CN113652399B - Method for separating exosomes in cerebrospinal fluid - Google Patents
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Abstract
The invention discloses a method for separating exosomes in cerebrospinal fluid, which belongs to the technical field of nano materials, and particularly relates to a method for capturing and separating exosomes in cerebrospinal fluid through functionalized magnetic nano particles obtained by grafting with streptavidin after modification treatment and carboxylation treatment of the magnetic nano particles, and then through the functionalized magnetic nano particles, tim4 protein and calcium element; in the modification treatment, oleic acid, pteroic acid and alkanol lactate are added into the magnetic nanoparticle dispersion liquid for modification treatment. The functionalized magnetic nano particles prepared by the invention have large effective hydraulic diameter and good grafting effect of streptavidin on the functionalized magnetic nano particles. The method has good effect of capturing exosomes in cerebrospinal fluid, and the capturing efficiency is 15-22%.
Description
Technical Field
The invention belongs to the technical field of nano materials, and particularly relates to a method for separating exosomes in cerebrospinal fluid.
Background
Exosomes are small, abundant vesicles containing complex nucleic acid types and proteins, found in a variety of body fluids, including blood, saliva, urine, cerebrospinal fluid and milk, and are considered important carriers of cellular communication.
Exosomes are regular in morphology, spherical, hemispheric or saucer-like structures, exosome membranes are lipid bilayer similar to cell membranes, exosomes secreted by different types of cells have a number of commonalities, including lipid-rich and abundant transmembrane proteins on the membrane, such as CD63, CD81, etc. of the four transmembrane protein family members, and various RNAs contained within the membrane. The exosomes carry a large number and various bioactive molecules such as proteins, lipids, short peptide chains, DNA, RNA, miRNA, circular RNA and the like, exist in a very stable manner in an extracellular environment, and are similar to a mobile signal carrier storage pool in vivo, the content components of the exosomes are closely related to the tissue sources of secretory cells and the pathophysiological states of the secretory cells, and serve as intercellular transport carriers, and the intercellular transport carriers transport the bioactive substances contained in the exosomes to the target cells through membrane fusion or endocytosis, so that the functions of the target cells are influenced, metabolism of the target cells is regulated, and the exosomes participate in important pathophysiological processes such as immune response, inflammatory reaction, cell communication, apoptosis, cell migration, angiogenesis, tumor cell growth and the like, so that the functions of remote information transmission and regulation are realized.
Exosomes have demonstrated great potential in the fields of liquid biopsy and targeted therapy. Exosome oncology research shows that exosomes play an important role in mediating tumor recurrence and metastasis, and the means of targeting exosome signal pathways is used for realizing tumor treatment, thereby providing a new way for creating effective clinical diagnosis and treatment strategies.
Disclosure of Invention
The invention aims to provide a method for separating exosomes in cerebrospinal fluid, which has good capturing efficiency on exosomes in cerebrospinal fluid.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a method of isolating exosomes in cerebrospinal fluid, comprising: after modification treatment and carboxylation treatment of the magnetic nano particles, grafting with streptavidin to obtain functionalized magnetic nano particles, and then capturing and separating exosomes in cerebrospinal fluid through the functionalized magnetic nano particles, tim4 protein and calcium element; in the modification treatment, oleic acid, pteroic acid and alkanol lactate are added into the magnetic nanoparticle dispersion liquid for modification treatment. The particle size of the magnetic nano particles is small, the specific surface area is large, and the magnetic particles are easy to agglomerate due to the dipole-dipole action, oleic acid, pteroic acid and alkanol lactic acid ester are added into the magnetic nano particle dispersion liquid together to modify the magnetic nano particles, water-soluble molecular chains are wound on the surfaces of the magnetic nano particles through the actions of Van der Waals force, hydrogen bond, coordination bond or covalent bond and the like to form coated modified magnetic nano particles, and the corresponding treatment effect can be improved in the subsequent hydroxylation treatment process, so that functionalized magnetic nano particles with high effective hydraulic diameter are obtained, the streptavidin grafting amount on the functionalized magnetic nano particles is high, and the capturing effect on exosomes in cerebrospinal fluid is good.
