CN115561147A

CN115561147A - Exosome protein detection method

Info

Publication number: CN115561147A
Application number: CN202211229134.1A
Authority: CN
Inventors: 徐颖湉; 崔大祥; 徐艳; 朱君; 杨迪诚
Original assignee: Shanghai National Engineering Research Center for Nanotechnology Co Ltd
Current assignee: Shanghai National Engineering Research Center for Nanotechnology Co Ltd
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2023-01-03

Abstract

The invention relates to an exosome protein detection method, in particular to an exosome extraction and protein detection method combining magnetic separation and flow cytometry of multicolor fluorescent microspheres. The invention belongs to the field of biological medicine. The nano-scale magnetic microspheres with large specific surface area are adopted to increase the capture efficiency of antibody binding sites and exosomes. Different exosome proteins are marked by multicolor fluorescent microspheres, and the flow cytometry is combined to rapidly and sensitively detect the different exosome proteins.

Description

Exosome protein detection method

Technical Field

The invention relates to an exosome protein detection method, and relates to an exosome separation and protein detection method based on magnetic nanoparticles and fluorescent microsphere labeling flow cytometry and application. The invention belongs to the field of biological medicine.

Background

Exosomes are membrane vesicle structures secreted by living cells, about 30-150 nm in diameter, derived from late endosomes (also known as multivesicular bodies), and naturally occur in body fluids such as blood, saliva, urine, and breast milk. Exosomes act as important communication mediators between cells, affecting their physiological activities to tissues. The functions of exosomes include: regulation of cell proliferation and apoptosis, metastasis and invasion, angiogenesis, immune response, cell differentiation, metabolic regulation, microbiome regulation, and viral infection. With the continuous and deep basic research on the secretion function, the potential of exosomes in liquid biopsy, precise medicine and regenerative medicine is widely accepted.

Currently, the existing product principles on exosome extraction on the market are: membranes with different pore sizes are used for separating extracellular vesicles; precipitating the precipitate exosomes using polymer reduction; separation by size exclusion chromatography; exosomes are isolated based on antigen-antibody or other similar reaction-specific immunoaffinity capture. The detection of the exosome protein marker is mainly carried out by western blot, immunohistochemistry and other methods, and the experiment is time-consuming and tedious, so that a simple, convenient and quick exosome separation and protein detection method is urgently needed.

Disclosure of Invention

Aiming at the existing requirements, the invention aims to provide an exosome protein detection method, namely an exosome separation extraction and protein detection method based on magnetic nanoparticles and fluorescent microsphere-labeled flow cytometry.

The purpose method of the invention is realized by the following scheme: an exosome protein detection method, a method for exosome extraction and protein detection combining magnetic separation and multicolor fluorescent microsphere flow cytometry, comprises the following steps:

1. the magnetic beads are connected with antibodies: weighing 100 mg of nano-magnetic particles and fully dissolving in 1 mL of phosphate buffer solution (PBS, 20 mM, pH 6); adding 0.5 mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 20 mM) and 0.5 mL of N-hydroxysuccinimide (NHS, 40 mM) to the above solution, mixing well, and activating at 25 deg.C for 30 min; after activation, the magnetic beads were magnetically attracted to remove the supernatant, washed thoroughly with PBS 3 times, and 1 mL PBS (10 mM, pH 7) was added; adding 10-100 mu L of antibody (1 mg/mL) into the activated magnetic beads, uniformly mixing, and reacting at 25 ℃ overnight; after completion of the reaction, the supernatant was removed by magnetic attraction, washed thoroughly 2 times with PBS (10 mM, pH 7) containing 0.1% Tween-20, then 0.1% BSA, 0.01% casein, and 0.1% Tween-20 in PBS (10 mM, pH 7) were added, reacted at 25 ℃ for 30 min, and then the supernatant was removed by magnetic attraction, washed thoroughly, then 1 mL of 0.1% BSA, and 0.1% Tween-20 in PBS (10 mM, pH 7) were added, and stored at 4 ℃ and labeled as antibody-labeled magnetic beads.

