CN109402052B - Preparation method and application of magnetic nanoparticles for capturing exosomes in blood - Google Patents
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Abstract
The invention discloses a preparation method and application of magnetic nanoparticles for capturing exosomes in blood, which are characterized by comprising the following steps: (1) mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate, dissolving in distilled water, stirring, uniformly mixing, heating to 70 ℃, then slowly dropwise adding an ammonia water solution, after hydrolysis is basically completed, slowly adding hexadecyltrimethylamine bromide into the solution, stirring, centrifuging, collecting a product, washing, taking a lower-layer precipitate, and drying in vacuum to obtain nano Fe3O4Particles; (2) taking nano Fe3O4Dispersing the particles in ethanol water solution, performing ultrasonic treatment, adding tetraethyl orthosilicate solution, gradually adding the mixture into ammonia water bath for reaction for 5 hours, centrifuging, collecting the product, washing and drying to obtain the product coated with SiO2And irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface of the porous membrane, so that the porous membrane has the advantages of maximally combining with exosomes without damage, small used sample amount, short treatment time and simple and convenient operation.
Description
Technical Field
The invention belongs to the field of tumor detection, and particularly relates to a preparation method and application of magnetic nanoparticles for capturing exosomes in blood.
Background
In recent years, the incidence of malignant tumors (also called cancers) in China has been remarkably increased due to changes in the environment and lifestyle. Meanwhile, the number of people dying from malignant tumor in China is increasing, and most of the phenomenon is attributed to that most patients have reached the middle and late stage of cancer when being diagnosed clearly and lose the best treatment opportunity. The survival rate of cancer patients in late stage after treatment is very low and the survival time is short, while the cure rate of cancer patients in early stage is high, about 80-90%. Therefore, early diagnosis is particularly important for cancer diagnosis and treatment. Currently, methods for diagnosing malignant tumors include endoscopic (EUS, PPS), imaging (B-ultrasound, CT, MRI, MRCP, PET, PIDUS, etc.), and Circulating Tumor Cell (CTCs) diagnosis. However, the position of part of malignant tumor is hidden, so that the recognition range of endoscope and imaging technology is limited, and the diagnosis effect is inaccurate. In addition, some traditional medical approaches and some invasive detection methods such as retrograde cholangiopancreatography (ERCP) have inherent drawbacks that are likely to cause complications. Therefore, there is a great need to find new, sensitive and easily applied detection methods and corresponding markers that can complement the existing diagnostic methods.
Detection of cellular exosomes may give new promise for early diagnosis of cancer. Exosomes are vesicles actively secreted by cells, originate from endosomes, have a diameter of about 40-200nm, and have a composition structure that a lipid bilayer membrane wraps a small amount of cytoplasm, and the cytoplasm contains various components of source cells, including a large amount of proteins, lipids, nucleic acids and the like. According to the statistics of exosome content database (http:// www.exocarta.org), it has been determined that 9769 proteins, 1116 lipids, 3408 mrnas and 2838 micro RNAs (micrornas, mirnas) exist in exosomes of different tissues and cell sources, including the common components common to exosomes and the components specific to exosome sources, and the contents play a key role in exosome-mediated intercellular substance and information exchange. In 2013, Cazzoli and the like take miRNAs (miR-378 a, miR-379, miR-139-5p and miR-200b-5 p) in detected plasma exosomes as a 'screening test' of lung cancer, and the results show that the sensitivity and specificity are 97.5% and 72% respectively, and the area under an ROC curve is 90.8%; 6 miRNAs (miR-151 a-5p, miR-30a-3p, miR-200b-5p, miR-629, miR-100 and miR-154-3 p) in exosomes are detected as a 'diagnosis test' of lung cancer, and the results show that the sensitivity and the specificity are 96.0% and 60.0% respectively, and the area under the ROC curve is 76.0%. In 2014, Ogata-Kawata et al compared the expression of 7 miRNAs (let-7 a, miR-1229, miR-1246, miR-150, miR-21, miR-223 and miR-23 a) in exosomes in the plasma of 88 colon cancer patients and 11 healthy control groups, and found that their expression in colon cancer patients was significantly higher than that in healthy control groups; for stage I colon cancer patients, miR-23a and miR-1246 also have high sensitivity, 95% and 90%, respectively, while the sensitivity of CA199 and CEA is only 10% and 15%, respectively; and 7 miRNAs of 29 colon cancer patients before and after the operation are detected, and the expression level after the operation is found to be remarkably reduced. Thus, the miRNAs expressed by the plasma exosomes can be used as a new early screening and diagnosis test.
