A fluorescence detection method for single extracellular vesicles

Abstract

The present invention provides a fluorescence detection method for a single extracellular vesicle, the steps are as follows: LPS is added to macrophages, incubated, polarized macrophages are obtained; EV is extracted from polarized macrophages; phosphatidylcholine is dissolved in chloroform-methanol mixed solvent, rotary evaporated, and liposome film is obtained; PBS and mixed liquid are added to the flask containing liposome film, mixed, and the film is hydrated; after hydration, the liposome is squeezed and centrifuged through a filter membrane, and the precipitate is collected; after the precipitate is dispersed with pbs, it is centrifuged again, the supernatant is taken, and the liposome solution is obtained; DIL is added to EV, centrifuged after incubation, the precipitate is collected, and resuspended with PBS; the resuspended liquid is mixed with the liposome solution, incubated; imaging is performed with an inverted fluorescence microscope. The present invention realizes fluorescence detection and analysis of the internal information of the vesicle by the membrane fusion of liposomes and EV vesicles, avoiding the problems such as membrane structure damage, content leakage, and difficulty in operation brought by the existing method.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and biomedicine, relates to extracellular vesicles, and specifically relates to a fluorescence detection method for single extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are nano-microvesicles that are actively released by cells into the body fluid circulation. They carry proteins and RNA that reflect the secreting cells, and these components can be used as detection markers. Therefore, analyzing the information of EVs has broad prospects in disease detection and treatment response monitoring.

[0003] In recent years, the commercial development of EVs has promoted their clinical application in liquid biopsy. Liquid biopsy technology has many advantages for EV determination, for example, EVs are present in high levels in biological fluids, and the proteins and nucleic acids inside them are not easily degraded.

[0004] However, the current EV liquid biopsy method mainly conducts batch detection of EV groups, and the results obtained reflect the characteristics of heterogeneous EV groups. In contrast, the analysis of a single EV level can provide richer information, such as the heterogeneity of potential biomarker expression and the separation of different EV subpopulations, which helps to identify EVs of disease origin, because the biomarkers carried by EVs will be upregulated or downregulated as the disease changes, which is of great significance for the early detection of the disease.

[0005] In the current analysis of single EVs, although surface proteins can be directly detected by immunoblotting, the detection of EV RNA and other information requires in-depth detection inside the EV, and sometimes the integrity of the overall structure of the EV must be maintained. In this regard, previous studies have used lysis buffer to lyse EVs and then detect and analyze the resulting contents. However, this approach has many problems: (1) Since the lysis buffer needs to destroy or dissolve part of the EV membrane to form pores, it is difficult to maintain the integrity of the overall structure of the EV, and it is difficult to avoid the possibility of loss of potential target biomarkers; (2) The process is extremely sensitive to the type of EV vesicles to be lysed, the type and concentration of the lysis agent, and the lysis conditions used, and is difficult to operate.

[0006] Therefore, it is urgent to develop a new method for detecting and analyzing single extracellular vesicles. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a fluorescence detection method for a single extracellular vesicle, which realizes fluorescence detection and analysis of the internal information of the vesicle through the fusion of the lipid complex and the lipid bilayer membrane of the EV vesicle, so as to avoid the problems of membrane structure damage, vesicle content leakage, and difficulty in operation caused by the existing lysis solution method.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] A fluorescence detection method for a single extracellular vesicle comprises the following steps:

[0010] (1) Polarization of macrophages

[0011] Add LPS (lipopolysaccharide) to macrophages and co-culture them to obtain polarized macrophages;

[0012] (2) Preparation of EVs (extracellular vesicles)

[0013] Extract EVs from polarized macrophages in step (1) using differential centrifugation;

[0014] (3) Preparation of liposome probes

[0015] Dissolving phosphatidylcholine in a chloroform-methanol mixed solvent and performing rotary evaporation to form a liposome film; adding PBS buffer and a mixed drug solution into a flask containing the liposome film, shaking and mixing to hydrate the film; the mixed drug solution includes NADPH, L-arginine, a nitric oxide fluorescent probe, and sodium hydroxide; the hydrated liposome solution is squeezed through a filter membrane, centrifuged, and precipitated; the precipitate is dispersed with PBS, centrifuged again, and the supernatant is taken to obtain the desired liposome solution;

[0016] (4) Fusion of liposome probes and EVs

[0017] Add DIL dye to the EV obtained in step (2), shake and incubate, then centrifuge, collect the precipitate, and resuspend it in PBS; then, take the resuspended solution and mix it with the liposome solution in step (3), shake and incubate;

[0018] (5) Fluorescence detection

[0019] Imaging was performed using an inverted fluorescence microscope.

[0020] As one of the preferred embodiments of the present invention, in step (1), LPS at a concentration of 200-500 ng / ml is added to macrophages, and the cells are incubated for 1-24 hours to obtain polarized macrophages.