2. Connecting the fluorescent microspheres with the antibody: 100 mg of fluorescent microspheres were weighed and dissolved well in 1 mL PBS (20 mM, pH 6); adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM EDC into the solution, mixing well, and activating at 25 ℃ for 30 min; after activation, the fluorescent microspheres were centrifuged to remove the supernatant, washed thoroughly with PBS 3 times, and 1 mL PBS (10 mM, pH 7) was added; adding 10-100 mu L of antibody (1 mg/mL) into the activated fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after completion of the reaction, the supernatant was removed by centrifugation, washed thoroughly 2 times with PBS (10 mM, pH 7) containing 0.1% Tween-20, followed by addition of 0.1% BSA, 0.01% casein and 0.1% Tween-20 PBS (10 mM, pH 7), reacted at 25 ℃ for 30 min, and then the supernatant was removed by centrifugation, washed thoroughly, then added 1 mL of 0.1% BSA and 0.1% Tween-20 PBS (10 mM, pH 7), and stored at 4 ℃ as fluorescent microspheres.

3. Exosome separation and fluorescent microsphere labeling: mixing the freshly extracted exosome and the antibody-labeled magnetic bead with PBS (10 mM, pH7) uniformly, reacting at 25 ℃ for 1-4 h, removing the supernatant by magnetic attraction, completely washing with PBS, and adding 100 μ L of PBS (10 mM, pH7); adding eluent into the solution, reacting at 25 deg.C for 1h, magnetically attracting, and retaining supernatant; the supernatant was added with fluorescent microspheres, reacted at 25 ℃ for 1 hour, centrifuged to remove the supernatant, washed thoroughly with PBS, and 100. Mu.L of PBS (10 mM, pH 7) was added.

4. Flow cytometry detection: the fluorescently labeled microspheres are detected with a flow cytometer. The instrument was calibrated using standard procedures with calibration samples. The 488 nm laser light source is adopted, and the fluorescent signals are respectively 530nm, 582 nm and 630 nm.

The size of the magnetic nano-particles is 100 to 500 nm.

The antibody provided by the invention is Ig G, CD9, CD63, CD81, TSG101 and the like.

The fluorescent microsphere adopts a 488 nm laser light source, and fluorescent signals are respectively 530nm, 580 nm and 630 nm.

The eluent of the invention comprises beta-mercaptoethanol (100 mM), SDS (2%), tris-HCl (62.5 mM).

The invention has the advantages that:

(1) The capture efficiency of antibody binding sites and exosomes is increased by adopting the nano-scale magnetic microspheres with large specific surface area.

(2) Different exosome proteins are marked by multicolor fluorescent microspheres, and the flow cytometry is combined to rapidly and sensitively detect various exosome proteins.

Drawings

FIG. 1 is a flow cytometer measurement of Ig G protein concentration 0.1 ng/mL;

FIG. 2 is a flow cytometric assay of the exosome membrane protein CD63 and CD9 proteins.

Detailed Description

The technical solution of the present invention is further described below by specific examples. The following examples are further illustrative of the present invention and do not limit the scope of the present invention.

Example 1

A method for detecting exosome protein, which adopts nano-scale magnetic microspheres with large specific surface area to be combined with an antibody so as to increase antibody combining sites; different exosome proteins are marked by multicolor fluorescent microspheres, and exosome extraction and protein detection are rapidly carried out by combining flow cytometry, and the method comprises the following steps:

(1) The magnetic beads are connected with antibodies:

weighing 100 mg of nano magnetic particles, and fully dissolving the nano magnetic particles in 1 mL of PBS (phosphate buffer solution) with the concentration of 20 mM and the pH value of 6 to obtain a magnetic sphere dispersion solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, mixing well, and activating the magnetic ball for 30 min at 25 ℃; magnetically absorbing the activated magnetic beads to remove supernatant, thoroughly washing the magnetic beads for 3 times by using PBS, and then adding 1 mL of 10 mM PBS buffer solution with the pH value of 7; adding 20 mu L of antibody Ig G antibody with the concentration of 1 mg/mL into the activated magnetic beads, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, removing the supernatant by magnetic attraction, thoroughly washing the mixture for 2 times by PBS (phosphate buffer solution) containing 0.1% Tween-20, 10 mM and pH7, adding 0.1% BSA, 0.01% casein and 0.1% Tween-20, reacting the mixture for 30 min at 25 ℃, then removing the supernatant by magnetic attraction, after thorough washing, adding 1 mL of 0.1% BSA and 0.1% Tween-20, 10 mM and pH7 PBS, and storing the mixture at 4 ℃ to obtain a solution I which is marked as Ig G antibody labeled magnetic beads;