Preferably, the magnetic nanoparticles are prepared from ferric chloride hexahydrate and ferrous chloride tetrahydrate.
Preferably, the carboxylation treatment is obtained by mixing and reacting an aqueous phase solution, an oil phase solution and other reagents.
More preferably, the aqueous solution comprises a tripropylene glycol ether, absolute ethanol and deionized water.
More preferably, styrene, acrylic acid, divinylbenzene are included in the oil phase solution.
More preferably, the other agent comprises an emulsifier and an initiator.
Preferably, the magnetic nanoparticle dispersion has a content of magnetic nanoparticles of 0.3 to 1.2wt%.
Preferably, the pteroic acid is added in an amount of 0.1 to 0.4wt% of the magnetic nanoparticle dispersion.
Preferably, the amount of the alkanol lactate added is 0.2-0.6wt% of the magnetic nanoparticle dispersion.
Preferably, the calcium element is from a calcium chloride solution.
Preferably, in the synthesis of the magnetic nano particles, ferric chloride hexahydrate and ferrous chloride tetrahydrate are added into deionized water, nitrogen is introduced to remove oxygen, then ammonia water is added, the mixture is reacted for 0.5 to 3 hours at the temperature of 40 to 60 ℃, and the mixture is subjected to magnet separation and washing to obtain the ferroferric oxide nano particles.
More preferably, the ferric chloride hexahydrate is added in an amount of 0.2 to 0.6wt% of deionized water.
More preferably, ferrous chloride tetrahydrate is used in an amount of 36.67-46.67wt% of ferric chloride hexahydrate.
More preferably, the ammonia is added in an amount of 4.15 to 12.45wt% of deionized water.
Preferably, in the preparation of the modified magnetic nano particles, the magnetic nano particles are magnetically separated, added into deionized water, ultrasonically dispersed to obtain a magnetic nano particle dispersion liquid, introduced with nitrogen to deoxidize, added with oleic acid, pteroic acid and alkanol lactate at the temperature of 60-80 ℃ for 2-6h, washed and dried with nitrogen to obtain the modified magnetic nano particles.
More preferably, the content of magnetic nanoparticles in the magnetic nanoparticle dispersion is 0.3 to 1.2wt%.
More preferably, oleic acid is added in an amount of 0.9 to 1.6wt% of the magnetic nanoparticle dispersion.
More preferably, the pteroic acid is added in an amount of 0.1 to 0.4wt% of the magnetic nanoparticle dispersion.
More preferably, the amount of alkanol lactate added is 0.2 to 0.6wt% of the magnetic nanoparticle dispersion.
Preferably, in the preparation of carboxylated magnetic nanoparticles, modified magnetic nanoparticles are dispersed in aqueous phase solution to obtain magnetic fluid, then oil phase solution is added, emulsifying agent and prenyl acetate are added, initiator is added, stirring and dispersing are carried out for 10-30min, reaction is carried out for 6-24h at 70-80 ℃ under the protection of nitrogen, and after the reaction is completed, magnetic separation and washing are carried out to obtain carboxylated magnetic nanoparticles.
More preferably, the content of tripropylene glycol ether in the aqueous solution is 10-15wt%.
More preferably, the content of absolute ethanol in the aqueous phase solution is 70-80wt%.
More preferably, the modified magnetic nanoparticles are used in an amount of 10-20wt% of the aqueous phase solution.
More preferably, the styrene content of the oil phase solution is 76-85wt%.
More preferably, the content of acrylic acid in the oil phase solution is 5 to 12wt%.
More preferably, the oil phase solution is used in an amount of 20 to 30wt% of the aqueous phase solution.
More preferably, the emulsifier is SDS and the amount of emulsifier used is 0.11-0.35wt% of the aqueous phase solution.
More preferably, the prenyl acetate is used in an amount of 0.1 to 0.6wt% of the aqueous phase solution. The prenyl acetate is used, through interaction with the modified magnetic nanoparticles, the carboxylation treatment effect is improved, the effective hydraulic diameter of the functionalized magnetic nanoparticles and the grafting amount of streptavidin are further improved, and when the functionalized magnetic nanoparticles are used for separating exosomes in cerebrospinal fluid, the capturing efficiency of the exosomes in the cerebrospinal fluid can be improved.