The exosome enrichment method comprises the following steps: ultracentrifugation (Ultracentrifugation), Gradient Ultracentrifugation (Gradient Ultracentrifugation), coprecipitation (Co-precipitation), Size-Exclusion Chromatography (Size-Exclusion Chromatography), and Field Flow separation (Field Flow Fractionation), and the like. However, these methods have various disadvantages according to their respective enrichment principles: the required sample amount is large; the treatment time is long; the repetition rate is low; low efficiency processing capacity and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of magnetic nanoparticles for capturing exosomes in blood and application thereof, wherein the magnetic nanoparticles are combined with exosomes with the particle size of less than 200nm to the greatest extent without damage, and are small in used sample amount, short in processing time and simple and convenient to operate.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for preparing magnetic nanoparticles for capturing exosomes in blood, comprising the steps of:
(1) mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate according to the molar ratio of 1.6-2.0: 1, dissolving the mixture in distilled water to obtain a mixed solution, stirring and uniformly mixing the mixed solution, heating the mixed solution to 55-85 ℃, and then dropwise adding NH with the concentration of 0.3-0.5 mol/L3·H2And (3) O solution, after the hydrolysis of the mixed solution is finished, slowly adding 1.5-2.5 mL of surfactant into the mixed solution, stirring, centrifuging to collect a product, repeatedly washing the product until the pH is =7.0, removing supernatant, taking the lower-layer precipitate, and drying in vacuum at 50-70 ℃ for more than 16h to obtain the nano Fe3O4Particles;
(2) taking the nano Fe obtained in the step (1)3O4Dispersing the particles in a solution prepared by mixing ethanol and distilled water according to the volume ratio of 3-5: 1, carrying out ultrasonic treatment, adding a tetraethyl orthosilicate solution with the concentration of 0.15-0.25 mol/L, and stirring while stirringGradually adding 6-7.5 mL of 0.2-0.4 mol/L ammonia water, reacting in a constant-temperature water bath for more than 4h, centrifugally collecting a product, respectively washing the product with ethanol and deionized water for a plurality of times, placing the product in a vacuum drying oven for constant-temperature drying, and taking out a dried product to obtain the SiO coated product2Irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface.
The surfactant in the step (1) is hexadecyl trimethylamine bromide.
The method for capturing exosomes by using the magnetic nanoparticles comprises the following steps:
(1) the method comprises the following steps of (1) after a blood sample is gently inverted and uniformly mixed for several times, centrifuging 1900 Xg for 8-15 min at the temperature of 3-5 ℃, carefully sucking a supernatant, centrifuging a precipitate at the temperature of 3-5 ℃ for 10-20 min at 3000 Xg, carefully sucking the supernatant, and combining the supernatants obtained by two-time centrifugation;
(2) will wrap up SiO2Slowly dripping irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm into the supernatant obtained in the step (1), shaking and uniformly mixing, obliquely fixing the mixture on a shaking table at an angle of 40-50 degrees, and shaking at room temperature to form a magnetic particle compound by physical adsorption of exosomes and the magnetic micro-nano particles;
(3) and (3) carrying out high-efficiency enrichment on the magnetic particle compound obtained in the step (2) by using an immune magnetic particle capture instrument, collecting the released magnetic particle compound and the free granular exosome vesicles, adding a proper amount of PBS (phosphate buffer solution) with the pH of 7.0 into the released magnetic particle compound, placing the released magnetic particle compound on a shaking table, carrying out shaking elution at room temperature, and centrifugally collecting the exosome vesicles, namely the exosomes captured by the magnetic nanoparticles.
Compared with the prior art, the invention has the advantages that: the invention relates to a preparation method and application of magnetic nanoparticles for capturing exosomes in blood, and the prepared irregular magnetic nanoparticles with the surface distribution of 40-200nm pore diameters can be combined with exosomes with the particle size of less than 200nm to the greatest extent without damage. Compared with other exosome enrichment methods, the method has the advantages of small sample amount, short processing time and simple and convenient operation, can efficiently sort exosomes in blood, and is favorable for early diagnosis of cancer.
