CN113358618A - Exosome capturing method based on surface multi-thorn coding microspheres - Google Patents
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Abstract
The invention discloses an exosome capturing method based on surface multi-thorn coding microspheres, the silicate colloidal crystal microspheres adopted by the method have bright structural color and stable coding, the specific surface area is greatly improved due to the unique surface with thorn structure, more antibody probes can be coupled, the sensitivity is higher than that of the conventional photonic crystal microspheres when the silicate colloidal crystal microspheres are used for detection, and the exosome capturing rate is extremely high due to the fact that the silicate colloidal crystal microspheres can adsorb exosomes due to the self structural characteristics.
Description
Technical Field
The invention relates to a silicate colloidal crystal microsphere based on a surface barbed structure and a method for capturing exosomes by taking the microsphere as an encoding vector, belonging to the fields of biomedical research and clinical detection.
Background
In recent years, multiplex analysis and high-throughput assay have attracted great research interest, which is very important for disease diagnosis, gene expression, drug screening, and the like. One successful biotechnology is the suspension array, which uses self-encoded microcarriers as detection elements, which has a higher sensitive reaction speed and greater flexibility than conventional planar microarrays. Many encoded microcarriers have been proposed for use in suspension arrays, including segmented nanorods, optical pattern-encoded particles, semiconductor Quantum Dots (QDs), fluorescent particles and photonic crystals. The photonic crystal particles in the suspension array have great potential due to the excellent optical performance. However, the surface of the photonic crystal in the existing report is flat and monotonous, and the quantity of probes which can be fixed, the reaction efficiency and the sensitivity are limited.
Exosomes are small vesicles of about 30-150nm in diameter secreted by living cells, present in cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid, and other biological fluids. The exosome membrane is a lipid bilayer similar to a cell membrane, and exosomes secreted by different types of cells have a lot of commonalities, including lipid-rich and abundant transmembrane proteins on the membrane, such as CD63, CD81 and the like which are members of the tetraspanin family, and various RNAs contained in the membrane. Exosomes carry a large number of bioactive molecules such as proteins, lipids, short peptide chains, DNA, RNA, miRNA, cyclic RNA, and the like, and exist in a very stable manner in the extracellular environment, and the content components are closely related to the tissue source of their secretory cells and the pathophysiological state of the secretory cells. As an intercellular transfer carrier, the bioactive substance is transferred to target cells through membrane fusion or endocytosis with the target cells, thereby influencing the functions of the target cells, regulating the metabolism of the target cells, participating in important pathophysiological processes such as immune response, inflammatory reaction, cell communication, apoptosis, cell migration, angiogenesis, tumor cell growth and the like, and realizing the functions of remote information transmission and regulation. The existing exosome capturing technology has many problems, the purity of the proposed exosome is not high, and the steps are complicated. The surface-barbed microsphere based on the pollen-like structure can effectively capture exosomes due to the structural advantages of the microsphere, and can realize multi-channel screening by coupling a specific probe.
Disclosure of Invention
The invention provides a silicate crystal reverse structure microsphere based on a surface barbed structure and a method for capturing exosomes by taking the microsphere as an encoding vector, and the silicate crystal reverse structure microsphere has a good application prospect.
In order to solve the problem that the capture efficiency of the existing photonic crystal microspheres for exosomes is low, the technical scheme provided by the invention is as follows: the method for capturing exosomes by using silicate microspheres with surface-barbed structures comprises the following steps:
(1) coupling of antibody probes:
soaking the barbed microspheres in an alcohol solution of APTES for several hours, then soaking the barbed microspheres in a succinic anhydride solution overnight, and then soaking the microspheres in an MES buffer solution system for 8 to 15 minutes by using EDC and NHS to activate carboxyl; washing with PBS buffer solution, and reacting the microsphere with the spine with the antibody probe at 37 ℃ overnight; finally, blocking unreacted carboxyl on the surface of the barbed microsphere by BSA (bovine serum albumin) in a constant temperature shaking table at 37 ℃, and then modifying the coded barbed microsphere with different colors by an antibody of a target exosome;
(2) capture of cancer cell-derived exosomes:
incubating the prepared antibody-modified coding barbed microspheres and a constant-temperature shaking table with an exosome PBS solution purified by ultracentrifugation at 37 ℃ overnight; after incubation, washing the encoded barbed microspheres with the captured exosomes by using PBS buffer solution, and then performing exosome fluorescent staining by using calcein at 37 ℃; finally, excess calcein was washed out thoroughly with PBS buffer and the fluorescence intensity of the spiked microspheres was read and recorded by a spectrometer and fluorescence microscope connected to a computer.
The proportion of the APTES alcohol solution in the step (1) is 5% v/v, and the proportion of the succinic anhydride solution is 1mg/mL of succinic anhydride/DMSO solution.