More preferably, the initiator is sodium persulfate and the amount of initiator used is 0.3 to 2.1wt% of the oil phase solution.
Preferably, in the preparation of the functionalized magnetic nano particles, the carboxylated magnetic nano particles are added into MES solution, ultrasonic dispersion is carried out to obtain carboxylated magnetic nano particle dispersion liquid, streptavidin is added, EDC solution is added, oscillation reaction is carried out for 2-5h, and after the reaction is completed, the MES solution is cleaned to obtain the functionalized magnetic nano particles.
More preferably, the solvent of the MES solution is deionized water, the content of MES in the MES solution is 0.13-0.39wt%, and the pH of the MES solution is 6-7.
More preferably, the carboxylated magnetic nanoparticle dispersion has a carboxylated magnetic nanoparticle content of 0.1 to 0.6wt%.
More preferably, streptavidin is used in an amount of 1-3wt% of carboxylated magnetic nanoparticles.
More preferably, the EDC solution is formulated by adding EDC to an MES solution, the EDC content of the EDC solution being 1.9-5.7wt%.
More preferably, the EDC solution is used in an amount of 1.2 to 3.6wt% of the carboxylated magnetic nanoparticle dispersion.
Preferably, in the separation of exosomes, cerebrospinal fluid is filtered by a filter membrane, functionalized magnetic nano particles, tim4 protein and calcium chloride solution are added into the cerebral spinal filtrate, the mixture is oscillated for 24-96 hours at the temperature of 1-5 ℃, the mixture is stood for 5-30min, the supernatant is removed, and the magnetic nano particles for capturing exosomes are obtained through separation. The captured exosomes can be used for further detection analysis.
More preferably, the functionalized magnetic nanoparticles are used in an amount of 0.3-1.2wt% of the brain ridge filtrate.
More preferably, the Tim4 protein is used in an amount of 0.1-0.6wt% of the cerebral spinal filtrate.
More preferably, the concentration of the calcium chloride solution is 0.01 to 0.08wt%.
More preferably, the calcium chloride solution is used in an amount of 30-50wt% of the brain ridge filtrate.
The functionalized magnetic nano particles are obtained by grafting with streptavidin after modification treatment and carboxylation treatment of the magnetic nano particles, and are used for separating exosomes in the gum ridge liquid, so that the method has the following beneficial effects: the effective hydraulic diameter of the functionalized magnetic nano particles is large, and is 1400-1750nm; the grafting effect of streptavidin on the functionalized magnetic nano particles is good, wherein the absorbance of the protein test is 0.35-0.5; the method has good effect of capturing exosomes in cerebrospinal fluid, and the capturing efficiency is 15-22%. Therefore, the method for separating the exosomes in the cerebrospinal fluid has good capturing efficiency on the exosomes in the cerebrospinal fluid.
Drawings
FIG. 1 is a graph of effective hydraulic diameter of functionalized magnetic nanoparticles;
FIG. 2 is a graph showing the results of a streptavidin graft assay on functionalized magnetic nanoparticles;
FIG. 3 is a graph of exosome capture efficiency in cerebrospinal fluid.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments and the attached drawings:
example 1:
a method for separating exosomes in cerebrospinal fluid,
synthesis of magnetic nanoparticles: adding ferric chloride hexahydrate and ferrous chloride tetrahydrate into deionized water, introducing nitrogen to remove oxygen, then adding ammonia water, reacting for 2 hours at 60 ℃, separating by a magnet, and washing to obtain the ferroferric oxide nano particles. The addition amount of ferric chloride hexahydrate is 0.6 weight percent of deionized water, the use amount of ferrous chloride tetrahydrate is 46.67 weight percent of ferric chloride hexahydrate, and the addition amount of ammonia water is 4.15 weight percent of deionized water.