Drawings
FIG. 1 is SiO in example 12Scanning electron micrographs of the magnetic micro-nano particles before and after coating are carried out; a is SiO2Scanning electron microscope images of the magnetic micro-nano particles before coating; b is SiO2Scanning electron microscope picture of magnetic micro-nano granule after the cladding, the size: 500 nm;
FIG. 2 is a schematic diagram of the principle of the immunomagnetic particle capture exosomes in the present invention;
FIG. 3 is a schematic structural view of a magnetic particle composite prepared in example 3; in the figure, NP represents the magnetic particles of the irregular magnetic micro-nano particles, and E represents an exosome.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
A method for preparing magnetic nanoparticles for capturing exosomes in blood, comprising the steps of:
(1) 1.8mol of ferric chloride hexahydrate (FeCl)3·6H2O) and 1mol of ferrous chloride tetrahydrate (FeCl)2·4H2O) are mixed and dissolved in 100 mL of distilled water, the mixture is heated to 70 ℃ after being evenly stirred, and then NH with the concentration of 0.4mol/L is slowly dripped3·H2O solution, when the pH of the solution is increased to 6-7, the iron salt is hydrolyzed to generate a large amount of Fe3O4Crystal particles; continuously dropping NH3·H2When the pH value of O is increased to 9-10, the mixed solution gradually becomes black, and the hydrolysis tends to be complete; after the hydrolysis was substantially completed, 2 mL of Cetyltrimethylammonium Bromide (CTAB), a surfactant, was slowly added to the solution, stirred for 30 min, and centrifuged to collect the product; repeatedly washing the product with distilled water until pH =7.0, removing supernatant, and vacuum drying the lower layer precipitate at 60 deg.C for 24 h to obtain nanometer Fe3O4Particles;
(2) taking 1.16 g of nano Fe obtained in the step (1)3O4The particles are dispersed in 100 mL of a mixture of ethanol and distilled water at a volume ratio of 4:1Adding 50 mL Tetraethyl orthosilicate (TEOS) solution with the concentration of 0.02 mol/L into the solution after ultrasonic treatment for 15min, gradually adding 6.67 mL ammonia water with the concentration of 0.3mol/L while stirring, reacting for more than 4h in a constant-temperature water bath, centrifugally collecting a product, respectively washing the product with ethanol and deionized water for a plurality of times, placing the product in a vacuum drying oven for constant-temperature drying for 12 h, and taking out a dried product, namely the SiO coated product2Irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface.
Mixing SiO2And (3) observing the magnetic micro-nano particles before and after coating by using a Scanning Electron Microscope (SEM).
Analysis of results As shown in FIG. 1, SiO was observed using a scanning electron microscope2Magnetic micro-nano particles before and after coating, uncoated SiO2The magnetic particles have poor dispersibility and are easy to agglomerate. This may be related to the large van der waals forces between the partial magnetization of the particles and the magnetic micro-nano particles before encapsulation. The wrapped magnetic micro-nano particles are spherical, the particle sizes are relatively consistent, the distribution is uniform, the agglomeration degree is relatively low compared with that of the magnetic micro-nano particles before wrapping, and the dispersibility is good.
Example 2
The difference from the above example 1 is that:
in the step (1): mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate in a molar ratio of 1.6:1, dissolving in distilled water to obtain a mixed solution, stirring, uniformly mixing, heating to 55 ℃, and then dropwise adding NH with the concentration of 0.3mol/L3·H2And (3) O solution, when the hydrolysis of the mixed solution is finished, slowly adding 1.5 mL of surfactant into the mixed solution, stirring, centrifuging to collect the product, repeatedly washing the product until the pH is =7.0, removing the supernatant, taking the lower layer precipitate, and drying in vacuum at 50 ℃ for more than 16h to obtain the nano Fe3O4Particles;
in the step (2): taking the nano Fe obtained in the step (1)3O4Dispersing the particles in a solution prepared by mixing ethanol and distilled water according to the volume ratio of 3:1, performing ultrasonic treatment, adding tetraethyl orthosilicate solution with the concentration of 0.15 mol/L, and stirring while gradually stirringGradually adding 6 mL of 0.4mol/L ammonia water, reacting in a constant temperature water bath for more than 4h, centrifuging to collect the product, washing the product with ethanol and deionized water for several times, drying in a vacuum drying oven at constant temperature, and taking out the dried product to obtain the SiO coated product2Irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface. The prepared product is observed by a scanning electron microscope, and the wrapped magnetic micro-nano particles are spherical, have relatively consistent granularity and uniform distribution, and have lower agglomeration degree and good dispersibility compared with the magnetic micro-nano particles before wrapping.
