA TGC TA-3'.
Example 11
The process for preparing the extracellular vesicle magnetic imprinting material is shown in figure 1, the extracellular vesicle of the template is connected to the surface of the magnetic particle by utilizing antigen-antibody specific binding, then a polymer recognition layer grows on the surface of the magnetic particle along the extracellular vesicle by utilizing molecular imprinting reaction and siloxane polymerization reaction, finally the extracellular vesicle of the template on the surface of the magnetic particle is removed by ultrasonic cleaning, and the extracellular vesicle magnetic imprinting material with extracellular vesicle imprinting is obtained by the following specific steps:
(1) Preparation of Fe 3 O 4 @SiO 2 Preparation of magnetic particles and surface functionalization:
dissolving 2.70g of ferric trichloride 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 Fe 3 O 4 A nanoparticle; centrifugal washing of Fe 3 O 4 Drying the nano particles in a vacuum drying oven at 60 ℃; 0.8mL of ethyl orthosilicate and 0.10g of Fe 3 O 4 Adding the nano particles into a mixture of 80mL of ethanol, 20mL of water and 1mL of ammonia water, stirring and reacting for 12h, washing with water, and drying to obtain Fe 3 O 4 @SiO 2 Magnetic particles;
fe prepared by the method 3 O 4 @SiO 2 The magnetic particles are uniformly dispersed in the water solution to obtain Fe with the concentration of 1mg/mL 3 O 4 @SiO 2 An aqueous solution of magnetic particles; adding 11 mu L of 3-aminopropyl triethoxysilane into 30mL of the solution, reacting at room temperature for 30min, washing with water, and drying to obtain aminated Fe 3 O 4 @SiO 2 Magnetic particles;
(2) Attachment of magnetic particles to extracellular vesicles by antigen-antibody specific binding
30mg of aminated Fe 3 O 4 @SiO 2 The magnetic particles are evenly dispersed in glutaraldehyde solution with the volume fraction of 30mL being 1%, reacted for 0.5 hour at room temperature, washed twice to obtain surface activated Fe 3 O 4 @SiO 2 Magnetic particles, i.e. hydroformylation magnetic particles;
100 mu L of CD63 antibody with the concentration of 1mg/mL is added into 10mL of aldehyde magnetic particles with the concentration of 1mg/mL, and is connected to the surfaces of the magnetic particles through aldehyde-amine condensation reaction;
the EVs are connected to the surface of the magnetic particles through antigen-antibody specific binding to obtain EVs-Fe 3 O 4 @SiO 2 A complex;
(3) At EVs-Fe 3 O 4 @SiO 2 Growing a polymer recognition layer on the composite
Will be 24mgEVs-Fe 3 O 4 @SiO 2 Dispersing into 5mL of phosphoric acid buffer solution, adding 32 mu L of tetraethoxysilane, allowing to react at room temperature for 2 hours, then adding 12 mu L of 3-aminopropyl triethoxysilane (APTES), 18 mu L of hydroxymethyl triethoxysilane (HMTEOS), 12 mu L of Propyltriethoxysilane (PTES) and 12 mu L of Benzyl Triethoxysilane (BTES), and then continuing the reaction at 10 ℃ for 20 hours; after the reaction is finished, removing the supernatant through magnetic separation, and washing with deionized water for three times to obtain magnetic particles growing with an extracellular vesicle recognition layer;
(4) Template extracellular vesicles from which magnetic particles having an extracellular vesicle recognition layer grown are removed
Placing the magnetic particles growing with the extracellular vesicle recognition layer in an eluent, carrying out water bath ultrasonic treatment for 30min to remove the extracellular vesicles of the template, wherein the eluent is an aqueous solution containing 0.1mol/L HCl+0.01% Txiton X-100, and the ultrasonic power is 45HZ; and (3) continuing ultrasonic treatment of the product for 20min, washing with deionized water, and drying to obtain the magnetic imprinting material with extracellular vesicle imprinting on the surface.
Example 12
This embodiment differs from embodiment 11 in that: the antibody used in step 3 was a CD9 protein antibody.
Example 13
This embodiment differs from embodiment 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, and the method is as follows:
the plasma of healthy people is diluted by 1000 times by PBS, 1mL of the plasma is taken and evenly mixed with 2mg of extracellular vesicle magnetic imprinting material, the reaction is carried out for 20min at room temperature by shaking, after the reaction is finished, the supernatant is removed by magnetic separation, the plasma is washed twice by phosphate buffer solution, the final precipitate is placed in phosphate buffer solution containing 2.5% glutaraldehyde for incubation overnight and fixation at 4 ℃, and then 30%,50%,70%,80%,90%,95% and 100% ethanol gradient dehydration and drying are carried out. The scanning electron microscope image of the resulting final product is shown in fig. 6, where it can be seen that the imprint on the surface of the magnetic imprinting material has disappeared and many extracellular vesicle particles appear, indicating that the extracellular vesicle magnetic imprinting material can rapidly capture and collect extracellular vesicles from plasma.