Modified magnetic nanoparticles: magnetic separation of magnetic nano particles is carried out, the magnetic nano particles are added into deionized water, ultrasonic dispersion is carried out to obtain magnetic nano particle dispersion liquid, nitrogen is introduced to remove oxygen, oleic acid, pteroic acid and alkanol lactate are added at the temperature of 80 ℃, after reaction is carried out for 4 hours, washing and nitrogen blow-drying are carried out, and modified magnetic nano particles are obtained. The content of the magnetic nanoparticles in the magnetic nanoparticle dispersion liquid is 0.9wt%, the addition amount of oleic acid is 1.6wt% of the magnetic nanoparticle dispersion liquid, the addition amount of pteroic acid is 0.14wt% of the magnetic nanoparticle dispersion liquid, and the addition amount of alkanol lactate is 0.26wt% of the magnetic nanoparticle dispersion liquid.
Carboxylated magnetic nanoparticles preparation: dispersing the modified magnetic nano particles in aqueous phase solution to obtain magnetic fluid, then adding oil phase solution, adding emulsifier, adding initiator, stirring and dispersing for 10min, reacting for 12h at 80 ℃ under the protection of nitrogen, magnetically separating and washing after the reaction is completed to obtain carboxylated magnetic nano particles. The water phase solution comprises tripropylene glycol ether, absolute ethyl alcohol and deionized water, wherein the content of tripropylene glycol ether in the water phase solution is 15wt%, the content of absolute ethyl alcohol in the water phase solution is 80wt%, and the use amount of the modified magnetic nano particles is 20wt% of the water phase solution; the oil phase solution comprises styrene, acrylic acid and divinylbenzene, the content of the styrene in the oil phase solution is 85wt%, the content of the acrylic acid in the oil phase solution is 9wt%, and the use amount of the oil phase solution is 26wt% of the water phase solution; the emulsifier is SDS, and the use amount of the emulsifier is 0.28wt% of the aqueous phase solution; the initiator is sodium persulfate, and the use amount of the initiator is 0.8 weight percent of the oil phase solution.
Functionalized magnetic nanoparticles: adding carboxylated magnetic nanoparticles into MES solution, performing ultrasonic dispersion to obtain carboxylated magnetic nanoparticle dispersion liquid, adding streptavidin, adding EDC solution, performing oscillation reaction for 3h, and cleaning the MES solution after the reaction is completed to obtain the functionalized magnetic nanoparticles. The solvent of the MES solution is deionized water, the content of MES in the MES solution is 0.28 weight percent, and the pH of the MES solution is 6.5; the carboxylated magnetic nanoparticle dispersion liquid contains 0.45 weight percent of carboxylated magnetic nanoparticles, the consumption of streptavidin is 2 weight percent of carboxylated magnetic nanoparticles, the EDC solution is prepared by adding EDC into MES solution, the EDC content in the EDC solution is 5.7 weight percent, and the consumption of the EDC solution is 3.6 weight percent of carboxylated magnetic nanoparticle dispersion liquid.
Exosome separation: filtering cerebrospinal fluid with 0.22 μm filter membrane, adding functional magnetic nanoparticle, tim4 protein and calcium chloride solution into the filtrate, oscillating at 4deg.C for 48 hr, standing for 20min, discarding supernatant, and separating to obtain magnetic nanoparticle for capturing exosome. The usage amount of the functionalized magnetic nano particles is 0.9wt% of the cerebral spinal filtrate, the usage amount of Tim4 protein is 0.4wt% of the cerebral spinal filtrate, the concentration of the calcium chloride solution is 0.06wt%, and the usage amount of the calcium chloride solution is 40wt% of the cerebral spinal filtrate.
Example 2:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 1 only in that in the modified magnetic nanoparticle preparation step, the amount of pteroic acid added was 0.21wt% of the magnetic nanoparticle dispersion, and the amount of alkanol lactate added was 0.36wt% of the magnetic nanoparticle dispersion.
Example 3:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 1 only in that in the modified magnetic nanoparticle preparation step, the amount of pteroic acid added was 0.32wt% of the magnetic nanoparticle dispersion, and the amount of alkanol lactate added was 0.49wt% of the magnetic nanoparticle dispersion.
Example 4:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 1 only in that in the modified magnetic nanoparticle preparation step, the amount of oleic acid added was 1.0wt% of the magnetic nanoparticle dispersion, the amount of pteroic acid added was 0.36wt% of the magnetic nanoparticle dispersion, and the amount of alkyl lactate added was 0.24wt% of the magnetic nanoparticle dispersion.