(2) Connecting the fluorescent microspheres with the antibody:

weighing 100 mg of fluorescent microspheres emitting light at 580 nm, and fully dissolving the fluorescent microspheres in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a fluorescent microsphere solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, fully and uniformly mixing, and activating the fluorescent microspheres for 30 min at 25 ℃; centrifuging the activated fluorescent microspheres to remove supernatant, thoroughly washing with PBS for 3 times, and adding 1 mL of 10 mM PBS buffer solution with pH of 7; adding 20 mu L of antibody Ig G with the concentration of 1 mg/mL into the activated fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, centrifuging to remove the supernatant, thoroughly washing for 2 times by using PBS (phosphate buffer solution) containing 0.1% Tween-20 and 10 mM and pH7, adding 0.1% BSA, 0.01% casein, 0.1% Tween-20 and 10 mM and pH7 PBS, reacting for 30 min at 25 ℃, centrifuging to remove the supernatant, after thorough washing, adding 1 mL of 0.1% BSA and 0.1% Tween-20 and 10 mM and pH7 PBS, and storing at 4 ℃ to obtain a solution II which is marked as an antibody labeled fluorescent microsphere;

(3) Exosome separation and fluorescent microsphere labeling:

uniformly mixing 10 mu L of antigen IgG with the concentration of 0-1 ng/mL, 10 mu L of IgG antibody labeled magnetic beads and 80 mu L of PBS with the concentration of 10 mM and the pH value of 7, reacting for 1-4 h at 25 ℃, magnetically absorbing to remove supernatant, thoroughly washing with PBS, and adding 100 mu L of PBS with the concentration of 10 mM and the pH value of 7; adding eluent into the solution, reacting for 1h at 25 ℃, magnetically attracting, and keeping supernatant; adding 10 μ L of fluorescent microspheres into the supernatant, reacting at 25 deg.C for 1h, centrifuging to remove the supernatant, washing with PBS, and adding 100 μ L of 10 mM PBS with pH 7;

(4) Flow cytometry detection: detecting the fluorescence-labeled microspheres by using a flow cytometer:

and (3) performing standard operation on the calibration instrument by using the calibration sample, and detecting the fluorescence-labeled microspheres by using a 488 nm laser light source and a fluorescence signal.

FIG. 1 is a flow cytometer map of Ig G protein concentration 0.1 ng/mL.

Example 2

An exosome protein detection method is similar to the steps in example 1, and comprises the following steps:

(1) The magnetic beads are connected with antibodies:

weighing 100 mg of nano magnetic particles, and fully dissolving the nano magnetic particles in 1 mL of PBS (phosphate buffer solution) with the concentration of 20 mM and the pH value of 6 to obtain a magnetic sphere dispersion solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, mixing well, and activating the magnetic ball for 30 min at 25 ℃; magnetically absorbing the activated magnetic beads to remove supernatant, thoroughly washing the magnetic beads for 3 times by using PBS, and then adding 1 mL of 10 mM PBS buffer solution with the pH value of 7; respectively adding 10 mu L of antibody CD63 or CD9 with the concentration of 1 mg/mL into the activated magnetic beads, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, removing the supernatant by magnetic attraction, thoroughly washing the mixture for 2 times by PBS (phosphate buffer solution) containing 0.1% Tween-20, 10 mM and pH7, adding 0.1% BSA, 0.01% casein and 0.1% Tween-20, reacting the mixture for 30 min at 25 ℃, then removing the supernatant by magnetic attraction, after thorough washing, adding 1 mL of 0.1% BSA and 0.1% Tween-20, 10 mM and pH7 PBS, and storing the mixture at 4 ℃ and respectively marking the mixture as CD63 antibody labeled magnetic beads and CD9 antibody labeled magnetic beads;