Example 15
The ability of the extracellular vesicle magnetic imprinting material to repeatedly capture extracellular vesicles was verified as follows:
the plasma of healthy people is diluted by 1000 times by PBS, 5mL of the plasma is taken and evenly mixed with 10mg of extracellular vesicle magnetic imprinting material, the reaction is carried out for 20min by shaking at room temperature, and after the reaction is finished, the precipitate is collected by magnetic separation. And measuring the concentration of extracellular vesicles in the solution before and after capturing by using an enzyme-linked immunosorbent method, and calculating the capturing efficiency. The resulting pellet was then washed with an eluent, which was an aqueous solution containing 0.1mol/L HCl+0.01% Txiton X-100, to remove extracellular vesicles, followed by three separate washes with ethanol and water, which were used for the next capture round. The whole process is carried out for 5 times, and the experimental result shows that after the magnetic imprinting material is repeatedly used for 5 times, the magnetic imprinting material still maintains high capturing efficiency, and the calculated capturing efficiency for 5 times is shown in fig. 7.
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Claims (7)
1. The preparation method of the extracellular vesicle magnetic imprinting material is characterized by comprising the following steps of:
(1) Preparation of magnetic particles Fe 3 O 4 @SiO 2 Magnetic particles with particle diameters of 450-600 nm;
(2) Magnetic particle Fe with 3-aminopropyl triethoxy silane 3 O 4 @SiO 2 Amination is carried out on the surface;
(3) Para-aminated magnetic particles Fe 3 O 4 @SiO 2 Carrying out hydroformylation on the surface;
(4) Adding antibody, and connecting with magnetic particle Fe through aldehyde-amine condensation reaction 3 O 4 @SiO 2 The surface of the antibody is one of a CD9 antibody, a CD63 antibody or a CD81 antibody;
(5) Connecting the extracellular vesicles of the template on the surfaces of the magnetic particles through antigen-antibody specific binding, wherein the mass ratio of the extracellular vesicles to the activated magnetic particles is (0.5-5 μg): 1mg;
(6) Adding tetraethoxysilane, 3-aminopropyl triethoxysilane (APTES), hydroxymethyl triethoxysilane (HMTEOS), propyl Triethoxysilane (PTES) and Benzyl Triethoxysilane (BTES), and growing a polymer recognition layer on the surface of the magnetic particle along extracellular vesicles through siloxane polymerization reaction;
(7) And (3) 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.
2. The method for preparing extracellular vesicle magnetic imprinting material according to claim 1, wherein the magnetic particles prepared in the step (1) are prepared by dissolving ferric trichloride hexahydrate and sodium acetate in ethylene glycol, stirring uniformly, and reacting in an oven to form Fe 3 O 4 A nanoparticle; centrifugal washing of Fe 3 O 4 Drying the nano particles in a vacuum drying oven; ethyl orthosilicate and Fe 3 O 4 Adding the nano particles into a mixture of ethanol, water and ammonia water, stirring for reaction, washing with water, and drying to obtain Fe 3 O 4 @SiO 2 Magnetic particles.
3. The method of claim 1, wherein the step (2) of aminating the magnetic particles comprises first aminating Fe 3 O 4 @SiO 2 Adding magnetic particles into dispersing water, adding 3-aminopropyl triethoxysilane with volume fraction of 1% ‒% to obtain mixed solution, reacting at room temperature, washing with water, and drying to obtain aminated Fe 3 O 4 @SiO 2 Magnetic particles.
4. The method for preparing extracellular vesicle magnetic imprinting material according to claim 1, wherein in the step (3), glutaraldehyde is used to activate amino groups on the surface of magnetic particles through non-covalent bond, thereby obtaining aldehyde-based magnetic particles.
5. The method of preparing an extracellular vesicle magnetic imprinting material according to claim 1, wherein the step (6) comprises the steps of:
(61) 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 1mg: 1-10 mug;
(62) Adding 3-aminopropyl triethoxysilane (APTES), hydroxymethyl triethoxysilane (HMTEOS), propyl Triethoxysilane (PTES) and Benzyl Triethoxysilane (BTES) into the reaction product, and performing polymerization growth on the surface of the magnetic particle compound along the surface of extracellular vesicles 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 1mg: 1-10 mug.
6. The method for preparing the extracellular vesicle magnetic imprinting material according to claim 1, wherein the power of the template extracellular vesicle removed by ultrasonic cleaning in the step (7) is 25-45 hz, and the time of the ultrasonic cleaning is 50min.
7. Use of the extracellular vesicle magnetic imprinting material of claim 1 for in vitro isolation of extracellular vesicles.
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