Example 5:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 1 only in that in the modified magnetic nanoparticle preparation step, the amount of oleic acid added was 1.3wt% of the magnetic nanoparticle dispersion, the amount of pteroic acid added was 0.26wt% of the magnetic nanoparticle dispersion, and the amount of alkyl lactate added was 0.46wt% of the magnetic nanoparticle dispersion.
Example 6:
a method for separating exosomes in cerebrospinal fluid,
synthesis of magnetic nanoparticles: adding ferric chloride hexahydrate and ferrous chloride tetrahydrate into deionized water, introducing nitrogen to remove oxygen, then adding ammonia water, reacting for 2 hours at 60 ℃, separating by a magnet, and washing to obtain the ferroferric oxide nano particles. The addition amount of ferric chloride hexahydrate is 0.6 weight percent of deionized water, the use amount of ferrous chloride tetrahydrate is 46.67 weight percent of ferric chloride hexahydrate, and the addition amount of ammonia water is 4.15 weight percent of deionized water.
Modified magnetic nanoparticles: magnetic separation of magnetic nano particles is carried out, the magnetic nano particles are added into deionized water, ultrasonic dispersion is carried out to obtain magnetic nano particle dispersion liquid, nitrogen is introduced to remove oxygen, oleic acid, pteroic acid and alkanol lactate are added at the temperature of 80 ℃, after reaction is carried out for 4 hours, washing and nitrogen blow-drying are carried out, and modified magnetic nano particles are obtained. The content of the magnetic nanoparticles in the magnetic nanoparticle dispersion liquid is 0.9wt%, the addition amount of oleic acid is 1.6wt% of the magnetic nanoparticle dispersion liquid, the addition amount of pteroic acid is 0.32wt% of the magnetic nanoparticle dispersion liquid, and the addition amount of alkanol lactate is 0.49wt% of the magnetic nanoparticle dispersion liquid.
Carboxylated magnetic nanoparticles preparation: dispersing the modified magnetic nano particles in aqueous phase solution to obtain magnetic fluid, then adding oil phase solution, adding emulsifier and prenyl acetate, adding initiator, stirring and dispersing for 10min, reacting for 12h at 80 ℃ under the protection of nitrogen, magnetically separating after the reaction is completed, and washing to obtain carboxylated magnetic nano particles. The water phase solution comprises tripropylene glycol ether, absolute ethyl alcohol and deionized water, wherein the content of tripropylene glycol ether in the water phase solution is 15wt%, the content of absolute ethyl alcohol in the water phase solution is 80wt%, and the use amount of the modified magnetic nano particles is 20wt% of the water phase solution; the oil phase solution comprises styrene, acrylic acid and divinylbenzene, the content of the styrene in the oil phase solution is 85wt%, the content of the acrylic acid in the oil phase solution is 9wt%, and the use amount of the oil phase solution is 26wt% of the water phase solution; the emulsifier is SDS, and the use amount of the emulsifier is 0.28wt% of the aqueous phase solution; the amount of prenyl acetate used was 0.16wt% of the aqueous phase solution; the initiator is sodium persulfate, and the use amount of the initiator is 0.8 weight percent of the oil phase solution.
Functionalized magnetic nanoparticles: adding carboxylated magnetic nanoparticles into MES solution, performing ultrasonic dispersion to obtain carboxylated magnetic nanoparticle dispersion liquid, adding streptavidin, adding EDC solution, performing oscillation reaction for 3h, and cleaning the MES solution after the reaction is completed to obtain the functionalized magnetic nanoparticles. The solvent of the MES solution is deionized water, the content of MES in the MES solution is 0.28 weight percent, and the pH of the MES solution is 6.5; the carboxylated magnetic nanoparticle dispersion liquid contains 0.45 weight percent of carboxylated magnetic nanoparticles, the consumption of streptavidin is 2 weight percent of carboxylated magnetic nanoparticles, the EDC solution is prepared by adding EDC into MES solution, the EDC content in the EDC solution is 5.7 weight percent, and the consumption of the EDC solution is 3.6 weight percent of carboxylated magnetic nanoparticle dispersion liquid.