(2) Connecting the fluorescent microspheres with the antibody:

weighing 100 mg of fluorescent microspheres with the emitted light of 530nm and 630 nm respectively, and fully dissolving the fluorescent microspheres in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a fluorescent microsphere solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, fully and uniformly mixing, and activating the fluorescent microspheres for 30 min at 25 ℃; centrifuging the activated fluorescent microspheres to remove supernatant, thoroughly washing with PBS for 3 times, and adding 1 mL of 10 mM PBS buffer solution with pH 7; adding 20 mu L of antibody CD63 with the concentration of 1 mg/mL into the activated green light fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, centrifuging to remove the supernatant, thoroughly washing for 2 times by using PBS (phosphate buffer solution) containing 0.1% Tween-20 and 10 mM and pH7, adding 0.1% BSA, 0.01% casein, 0.1% Tween-20 and 10 mM and pH7 PBS, reacting for 30 min at 25 ℃, centrifuging to remove the supernatant, after thorough washing, adding 1 mL of 0.1% BSA, 0.1% Tween-20 and 10 mM and pH7 PBS, storing at 4 ℃, and marking as CD63 antibody labeled fluorescent microspheres and CD9 antibody labeled fluorescent microspheres;

(3) Exosome separation and fluorescent microsphere labeling:

taking 10 mu L of fresh extracted exosome, 10 mu L of magnetic bead labeled with an LCD63 antibody, 10 mu L of magnetic bead labeled with a CD9 antibody and 70 mu L of PBS with concentration of 10 mM and pH value of 7, uniformly mixing, wherein the total volume is 100 mu L, reacting at 25 ℃ for 1-4 h, magnetically absorbing to remove supernatant, thoroughly washing with PBS, and adding 100 mu L of PBS with concentration of 10 mM and pH value of 7; adding eluent into the solution, reacting for 1h at 25 ℃, magnetically attracting, and keeping supernatant; adding 10 mu of LCD63 antibody-labeled fluorescent microspheres and CD9 antibody-labeled fluorescent microspheres into the supernatant, reacting for 1h at 25 ℃, centrifuging to remove the supernatant, thoroughly washing with PBS, and adding 100 mu of L of 10 mM PBS with pH 7;

and (3) performing standard operation on the calibration instrument by adopting a calibration sample, and detecting the fluorescence-labeled microspheres by adopting a 488 nm laser light source and a fluorescence signal.

Figure 2 is a flow cytometric assay of the exosome membrane proteins CD63 and CD9 proteins.

Claims

1. The method for detecting the exosome protein is characterized in that a nano-scale magnetic microsphere with large specific surface area is combined with an antibody to increase the binding sites of the antibody; the method for fast extracting exosomes and detecting proteins by using the multicolor fluorescent microspheres to mark different exosome proteins and combining flow cytometry comprises the following steps:

(1) Magnetic bead and antibody connection:

weighing 100 mg of nano magnetic particles, and fully dissolving the nano magnetic particles in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a magnetic sphere dispersion solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, mixing well, and activating the magnetic ball for 30 min at 25 ℃; magnetically absorbing the activated magnetic beads to remove supernatant, thoroughly washing with PBS, and adding 1 mL of 10 mM PBS buffer solution with pH 7; adding 10 to 100 mu L of antibody with the concentration of 1 mg/mL into the activated magnetic beads, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction, removing the supernatant by magnetic attraction, thoroughly washing with PBS containing 0.1% Tween-20, 10 mM in concentration and pH7, adding 0.1% BSA, 0.01% casein, and 0.1% Tween-20, 10 mM in concentration and pH7 PBS buffer, reacting at 25 ℃ for 30 min, then removing the supernatant by magnetic attraction, thoroughly washing, adding 1 mL of 0.1% BSA and 0.1% Tween-20 in PBS (10 mM, pH 7), storing at 4 ℃ and marking as antibody-labeled magnetic beads;