Example 3
The difference from the above example 1 is that:
in the step (1): mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate in a molar ratio of 2.0:1, dissolving in distilled water to obtain a mixed solution, stirring, uniformly mixing, heating to 85 ℃, and then dropwise adding NH with the concentration of 0.5mol/L3·H2And (3) O solution, when the hydrolysis of the mixed solution is finished, slowly adding 2.5mL of surfactant into the mixed solution, stirring, centrifuging to collect the product, repeatedly washing the product until the pH is =7.0, removing the supernatant, taking the lower layer precipitate, and drying in vacuum at 70 ℃ for more than 16h to obtain the nano Fe3O4Particles;
in the step (2): taking the nano Fe obtained in the step (1)3O4Dispersing the particles in a solution prepared by mixing ethanol and distilled water according to the volume ratio of 5:1, carrying out ultrasonic treatment, adding tetraethyl orthosilicate solution with the concentration of 0.25mol/L, gradually adding 7.5mL ammonia water with the concentration of 0.2 mol/L while stirring, reacting for more than 4h in a constant-temperature water bath, centrifugally collecting a product, washing the product with ethanol and deionized water for a plurality of times, placing the product in a vacuum drying oven for constant-temperature drying, and taking out a dried product, namely the SiO coated product2Irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface. The prepared product is observed by a scanning electron microscope, and the wrapped magnetic micro-nano particles are spherical, have relatively consistent granularity and uniform distribution, and have lower agglomeration degree and good dispersibility compared with the magnetic micro-nano particles before wrapping.
Example 4
The method for detecting exosomes in blood using the magnetic nanoparticles of example 1 above, as shown in fig. 2, includes the following steps
(1) Liposomes of different particle sizes were selected as exosome mimics, set as follows: 1. the grain size is less than 200 nm; 2. the particle size is 200-400 nm;
(2) irregular magnetic micro-nano particles with surface distribution pore diameters of 40-200nm prepared in examples 1-3 were slowly dropped into a centrifuge tube containing an exosome mimetic, and shaken to be sufficiently mixed. Fixing the centrifugal tube on a shaker in an inclined way at an angle of 40-50 degrees (any angle within 40-50 degrees), shaking at room temperature and 120 rpm for 20min, and forming a magnetic particle compound by physical adsorption of the centrifugal tube and the shaker;
(3) performing high-efficiency enrichment on the formed magnetic particle compound by using an immune magnetic particle capture instrument, collecting the released magnetic particle compound and a free exosome analog (an exosome analog which is not combined with magnetic micro-nano particles), adding a proper amount of PBS (phosphate buffer solution) with the pH of 7.0 into the released magnetic particle compound, placing the compound on a shaking table for shaking elution at room temperature, and centrifugally collecting exosome vesicles, namely the exosomes captured by the magnetic nano particles;
(4) identifying the released magnetic particle compound and the exosome simulant by using a scanning electron microscope, and calculating the combination efficiency and the release efficiency of the exosome simulant with different particle sizes and the magnetic micro-nano particles;
(5) the number of the exosome mimics combined with the magnetic micro-nano particles = the total number of the exosome mimics-the number of free exosome mimics;
the binding efficiency formula = (number of exosome mimics bound to magnetic micro-nano particles/total number of exosome mimics) × 100%;
release efficiency formula = (number of exosomes mimics released by elution/number of exosomes mimics bound to magnetic micro-nano particles) × 100%;
and (4) analyzing results: irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm can be combined with an exosome simulant with the particle diameter less than 200nm, and the combination rate of the two is more than 60%. The irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm can only be combined with a small amount of exosome simulants with the particle diameter of 200-400 nm, and the combination rate of the two is about 15%. In addition, irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm can release particles with the particle diameter less than 200nm after elution of eluent, so that subsequent detection is facilitated.