Exosome separation: filtering cerebrospinal fluid with 0.22 μm filter membrane, adding functional magnetic nanoparticle, tim4 protein and calcium chloride solution into the filtrate, oscillating at 4deg.C for 48 hr, standing for 20min, discarding supernatant, and separating to obtain magnetic nanoparticle for capturing exosome. The usage amount of the functionalized magnetic nano particles is 0.9wt% of the cerebral spinal filtrate, the usage amount of Tim4 protein is 0.4wt% of the cerebral spinal filtrate, the concentration of the calcium chloride solution is 0.06wt%, and the usage amount of the calcium chloride solution is 40wt% of the cerebral spinal filtrate.
Example 7:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 6 only in that in the carboxylated magnetic nanoparticle preparation, prenyl acetate is used in an amount of 0.48wt% of the aqueous phase solution.
Example 8:
a method for separating exosomes in cerebrospinal fluid,
the present example differs from example 7 only in that in the preparation of carboxylated magnetic nanoparticles, the aqueous solution includes tripropylene glycol ether, absolute ethyl alcohol and deionized water, the content of tripropylene glycol ether in the aqueous solution is 13wt%, the content of absolute ethyl alcohol in the aqueous solution is 75wt%, and the amount of modified magnetic nanoparticles used is 16wt% of the aqueous solution.
Example 9:
a method for separating exosomes in cerebrospinal fluid,
the present example differs from example 7 only in that in the preparation of carboxylated magnetic nanoparticles, styrene, acrylic acid, divinylbenzene were included in the oil phase solution, the content of styrene in the oil phase solution was 76wt%, the content of styrene in the oil phase solution was 6wt%, and the amount of use of the oil phase solution was 25wt% of the aqueous phase solution.
Example 10:
a method for separating exosomes in cerebrospinal fluid,
this example differs from example 7 only in that the content of tripropylene glycol ether in the aqueous solution in carboxylated magnetic nanoparticle preparation is 18wt% and the prenyl acetate is used in an amount of 0.36wt% of the aqueous solution.
Comparative example 1:
a method for separating exosomes in cerebrospinal fluid,
this comparative example differs from example 3 only in that no alkanol lactate was added in the modified magnetic nanoparticle preparation step.
Comparative example 2:
a method for separating exosomes in cerebrospinal fluid,
this comparative example differs from example 3 only in that no pteroic acid was added in the modified magnetic nanoparticle preparation step.
Comparative example 3:
a method for separating exosomes in cerebrospinal fluid,
this comparative example differs from example 3 only in that pteroic acid and alkanol lactate were not added in the modified magnetic nanoparticle preparation step.
Test example 1:
1. particle size of nanoparticle
Test sample: the functionalized magnetic nanoparticles prepared in each example and comparative example.
The testing method comprises the following steps: dynamic light scattering DLS characterizes the particle size of the test sample.
The effective hydraulic diameter test result of the functionalized magnetic nano particles is shown in fig. 1, wherein the effective hydraulic diameter of the functionalized magnetic nano particles prepared by the method of the embodiment 3 is 1579.64nm, the effective hydraulic diameter of the functionalized magnetic nano particles prepared by the method of the comparative embodiment 3 is 1267.34nm, and compared with the comparative embodiment 3, the embodiment 3 shows that in the preparation of the modified magnetic nano particles, the effective hydraulic diameter of the functionalized magnetic nano particles prepared by the subsequent steps is greatly improved after the magnetic nano particles are treated by the common use of the pteroic acid, the alkanol lactate and the oleic acid, and the use of the pteroic acid and the alkanol lactate has remarkable effects; the effective hydraulic diameter of the functionalized magnetic nanoparticle prepared by the method of comparative example 1 is 1277.16nm, and example 3 compared with comparative example 1 shows that when the modified magnetic nanoparticle is prepared by the method of the invention, the joint use of the pteroic acid and the alkanol lactate is better than the single use of the pteroic acid; the effective hydraulic diameter of the functionalized magnetic nanoparticle prepared by the method of comparative example 2 is 1283.78nm, which shows that when the modified magnetic nanoparticle is prepared by the method of the invention, the common use of the pteroic acid and the alkanol lactate is superior to the single use of the alkanol lactate; example 3 shows that the improvement effect on the effective hydraulic diameter of the obtained functionalized magnetic nanoparticles is very weak when the pteroic acid and the alkanol lactate are replaced with either one of the two substances, compared with comparative examples 1-3, and the improvement on the effective hydraulic diameter of the obtained functionalized magnetic nanoparticles is very remarkable when the pteroic acid and the alkanol lactate are used together and the oleic acid exists. Examples 6-7 compared with example 3 demonstrate that the use of prenyl acetates in the preparation of carboxylated magnetic nanoparticles has an increasing effect on the effective hydraulic diameter of the functionalized magnetic nanoparticles obtained by subsequent preparation.