(2) Connecting the fluorescent microspheres with the antibody:

weighing 100 mg of fluorescent microspheres, and fully dissolving the fluorescent microspheres in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a fluorescent microsphere solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, fully and uniformly mixing, and activating the fluorescent microspheres for 30 min at 25 ℃; centrifuging the activated fluorescent microspheres to remove supernatant, thoroughly washing with PBS, and adding 1 mL of 10 mM PBS buffer solution with pH 7; adding 10 to 100 mu L of antibody with the concentration of 1 mg/mL into the activated fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction, centrifuging to remove the supernatant, thoroughly washing with 10 mM PBS (pH 7) containing 0.1% Tween-20, adding 0.1% BSA, 0.01% casein, 10 mM PBS (pH 7) containing 0.1% Tween-20, reacting at 25 ℃ for 30 min, centrifuging to remove the supernatant, thoroughly washing, adding 1 mL PBS (10 mM, pH 7) containing 0.1% BSA and 0.1% Tween-20, storing at 4 ℃ and marking as antibody-labeled fluorescent microspheres;

(3) Exosome separation and fluorescent microsphere labeling:

mixing the fresh extract exosome and the solution I with 10 mM PBS (pH 7) uniformly, reacting at 25 ℃ for 1-4 h with a total volume of 100 mu L, magnetically sucking to remove supernatant, thoroughly washing with PBS, and adding 100 mu L PBS (10 mM PBS and pH 7); adding eluent into the solution, reacting for 1h at 25 ℃, magnetically attracting, and keeping supernatant; adding solution II into the supernatant, reacting at 25 deg.C for 1h, centrifuging to remove supernatant, washing with PBS, and adding 100 μ L10 mM PBS with pH 7;

2. The exosome protein detection method according to claim 1, characterized in that the size of the magnetic nanoparticles is 100 to 500 nm.

3. An exosome protein detection method according to claim 1, characterized in that the antibody is one or more of Ig G, CD9, CD63, CD81, TSG 101.

4. The method for detecting exosome protein according to claim 1, characterized in that the fluorescent microsphere adopts a 488 nm laser light source, and the fluorescent signals are 530nm, 580 nm and 630 nm respectively.

5. The method for detecting an exosome protein according to claim 1, wherein the eluent comprises 100 mM of β -mercaptoethanol, 2% of SDS, 62.5 mM of Tris-HCl.

6. An exosome protein detection method according to any one of claims 1 to 5, characterised by the steps of:

(1) Magnetic bead and antibody connection:

(2) Connecting the fluorescent microspheres with the antibody:

weighing 100 mg of fluorescent microspheres emitting light at 580 nm, and fully dissolving the fluorescent microspheres in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a fluorescent microsphere solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, fully and uniformly mixing, and activating the fluorescent microspheres for 30 min at 25 ℃; centrifuging the activated fluorescent microspheres to remove supernatant, thoroughly washing with PBS for 3 times, and adding 1 mL of 10 mM PBS buffer solution with pH 7; adding 20 mu L of antibody Ig G with the concentration of 1 mg/mL into the activated fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, centrifuging to remove the supernatant, thoroughly washing for 2 times by using PBS (phosphate buffer solution) containing 10 mM of 0.1% Tween-20 and having a pH of 7, adding 0.1% BSA, 0.01% casein, 10 mM of 0.1% Tween-20 and having a pH of 7, reacting for 30 min at 25 ℃, centrifuging to remove the supernatant, thoroughly washing, adding 1 mL of 0.1% BSA, 10 mM of 0.1% Tween-20 and having a pH of 7, and storing at 4 ℃ to obtain a solution II which is marked as an antibody labeled fluorescent microsphere;

(3) Exosome separation and fluorescent microsphere labeling:

uniformly mixing 10 mu L of antigen IgG with the concentration of 0-1 ng/mL, 10 mu L of IgG antibody labeled magnetic beads and 80 mu L of PBS with the concentration of 10 mM and the pH value of 7, reacting for 1-4 h at 25 ℃, magnetically sucking to remove supernatant, completely washing the mixture with PBS, and adding 100 mu L of PBS with the concentration of 10 mM and the pH value of 7; adding eluent into the solution, reacting for 1h at 25 ℃, magnetically attracting, and keeping supernatant; adding 10 μ L of fluorescent microspheres into the supernatant, reacting at 25 deg.C for 1h, centrifuging to remove the supernatant, washing with PBS, and adding 100 μ L of 10 mM PBS with pH 7;