Example 5
(1) Selecting supernatant secreted by a cell line, and incubating and combining the supernatant with the magnetic micro-nano particles to form a magnetic particle compound;
(2) using an immunomagnetic particle capture instrument (CellRich as capture instrument)TMAn immune magnetic particle capture instrument, the model of which is NLS-CR-001R 2), efficiently enriches the formed magnetic particle compound, and collects the released magnetic particle compound and free granular vesicles (vesicles not combined with magnetic micro-nano particles); eluting the released magnetic particle compound by using PBS buffer solution, and centrifugally collecting vesicles;
(3) the vesicles released after elution were identified using a scanning electron microscope (femina, model Pro-SE), a particle size analyzer Nanoparticle tracking analysis, NTA (malvern, uk, model Zetasizer Nano ZS) and Western Blotting (WB);
(4) and (4) SEM identification: respectively sucking 20-40 μ L of magnetic particle complex and vesicle released after elution, spotting on copper mesh, standing for 2 min, and sucking with filter paper from side; adding 20-40 μ L phosphotungstic acid on a copper net for negative dyeing, standing for 2 min, and sucking dry from the side by using filter paper; washing with 20-40 μ L PBS buffer solution for 1 time, and sucking from the side with filter paper; baking under an incandescent lamp for 20min, placing on a scanning electron microscope for magnifying observation, and taking a picture for recording;
(5) NTA identification: sucking 100 mu L of vesicles released after elution by a liquid transfer machine, slowly adding the vesicles to the bottom of an optical platform along the side wall of the optical measurement platform (so as to avoid interference of measurement results due to bubbles), placing the optical platform in a measuring instrument for starting measurement, and obtaining a measurement result by adopting a number average particle size counting method for reading;
(6) WB identification: taking 100 mu L of vesicle released after elution, adding equal volume of cell lysate, ultrasonically crushing for 30 min, centrifuging at 12000 rpm for 20min, taking supernate, adding a proper amount of 6 multiplied by sample loading buffer solution, and boiling for 10 min; taking 20 mu L of sample to carry out electrophoresis in 10SDS-PAGE gel, transferring protein to a PDVF membrane after the electrophoresis is finished, sealing the PDVF membrane by 3wt% BSA at 4 ℃ overnight, adding monoclonal antibodies such as mouse anti-human CD63, mouse anti-human CD9, mouse anti-human TSG101 and the like, and shaking the mixture overnight at 4 ℃; washing with 0.5wt% Tween-20 Tris-HCl buffer solution for 5min for 3 times; adding a horseradish peroxidase-labeled mouse secondary antibody, incubating at room temperature for 2 h, washing for 3 times with 0.5wt% Tween-20 Tris-HCl buffer solution for 5min each time, carrying out ECL chemiluminescence color development, and observing an experimental result.
As a result, as shown in fig. 3, when the magnetic microparticle composite is observed by using a scanning electron microscope, it can be seen that particles with different sizes are adsorbed on the irregular surface of the magnetic micro-nano particles, and the particles may be exosomes. And (4) carrying out SEM detection on the vesicles released after elution, and observing that the particle sizes of the particles are less than 200nm under a mirror. Meanwhile, the particle size of the vesicles released after elution was analyzed by NTA, and it was observed that they had a peak in the range of 40-200 nm. In addition, WB was used to detect that CD63, ALIX, CD9, CD81, TSG101 and HSP70 proteins in vesicles were able to be expressed. The results show that the magnetic micro-nano particles can adsorb exosomes with the particle size of 40-200 nm.
Example 6
(1) Peripheral blood of 6 tumor patients is extracted, each sample is subpackaged and detected within 24 hours, 5 mL/tube and 3 tubes are respectively obtained;
(2) the method comprises the following steps of (1) after a blood sample is gently inverted and uniformly mixed for several times, centrifuging 1900 Xg for 8-15 min at the temperature of 3-5 ℃, carefully sucking a supernatant, centrifuging a precipitate at the temperature of 3-5 ℃ for 10-20 min at 3000 Xg, carefully sucking the supernatant, and combining the supernatants obtained by two-time centrifugation;
(3) slowly dripping irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm prepared in the embodiment 1-3 into the extracted supernatant, and shaking and uniformly mixing; fixing the magnetic micro-nano particles on a shaking table in an inclined way at an angle of 40-45 degrees, shaking at room temperature and 120 rpm for 20min, and forming a magnetic particle compound by the exosome and the magnetic micro-nano particles through physical adsorption;
(4) performing high-efficiency enrichment on the formed magnetic particle compound by using an immune magnetic particle capture instrument, and collecting the released magnetic particle compound and free granular vesicles (vesicles not combined with the magnetic micro-nano particles); eluting the released magnetic particle compound and collecting the vesicles;