The effective hydraulic diameter of the functionalized magnetic nano particles prepared by the invention is 1400-1750nm.
2. Grafting amount of functionalized magnetic nanoparticles
Test sample: the functionalized magnetic nanoparticles prepared in each example and comparative example.
The testing method comprises the following steps: the grafting amount on the test sample was characterized by using the BCA protein concentration assay kit. The amount of grafted streptavidin was characterized by absorbance. Carboxylated magnetic nanoparticles without grafted streptavidin were set as a blank, and the blank absorbance was subtracted from the protein absorbance characterizing the amount of streptavidin grafted, below.
The value of the streptavidin grafted on the functionalized magnetic nanoparticle is characterized by protein absorbance, the test result of the protein absorbance is shown in figure 2, wherein the protein absorbance of the functionalized magnetic nanoparticle prepared by the method of example 3 is 0.41, the protein absorbance of the functionalized magnetic nanoparticle prepared by the method of comparative example 3 is 0.32, and compared with comparative example 3, example 3 shows that in the preparation of the modified magnetic nanoparticle, the modified magnetic nanoparticle is treated by using pteroic acid, alkanol lactate and oleic acid together, and the grafting amount of the streptavidin prepared by the subsequent steps is improved, thus showing that the use of pteroic acid and alkanol lactate has remarkable effects; the absorbance of the functionalized magnetic nanoparticle prepared by the method of comparative example 1 is 0.33, and example 3 compared with comparative example 1 shows that the co-use of pteroic acid and alkanol lactate is superior to the single use of pteroic acid when preparing modified magnetic nanoparticles by the method of the invention; the absorbance of the functionalized magnetic nanoparticle prepared by the method of comparative example 2 is 0.34, which indicates that the common use of pteroic acid and alkanol lactate is better than the single use of alkanol lactate when preparing the modified magnetic nanoparticle according to the method of the invention; example 3 shows that the effect of increasing the amount of streptavidin grafted on the functionalized magnetic nanoparticles obtained was very weak when the pteroic acid and the alkanol lactate were replaced with either of the two substances, compared with comparative examples 1-3, whereas the amount of streptavidin grafted on the functionalized magnetic nanoparticles obtained was very significant when the pteroic acid and the alkanol lactate were used together and in the presence of oleic acid. Examples 6-7 compared with example 3 demonstrate that the use of prenyl acetates in the preparation of carboxylated magnetic nanoparticles has an improved effect on the amount of streptavidin grafted on the functionalized magnetic nanoparticles obtained by subsequent preparation.
The grafting amount of streptavidin on the functionalized magnetic nano particles prepared by the invention is expressed by protein absorbance, and the absorbance is 0.35-0.5.
3. Exosome capture efficiency
The testing method comprises the following steps: the methods of the examples and comparative examples.
When the content of exosomes in cerebrospinal fluid is detected by the method of the invention, functionalized magnetic nano-particles and Tim4 protein are added to be subjected to oscillation treatment, and then the exosomes can be captured by the functionalized magnetic nano-particles and the Tim4 protein.
Protein concentration test method after capture: the exosomes are eluted by the test sample with an eluent, and then the protein concentration is detected by using a method of BCA protein concentration determination kit.
Protein concentration test method before capture: the filtered cerebrospinal fluid is used for detecting the protein concentration by adopting a BCA protein concentration detection kit method.