Filtering the cell culture supernatant by using a 0.22 micron ultrafiltration membrane before ultracentrifugation purification in the step (2), wherein the centrifugation speed is 100000 rpm.
In order to make the calcein enter the cells more easily, the calcein in the step (2) is AM modified calcein, and the fluorescence is green fluorescence and is excited by blue light.
And (3) capturing the exosomes in the step (2) by using the barbed microspheres modified by antibody probes responding to the exosomes of the specific cancer cells, and after the antibody probes are incubated in a solution containing corresponding targets, staining the microspheres by calcein to observe obvious fluorescence.
Compared with the scheme of the colloidal crystal microspheres prepared by the prior art, the invention has the advantages that:
the silicate colloidal crystal microsphere with the spines on the surface solves the problem that the traditional photonic crystal is low in exosome capture efficiency, can capture a large number of exosomes through simple steps, can realize multi-channel screening, and has wide application prospect in the field of biomedicine. The silicate colloidal crystal microspheres used in the invention have the advantages of simple preparation method, easily available materials, low cost, high preparation efficiency and the like, and are beneficial to clinical application of exosomes.
Drawings
The invention is further described with reference to the following figures and examples.
FIG. 1 is a schematic diagram of exosome capture by silicate colloidal crystal microspheres;
FIG. 2 is the surface structure of silicate colloidal crystal microsphere with exosome captured under scanning electron microscope, with scale bar of 200 nm.
Detailed Description
The above-described scheme is further illustrated below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. The conditions employed in the examples may be adjusted to specific conditions, and the conditions not specified are generally those in routine experiments.
Example 1 Capture of pancreatic cancer cell-derived exosomes
The antibody probe used was an exosome universal probe: CD9
(1) Preparing surface barbed microspheres and coupling probes:
taking a proper amount of silicate microspheres with surface spines as a coupled antibody probe, soaking the silicate microspheres with spines in 5% APTES alcohol solution (v/v) for 4 hours, then soaking the silicate microspheres with surface spines in succinic anhydride DMSO solution (1mg/mL) overnight, then activating carboxyl groups in an EDC and NHS buffer system in an MES buffer system, and soaking for 10 minutes. After washing 3 times with PBS buffer, the spiked microspheres were reacted with the antibody probes at 37 ℃ overnight. Finally, unreacted carboxyl groups on the surface of the barbed microspheres were blocked with 5% BSA for 2h at 37 ℃ in a constant temperature shaker, and then the barbed microspheres were modified with the antibody to the target exosomes.
(2) Cancer cell-derived exosome capture
The prepared antibody-modified barbed microspheres were incubated with ultracentrifuged and purified exosome PBS solution at 37 ℃ overnight in a constant temperature shaker. After incubation, the encoded barbed microspheres with captured exosomes were washed 3 times with PBS buffer, followed by fluorescent staining of exosomes with 0.1% calcein (v/v) at 37 ℃. Finally, excess calcein was washed out thoroughly with PBS buffer and the fluorescence intensity of the spiked microspheres was read and recorded by a spectrometer and fluorescence microscope connected to a computer. Under the excitation of blue light, the microspheres have bright green fluorescence, and exosomes with captured surfaces can be seen under a scanning electron microscope.
Example 2 exosome capture from hepatoma cells
The antibody probe used was an exosome universal probe: CD63
(1) Preparing surface barbed microspheres and coupling probes:
taking a proper amount of silicate microspheres with surface spines as a coupled antibody probe, soaking the silicate microspheres with spines in 5% APTES alcohol solution (v/v) for 4 hours, then soaking the silicate microspheres with surface spines in succinic anhydride DMSO solution (1mg/mL) overnight, then activating carboxyl groups in an EDC and NHS buffer system in an MES buffer system, and soaking for 10 minutes. After washing 3 times with PBS buffer, the spiked microspheres were reacted with the antibody probes at 37 ℃ overnight. Finally, unreacted carboxyl groups on the surface of the barbed microspheres were blocked with 5% BSA for 2h at 37 ℃ in a constant temperature shaker, and then the barbed microspheres were modified with the antibody to the target exosomes.
(2) Cancer cell-derived exosome capture
The prepared antibody-modified barbed microspheres were incubated with ultracentrifuged and purified exosome PBS solution at 37 ℃ overnight in a constant temperature shaker. After incubation, the encoded barbed microspheres with captured exosomes were washed 3 times with PBS buffer, followed by fluorescent staining of exosomes with 0.1% calcein (v/v) at 37 ℃. Finally, excess calcein was washed out thoroughly with PBS buffer and the fluorescence intensity of the spiked microspheres was read and recorded by a spectrometer and fluorescence microscope connected to a computer. Under the excitation of blue light, the microspheres have bright green fluorescence, and exosomes with captured surfaces can be seen under a scanning electron microscope.