(5) identifying the vesicles released after elution by using a scanning electron microscope, a particle size analyzer and a western blotting method;
(6) and (4) SEM identification: respectively sucking 20-40 μ L of magnetic particle complex and vesicle released after elution, spotting on copper mesh, standing for 2 min, and sucking with filter paper from side; adding 20-40 μ L phosphotungstic acid on a copper net for negative dyeing, standing for 2 min, and sucking dry from the side by using filter paper; washing with 20-40 μ L PBS for 1 time, and sucking from the side with filter paper; baking under an incandescent lamp for 20min, placing on a scanning electron microscope for magnifying observation, and taking a picture for recording;
(7) NTA identification: sucking 100 mu L of vesicles released after elution by a liquid transfer machine, slowly adding the vesicles to the bottom of an optical platform along the side wall of the optical measurement platform (so as to avoid interference of measurement results due to bubbles), placing the optical platform in a measuring instrument for starting measurement, and obtaining a measurement result by adopting a number average particle size counting method for reading;
(8) WB identification: taking 100 mu L of vesicle released after elution, adding equal volume of cell lysate, ultrasonically crushing for 30 min, centrifuging at 12000 rpm for 20min, taking supernate, adding a proper amount of 6 multiplied by sample loading buffer solution, and boiling for 10 min; taking 20 mu L of sample to carry out electrophoresis in 10SDS-PAGE gel, transferring protein to a PDVF membrane after the electrophoresis is finished, sealing the PDVF membrane by 3 percent BSA at 4 ℃ for one night, adding monoclonal antibodies such as mouse anti-human CD63, mouse anti-human CD9, mouse anti-human TSG101 and the like, and shaking the mixture at 4 ℃ for one night; washing with 0.5% Tween 20 Tris-HCl buffer solution for 5min for 3 times; adding a horseradish peroxidase-labeled mouse secondary antibody, incubating at room temperature for 2 h, washing for 3 times with 0.5% Tween 20 Tris-HCl buffer solution for 5min each time, carrying out ECL chemiluminescence color development, and observing an experimental result; among them, blood sample information of 6 tumor patients is shown in table 1.
TABLE 1
And (4) analyzing results: after blood samples of 6 tumor patients and the magnetic micro-nano particles are incubated and combined, efficient enrichment is carried out through an immune magnetic particle capture instrument, and vesicles eluted and released by eluent are obtained. The released vesicle is an exosome and has the particle size less than 200nm, which is identified by a scanning electron microscope, a particle size analyzer and a western blot method.
According to the method for detecting exosomes in blood, the irregular magnetic micro-nano particles with the pore diameters of 40-200nm distributed on the surface can be combined with exosomes with the particle diameters of less than 200nm to the greatest extent without damage. Compared with other exosome enrichment methods, the method has the advantages of small sample amount, short processing time and simplicity and convenience in operation.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that such changes, modifications, additions and substitutions are within the true spirit of the invention.
Claims (2)
1. A method for preparing magnetic nanoparticles for capturing exosomes in blood, characterized by comprising the following steps:
(1) mixing ferric chloride hexahydrate and ferrous chloride tetrahydrate according to the molar ratio of 1.6-2.0: 1, dissolving the mixture in distilled water to obtain a mixed solution, stirring and uniformly mixing the mixed solution, heating the mixed solution to 55-85 ℃, and then dropwise adding NH with the concentration of 0.3-0.5 mol/L3·H2And (3) adding 1.5-2.5 mL of surfactant slowly into the mixed solution after the mixed solution is hydrolyzed, centrifuging and collecting a product after stirring, repeatedly washing the product until the pH value is 7.0, removing the supernatant, taking the lower-layer precipitate, and drying in vacuum at 50-70 ℃ for more than 16h to obtain the nano Fe3O4Particles; wherein the surfactant is cetyltrimethylammonium bromide;
(2) taking the nano Fe obtained in the step (1)3O4Dispersing the particles in a solution formed by mixing ethanol and distilled water according to the volume ratio of 3-5: 1, after ultrasonic treatment, adding tetraethyl orthosilicate solution with the concentration of 0.15-0.25 mol/L, and gradually adding 6-7.5 mL of concentrated solution while stirringReacting ammonia water with the temperature of 0.2-0.4 mol/L in a constant-temperature water bath for more than 4 hours, centrifugally collecting a product, respectively washing the product with ethanol and deionized water for a plurality of times, placing the product in a vacuum drying oven for constant-temperature drying, and taking out a dried product, namely the product wrapped with SiO2Irregular magnetic micro-nano particles with the pore diameter of 40-200nm are distributed on the surface.