[0021] As one of the preferred embodiments of the present invention, in step (2), the specific process of extracting EVs by differential centrifugation is as follows:

[0022] The collected polarized macrophage culture fluid was centrifuged at 600g for 10 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was centrifuged at 14000-15000g for 2 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was further centrifuged at 14000-15000g for 1 h at 4°C, the supernatant was discarded, and the precipitate was collected to obtain EV.

[0023] As one of the preferred embodiments of the present invention, in step (3), the specific preparation process of the liposome film is as follows:

[0024] In a round-bottom flask, dissolve 10-20 mg of phosphatidylcholine in a chloroform-methanol mixed solvent and mix thoroughly until it is completely dissolved and the solution is clear and transparent; then, use a rotary evaporator to evaporate at 40-45°C until the organic solvent is removed and a liposome film is formed.

[0025] As one of the preferred embodiments of the present invention, in the chloroform-methanol mixed solvent, the volume ratio of chloroform to methanol is 3:1.

[0026] As one of the preferred embodiments of the present invention, in step (3), the specific preparation process of the liposome solution is:

[0027] The flask was placed in a vacuum at room temperature for at least 2 h. Then, PBS buffer and the mixed drug solution were added to the flask containing the liposome film, and then the film was shaken and mixed using a high-speed vortex oscillator at 37°C for 30 min to completely hydrate the film. Then, the hydrated liposome solution was squeezed through a filter membrane with a pore size of 800 nm for 3 to 5 times to obtain a uniform liposome size. Finally, the squeezed solution was centrifuged at 5000~6000g for 10 min at 4°C, the supernatant was discarded, and the precipitate was collected. After the precipitate was dispersed with PBS buffer, it was centrifuged again at 1000~1500g for 10 min at 4°C, and the supernatant was taken to obtain the desired liposome solution.

[0028] As one of the preferred embodiments of the present invention, in the flask containing the liposome film, the amount of PBS buffer added is 3.5 ml, and the amount of the mixed drug solution added is 1.5 ml.

[0029] As one of the preferred embodiments of the present invention, the composition of the mixed drug solution is: 250ul of NADPH with a concentration of 3.36mM+1ml of L-arginine with a concentration of 5mM+50ul of nitric oxide fluorescent probe+200ul of sodium hydroxide.

[0030] As one of the preferred embodiments of the present invention, in step (4), the specific fusion process of the plastid probe and EV is:

[0031] EV and DIL dye were mixed and placed in a thermostatic oscillator at 37°C for 30 min. Next, the resulting solution was centrifuged at 10,000-11,000 g for 10 min at 4°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended with PBS, and the resuspended solution was mixed with the liposome solution and incubated in a thermostatic oscillator at 37°C for 90 min.

[0032] As one of the preferred embodiments of the present invention, when dyeing the EV, 1 ml of EV is mixed with 1 ul of DIL dye; when fusing EV with liposomes, 25 ul of the resuspension is mixed with 25 ul of liposome solution.

[0033] Detection principle:

[0034] The detection marker of the present invention is "nitric oxide synthase (iNOS)", which mainly utilizes membrane fusion detection method to allow iNOS (on EV) and L-arginine to react to generate NO. A large amount of NO triggers the NO fluorescent probe, thereby enhancing the fluorescence and amplifying the signal. Moreover, after fusion, the vesicle particle size increases and can be captured by a fluorescence microscope, so that a single EV can be clearly observed, and then clinical samples can be used to perform EV analysis at the single EV level.

[0035] The advantages of the present invention compared to the prior art are:

[0036] The detection technology of the present invention uses the "lipid bilayer membrane fusion" of the lipid complex and the EV vesicle to detect and analyze the internal information of the vesicle, demonstrating its ability to visualize a single EV and simultaneously analyze and detect the information inside the EV. After fusion, a single fusion vesicle is formed, accompanied by the combination and redistribution of the respective membrane components and the mixing of the internal encapsulated substances; then, an inverted fluorescence microscope is used to carry out rapid and high-resolution detection, and Image J is used for automatic data processing to improve the accuracy of the results. The method of the present invention avoids the problems of membrane structure damage, vesicle content leakage, and difficulty in operation caused by the existing lysis solution method. At the same time, since the probe and the target detection object are transmitted through fusion, nonspecific adsorption is reduced, and the interference of the sample environment is also reduced, which helps to improve the quality of the inverted image during detection and the accuracy of the results. The detection technology of the present invention can provide a reliable liquid biopsy for early screening and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a schematic diagram of the detection principle of EV in Experimental Example 1;

[0038] Figure 2It is the fluorescence comparison and quantitative analysis after the fusion of M1 / M2-EV and liposome probe in Experimental Example 1 (in the figure, Figure A is the fluorescence comparison of M1 / M2-EV and liposome probe, and Figure B is the corresponding quantitative analysis result);