7. An exosome protein detection method according to any one of claims 1 to 5, characterised by the steps of:

(1) Magnetic bead and antibody connection:

weighing 100 mg of nano magnetic particles, and fully dissolving the nano magnetic particles in 1 mL of PBS (phosphate buffer solution) with the concentration of 20 mM and the pH value of 6 to obtain a magnetic sphere dispersion solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, mixing well, and activating the magnetic ball for 30 min at 25 ℃; magnetically absorbing the activated magnetic beads to remove supernatant, thoroughly washing the magnetic beads for 3 times by using PBS, and then adding 1 mL of PBS buffer solution with the concentration of 10 mM and the pH value of 7; respectively adding 10 mu L of antibody CD63 or CD9 with the concentration of 1 mg/mL into the activated magnetic beads, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, removing the supernatant by magnetic attraction, thoroughly washing the mixture for 2 times by PBS (phosphate buffer solution) containing 0.1% Tween-20, 10 mM and pH7, adding 0.1% BSA, 0.01% casein and 0.1% Tween-20, reacting the mixture for 30 min at 25 ℃, then removing the supernatant by magnetic attraction, after thorough washing, adding 1 mL of 0.1% BSA and 0.1% Tween-20, 10 mM and pH7 PBS, and storing the mixture at 4 ℃ and respectively marking the mixture as CD63 antibody labeled magnetic beads and CD9 antibody labeled magnetic beads;

(2) Connecting the fluorescent microspheres with the antibody:

weighing 100 mg of fluorescent microspheres with the emitted light of 530nm and 630 nm respectively, and fully dissolving the fluorescent microspheres in 1 mL of PBS buffer solution with the concentration of 20 mM and the pH value of 6 to obtain a fluorescent microsphere solution; adding 0.5 mL of 20 mM EDC and 0.5 mL of 40 mM NHS into the solution, fully and uniformly mixing, and activating the fluorescent microspheres for 30 min at 25 ℃; centrifuging the activated fluorescent microspheres to remove supernatant, thoroughly washing with PBS for 3 times, and adding 1 mL of 10 mM PBS buffer solution with pH of 7; adding 20 mu L of antibody CD63 with the concentration of 1 mg/mL into the activated green light fluorescent microspheres, uniformly mixing, and reacting at 25 ℃ overnight; after the reaction is finished, centrifuging to remove the supernatant, thoroughly washing for 2 times by using PBS (phosphate buffer solution) containing 0.1% Tween-20 and 10 mM and pH7, adding 0.1% BSA, 0.01% casein, 0.1% Tween-20 and 10 mM and pH7 PBS, reacting for 30 min at 25 ℃, centrifuging to remove the supernatant, after thorough washing, adding 1 mL of 0.1% BSA, 0.1% Tween-20 and 10 mM and pH7 PBS, storing at 4 ℃, and marking as CD63 antibody labeled fluorescent microspheres and CD9 antibody labeled fluorescent microspheres;

(3) Exosome separation and fluorescent microsphere labeling:

taking 10 mu L of fresh extracted exosome, 10 mu L of magnetic bead labeled with an LCD63 antibody, 10 mu L of magnetic bead labeled with a CD9 antibody and 70 mu L of PBS with concentration of 10 mM and pH value of 7, uniformly mixing, wherein the total volume is 100 mu L, reacting at 25 ℃ for 1-4 h, magnetically absorbing to remove supernatant, thoroughly washing with PBS, and adding 100 mu L of PBS with concentration of 10 mM and pH value of 7; adding eluent into the solution, reacting for 1h at 25 ℃, magnetically attracting, and keeping supernatant; adding 10 mu of LCD63 antibody-labeled fluorescent microspheres and CD9 antibody-labeled fluorescent microspheres into the supernatant, reacting for 1h at 25 ℃, centrifuging to remove the supernatant, thoroughly washing with PBS, and adding 100 mu of 10 mM PBS with pH 7;