2. A method for capturing exosomes using the magnetic nanoparticles of claim 1, the method not being directly used for diagnosis or therapy, characterized by comprising the steps of:
(1) the method comprises the following steps of (1) after a blood sample is gently inverted and uniformly mixed for several times, centrifuging 1900 Xg for 8-15 min at the temperature of 3-5 ℃, carefully sucking a supernatant, centrifuging a precipitate at the temperature of 3-5 ℃ for 10-20 min at 3000 Xg, carefully sucking the supernatant, and combining the supernatants obtained by two-time centrifugation;
(2) will wrap up SiO2Slowly dripping irregular magnetic micro-nano particles with the surface distribution pore diameter of 40-200nm into the supernatant obtained in the step (1), shaking and uniformly mixing, obliquely fixing the mixture on a shaking table at an angle of 40-50 degrees, and shaking at room temperature to form a magnetic particle compound by physical adsorption of exosomes and the magnetic micro-nano particles;
(3) and (3) carrying out high-efficiency enrichment on the magnetic particle compound obtained in the step (2) by using an immune magnetic particle capture instrument, collecting the released magnetic particle compound and the free granular exosome vesicles, adding a proper amount of PBS (phosphate buffer solution) with the pH of 7.0 into the released magnetic particle compound, placing the released magnetic particle compound on a shaking table, carrying out shaking elution at room temperature, and centrifugally collecting the exosome vesicles, namely the exosomes captured by the magnetic nanoparticles.
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| CN111358957B (en) * | 2020-03-06 | 2024-03-26 | 西安组织工程与再生医学研究所 | Magnetic nanoparticles |
| CN112717844B (en) * | 2020-12-10 | 2021-07-23 | 南京紫金山分子医学技术研究院有限公司 | Magnetic nano material for extracellular vesicle enrichment, preparation method and application thereof, and extracellular vesicle enrichment material |
| CN115992092B (en) * | 2023-02-18 | 2023-11-17 | 湖州科元生物科技有限公司 | Method for extracting exosomes based on transition metal oxyhydroxide |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1421455A (en) * | 2002-12-11 | 2003-06-04 | 湖南大学 | Fast DNA extracting and purifying process with shell magnetic nanoscale particle of aminated silica |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
| CN103908945A (en) * | 2014-03-31 | 2014-07-09 | 洛阳惠尔纳米科技有限公司 | Preparation method of magnetic beads by extracting nucleic acid and application of method |
| CN105628672A (en) * | 2015-12-17 | 2016-06-01 | 东南大学 | Method for quickly detecting exosomes through SERS signal |
| CN105642239A (en) * | 2016-03-17 | 2016-06-08 | 黑龙江省科学院高技术研究院 | Preparation method of magnetic micro-beads for extracting nucleic acid |
| WO2016015172A8 (en) * | 2014-07-29 | 2016-07-28 | 北京福纳康生物技术有限公司 | A method for treating cancer based on metallofullerene monocrystalline nanoparticles that specifically disrupt tumor blood vessels |
| WO2016201220A1 (en) * | 2015-06-10 | 2016-12-15 | Children's National Medical Center | Adipocyte-derived exosomes, and compositions, kits, and methods of using the same for detection and screening |
| CN106289926A (en) * | 2016-07-26 | 2017-01-04 | 华东理工大学 | A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body |
| CN107254430A (en) * | 2017-08-11 | 2017-10-17 | 上海浦美生物医药科技有限公司 | A kind of method based on positive charge adsorbing separation excretion body |
| CN107446797A (en) * | 2016-05-31 | 2017-12-08 | 陈欲超 | Excretion body process chip and processing method |
| CN110302752A (en) * | 2019-06-18 | 2019-10-08 | 湖南工业大学 | A kind of graphene oxide cladding Fe3O4Nano-cluster composite material and preparation method |
| CN112129939A (en) * | 2020-08-05 | 2020-12-25 | 宁波大学 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
| CN112121768A (en) * | 2020-09-30 | 2020-12-25 | 武汉儿童医院 | Amino magnetic bead, preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012121528A2 (en) * | 2011-03-04 | 2012-09-13 | Samsung Life Public Welfare Foundation | Magnetic nanocomposite specific for thyroid cancer and use thereof |