Example 3 lymphoma cell-derived exosome capture
The antibody probe used was an exosome universal probe: CD81
(2) Preparing surface barbed microspheres and coupling probes:
taking a proper amount of silicate microspheres with surface spines as a coupled antibody probe, soaking the silicate microspheres with spines in 5% APTES alcohol solution (v/v) for 4 hours, then soaking the silicate microspheres with surface spines in succinic anhydride DMSO solution (1mg/mL) overnight, then activating carboxyl groups in an EDC and NHS buffer system in an MES buffer system, and soaking for 10 minutes. After washing 3 times with PBS buffer, the spiked microspheres were reacted with the antibody probes at 37 ℃ overnight. Finally, unreacted carboxyl groups on the surface of the barbed microspheres were blocked with 5% BSA for 2h at 37 ℃ in a constant temperature shaker, and then the barbed microspheres were modified with the antibody to the target exosomes.
(2) Cancer cell-derived exosome capture
The prepared antibody-modified barbed microspheres were incubated with ultracentrifuged and purified exosome PBS solution at 37 ℃ overnight in a constant temperature shaker. After incubation, the encoded barbed microspheres with captured exosomes were washed 3 times with PBS buffer, followed by fluorescent staining of exosomes with 0.1% calcein (v/v) at 37 ℃. Finally, excess calcein was washed out thoroughly with PBS buffer and the fluorescence intensity of the spiked microspheres was read and recorded by a spectrometer and fluorescence microscope connected to a computer. Under the excitation of blue light, the microspheres have bright green fluorescence, and exosomes with captured surfaces can be seen under a scanning electron microscope.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.
Claims (5)
1. An exosome capturing method based on surface multi-thorn coding microspheres is characterized by comprising the following steps:
(1) coupling of antibody probes:
soaking the barbed microspheres in an alcohol solution of APTES for several hours, then soaking the barbed microspheres in a succinic anhydride solution overnight, and then soaking the microspheres in an MES buffer solution system for 8 to 15 minutes by using EDC and NHS to activate carboxyl; washing with PBS buffer solution, and reacting the microsphere with the spine with the antibody probe at 37 ℃ overnight; finally, blocking unreacted carboxyl on the surface of the barbed microsphere by BSA (bovine serum albumin) in a constant temperature shaking table at 37 ℃, and then modifying the coded barbed microsphere with different colors by an antibody of a target exosome;
(2) capture of cancer cell-derived exosomes:
incubating the prepared antibody-modified coding barbed microspheres and a constant-temperature shaking table with an exosome PBS solution purified by ultracentrifugation at 37 ℃ overnight; after incubation, washing the encoded barbed microspheres with the captured exosomes by using PBS buffer solution, and then performing exosome fluorescent staining by using calcein at 37 ℃; finally, excess calcein was washed out thoroughly with PBS buffer and the fluorescence intensity of the spiked microspheres was read and recorded by a spectrometer and fluorescence microscope connected to a computer.
2. The method for capturing exosomes based on surface-thorny coded microspheres according to claim 1, wherein the ratio of APTES alcohol solution in the step (1) is 5% v/v, and the ratio of succinic anhydride in succinic anhydride solution is 1 mg/mL.
3. The method for capturing exosomes based on surface-spiny coded microspheres according to claim 1, wherein a 0.22 micron ultrafiltration membrane is used for filtering cell culture supernatant before ultracentrifugation purification in step (2), and the centrifugation speed is 100000 rpm.
4. The method for capturing exosomes based on surface-thorny coded microspheres according to claim 1, wherein in order to make calcein enter cells more easily, the calcein in the step (2) is AM-modified calcein, and fluorescence is green fluorescence and is excited by blue light.
5. The method for capturing exosomes based on surface-spiny coded microspheres according to claim 1, wherein the exosomes captured in the step (2) are antibody probe-modified spiny microspheres responding to exosomes of specific cancer cells, and after the exosomes are incubated in a solution containing corresponding targets, obvious fluorescence is observed through calcein staining.
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| CN114720683A (en) * | 2022-05-20 | 2022-07-08 | 南京鼓楼医院 | Preparation method and application of magnetic Janus microcarrier for bladder cancer exosome multivariate analysis |
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| CN112444629A (en) * | 2020-12-09 | 2021-03-05 | 青岛科技大学 | Exosome detection method based on hairpin nucleic acid molecule signal amplification circuit and application |
| CN112755925A (en) * | 2020-12-25 | 2021-05-07 | 南通大学 | Method for extracting exosome from nervous tissue |
| CN113174359A (en) * | 2021-03-31 | 2021-07-27 | 中山大学 | Exosome capturing paper chip and preparation method and application thereof |
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| CN114720683A (en) * | 2022-05-20 | 2022-07-08 | 南京鼓楼医院 | Preparation method and application of magnetic Janus microcarrier for bladder cancer exosome multivariate analysis |
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