Exosome capture efficiency = protein absorbance of captured exosomes/protein absorbance in pre-capture brain ridge filtrate x 100%.
The test result of the method for capturing the cerebrospinal fluid exosome is shown in fig. 3, wherein the capturing efficiency of the method of the embodiment 3 for capturing the cerebrospinal fluid exosome is 18.08%, and the capturing efficiency of the method of the comparative embodiment 3 for capturing the cerebrospinal fluid exosome is 13.65%, compared with the method of the comparative embodiment 3, the embodiment 3 shows that in the preparation of the modified magnetic nano particles, the magnetic nano particles are treated by the common use of the pteroic acid, the alkanol lactate and the oleic acid, and then the functionalized magnetic nano particles are prepared through the subsequent steps, and the capturing efficiency of the obtained functionalized magnetic nano particles, tim4 protein and calcium chloride for capturing the cerebrospinal fluid exosome is greatly improved, so that the use of the pteroic acid and the alkanol lactate has a remarkable effect; the capturing efficiency of the method of comparative example 1 on the cerebrospinal fluid exosome is 14.18%, and example 3 compared with comparative example 1 shows that when the modified magnetic nanoparticles are prepared according to the method of the invention, the co-use of the pteroic acid and the alkanol lactate is superior to the single use of the pteroic acid; the capturing efficiency of the method of comparative example 2 on the cerebrospinal fluid exosome is 13.8%, which shows that when the modified magnetic nanoparticle is prepared according to the method of the invention, the joint use of the pteroic acid and the alkanol lactate is superior to the single use of the alkanol lactate; example 3 shows that when the pteroic acid and the alkanol lactate are replaced by either one of the two substances, the capturing rate of the obtained functionalized magnetic nano particles for capturing exosomes in cerebrospinal fluid according to the method of the invention is improved, and the capturing rate of the obtained functionalized magnetic nano particles for capturing exosomes in cerebrospinal fluid according to the method of the invention is obviously improved when the pteroic acid and the alkanol lactate are used together and oleic acid exists. Examples 6-7 compared with example 3 demonstrate that the use of prenyl acetates in the preparation of carboxylated magnetic nanoparticles has an improved effect on the efficiency of capturing exosomes in cerebrospinal fluid by the method of the invention for the subsequent preparation of functionalized magnetic nanoparticles.
The method has good effect of capturing exosomes in cerebrospinal fluid, and the capturing efficiency is 15-22%.
The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.
Claims (5)
1. A method of isolating exosomes in cerebrospinal fluid, comprising: after modification treatment and carboxylation treatment of the magnetic nano particles, grafting with streptavidin to obtain functionalized magnetic nano particles, and then capturing and separating exosomes in cerebrospinal fluid through the functionalized magnetic nano particles, tim4 protein and calcium element; in the modification treatment, oleic acid, pteroic acid and alkanol lactate are added into a magnetic nanoparticle dispersion liquid to carry out modification treatment; the magnetic nano particles are prepared from ferric chloride hexahydrate and ferrous chloride tetrahydrate; the carboxylation is carried out by mixing aqueous phase solution, oil phase solution and other reagents, reacting, wherein the aqueous phase solution is tripropylene glycol ether, absolute ethyl alcohol and deionized water, the oil phase solution is styrene, acrylic acid and divinylbenzene, and the other reagents are emulsifying agent and initiator.
2. A method for separating exosomes in cerebrospinal fluid according to claim 1, wherein: the content of the magnetic nano particles in the magnetic nano particle dispersion liquid is 0.3-1.2wt%.
3. A method for separating exosomes in cerebrospinal fluid according to claim 1, wherein: the addition amount of the pteroic acid is 0.1-0.4wt% of the magnetic nanoparticle dispersion liquid.
4. A method for separating exosomes in cerebrospinal fluid according to claim 1, wherein: the addition amount of the alkanol lactate is 0.2-0.6wt% of the magnetic nanoparticle dispersion liquid.
5. A method for separating exosomes in cerebrospinal fluid according to claim 1, wherein: the calcium element is from a calcium chloride solution.
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