| CN111505264B (en) * | 2019-01-30 | 2024-05-17 | 上海思路迪生物医学科技有限公司 | Exosome separation method, immunomagnetic beads and kit |
-
2018
- 2018-10-15 CN CN201811196530.2A patent/CN109402052B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1421455A (en) * | 2002-12-11 | 2003-06-04 | 湖南大学 | Fast DNA extracting and purifying process with shell magnetic nanoscale particle of aminated silica |
| CN102229925A (en) * | 2011-05-13 | 2011-11-02 | 薛昱 | Enhanced magnetic-bead-based nucleic acid extraction method |
| CN103908945A (en) * | 2014-03-31 | 2014-07-09 | 洛阳惠尔纳米科技有限公司 | Preparation method of magnetic beads by extracting nucleic acid and application of method |
| WO2016015172A8 (en) * | 2014-07-29 | 2016-07-28 | 北京福纳康生物技术有限公司 | A method for treating cancer based on metallofullerene monocrystalline nanoparticles that specifically disrupt tumor blood vessels |
| WO2016201220A1 (en) * | 2015-06-10 | 2016-12-15 | Children's National Medical Center | Adipocyte-derived exosomes, and compositions, kits, and methods of using the same for detection and screening |
| CN105628672A (en) * | 2015-12-17 | 2016-06-01 | 东南大学 | Method for quickly detecting exosomes through SERS signal |
| CN105642239A (en) * | 2016-03-17 | 2016-06-08 | 黑龙江省科学院高技术研究院 | Preparation method of magnetic micro-beads for extracting nucleic acid |
| CN107446797A (en) * | 2016-05-31 | 2017-12-08 | 陈欲超 | Excretion body process chip and processing method |
| CN106289926A (en) * | 2016-07-26 | 2017-01-04 | 华东理工大学 | A kind of method using immuno magnetic cell separation serum China and foreign countries to secrete body |
| CN107254430A (en) * | 2017-08-11 | 2017-10-17 | 上海浦美生物医药科技有限公司 | A kind of method based on positive charge adsorbing separation excretion body |
| CN110302752A (en) * | 2019-06-18 | 2019-10-08 | 湖南工业大学 | A kind of graphene oxide cladding Fe3O4Nano-cluster composite material and preparation method |
| CN112129939A (en) * | 2020-08-05 | 2020-12-25 | 宁波大学 | Based on Fe3O4@SiO2@TiO2Method for detecting prostate cancer exosomes by using nanoparticle enrichment and PSMA sensor |
| CN112121768A (en) * | 2020-09-30 | 2020-12-25 | 武汉儿童医院 | Amino magnetic bead, preparation method and application thereof |
Non-Patent Citations (13)
| Title |
|---|
| A novel strategy for facile serum exosome isolation based on specific interactions between phospholipid bilayers and TiO2;Fangyuan Gao等;《Chemical science》;20181203;第10卷(第6期);第1579-1588页 * |
| Blood Exosomes Endowed with Magnetic and Targeting Properties for Cancer Therapy;Qi, Hongzhao等;《ACS NANO》;20160331;第10卷(第3期);第3323-3333页 * |
| Exosome purification based on PEG-coated Fe3O4 nanoparticles;Ming Chang等;《PLoS One》;20180622;第13卷(第6期);第1-13页 * |
| Microchip-based Immunomagnetic Detection of Circulating Tumor Cell;Kazunori Hoshino等;《Lab on a chip》;20110824;第11卷(第20期);第3449-3457页 * |
| Novel Magnetic Nanocluster@Graphene Oxide Composites for Potential Application in Environmental Adsorption;Zhongliang Hu等;《Journal of nanoscience and nanotechnology》;20200301;第20卷(第3期);第1814-1821页 * |
| PEG修饰降低磁性纳米球与细胞的非特异性吸附;董小娟;《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》;20200615(第6期);A006-204页 * |
| Personalized detection of circling exosomal PD-L1 based on Fe3O4@TiO2 isolation and SERS immunoassay，Yuanfeng Pang等，Biosensors and Bioelectronics;Yuanfeng Pang等;《Biosens Bioelectron》;20200115;第148卷;第1-9页 * |
| Treatment of infarcted heart tissue via the capture and local delivery of circulating exosomes through antibody-conjugated magnetic nanoparticles;LiuS, Chen X等;《Nature Biomedical Engineering》;20201130;第4卷(第11期);第1063-1075页 * |
| 功能化四氧化三铁的合成和表征及其对钙离子的吸附;周红等;《武汉工程大学学报》;20130619;第35卷(第4期);第1.2.1节 * |
| 四氧化三铁/二氧化硅复合磁性纳米粒子的制备与表征;朱霞萍等;《精细石油化工》;20110217;第27卷(第4期);第57页右栏第4段-第58页左栏第1段 * |
| 四氧化三铁纳米粒子合成及表征;张鑫等;《化学工业与工程》;20060323;第23卷(第1期);第46页左栏第2-4段 * |
| 基于环境***的Fe3O4@SiO2磁性分子印迹聚合物的制备及其性能研究;门海芬;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20130715(第7期);B014-86页 * |
| 外泌体分离纯化评估方法的建立;龚曼菲;《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》;20190715(第7期);A006-25页 * |
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