[0039] Figure 3 This is the fluorescence comparison diagram after the liposome probe was fused with free iNOS and EV membrane in Experimental Example 2 (using the liposome probe and EV alone as controls, scale bar 20 μm);

[0040] Figure 4 The membrane fusion detection images and quantitative data analysis of EVs obtained by co-incubating macrophages with different concentrations of lipopolysaccharide LPS in Experimental Example 3 (Figure A is the membrane fusion detection images of different concentrations of LPS, and Figure B is the corresponding quantitative analysis results);

[0041] Figure 5 These are the membrane fusion detection images at different lipopolysaccharide LPS polarization times in Experimental Example 4 and the corresponding iNOS content and nitric oxide fluorescence changes (in the figure, Figure A shows the liposome probe, cell membrane red fluorescent probe and the merged detection image at different polarization times, Figure B shows the nitric oxide fluorescence signal changes at different lipopolysaccharide LPS polarization times, and Figure C shows the nitric oxide synthase iNOS content changes at different lipopolysaccharide LPS polarization times). DETAILED DESCRIPTION

[0042] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0043] Meanwhile, in the following embodiments and experimental examples, the main drugs and production companies involved are shown in Table 1, and the related experimental instruments and production companies involved are shown in Table 2. Other reagents used, unless otherwise specified, are commercially available products; the methods and equipment used are conventional methods and equipment in the art and will not be described in detail.

[0044] Table 1 Experimental drugs and production companies

[0045]

[0046] Table 2 Experimental instruments and production companies

[0047]

[0048] Example 1

[0049] A fluorescence detection method for a single extracellular vesicle in this embodiment includes the following steps:

[0050] (1) Polarization of macrophages

[0051] LPS (lipopolysaccharide) at a concentration of 200 ng / ml was added to the macrophages and incubated for 1 hour to obtain polarized macrophages.

[0052] (2) Preparation of EVs (extracellular vesicles)

[0053] The collected polarized macrophage culture fluid was centrifuged at 600g for 10 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was centrifuged at 14000g for 2 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was further centrifuged at 14000g for 1 h at 4°C, the supernatant was discarded, and the precipitate (i.e., EV) was collected; the obtained EV precipitate was resuspended in PBS and stored at -20°C for later use.

[0054] (3) Preparation of liposome probes

[0055] In a round-bottom flask, 10 mg of phosphatidylcholine was dissolved in a chloroform-methanol mixed solvent (chloroform: methanol volume ratio = 3:1) and mixed thoroughly until it was completely dissolved and the solution was clear and transparent; then, a rotary evaporator was used to evaporate the organic solvent at 40°C to form a liposome film.

[0056] At room temperature, place the flask in a vacuum for at least 2 hours to ensure drying; then, slowly add 3.5 ml PBS buffer and 1.5 ml mixed solution preheated to 37°C to the flask containing the liposome film, and then use a high-speed vortex oscillator to shake and mix at 37°C for 30 minutes to completely hydrate the film. Among them, the 1.5 ml mixed solution consists of: 250ul NADPH with a concentration of 3.36mM + 1ml L-arginine with a concentration of 5mM + 50ul nitric oxide fluorescent probe + 200ul sodium hydroxide.

[0057] The hydrated liposome solution was squeezed through a filter membrane with a pore size of 800 nm three times, gently and slowly squeezing to obtain a uniform liposome size; finally, the solution after squeezing was centrifuged at 5000g for 10 min at 4°C, the supernatant was discarded (to remove small particles), and the precipitate was collected; after the precipitate was dispersed with PBS buffer, it was centrifuged again at 1000g for 10 min at 4°C, and the supernatant was taken to obtain the desired liposome solution.

[0058] (4) Fusion of liposome probes and EVs

[0059] Mix 1 ml of the EV obtained in step (2) and 1 ul of the DIL dye, and place in a thermostatic oscillator at 37°C for 30 min. Then, centrifuge the resulting solution at 10,000 g for 10 min at 4°C, discard the supernatant, and collect the precipitate. Resuspend the precipitate with PBS, then take 25 ul of the resuspended solution and mix it with 25 ul of the liposome solution in step (3), and incubate in a thermostatic oscillator at 37°C for 90 min.

[0060] (5) Fluorescence detection

[0061] Imaging was performed using an inverted fluorescence microscope.

[0062] Example 2

[0063] A fluorescence detection method for a single extracellular vesicle in this embodiment includes the following steps:

[0064] (1) Polarization of macrophages

[0065] LPS (lipopolysaccharide) at a concentration of 300 ng / ml was added to the macrophages and incubated for 8 hours to obtain polarized macrophages.

[0066] (2) Preparation of EVs (extracellular vesicles)

[0067] The collected polarized macrophage culture fluid was centrifuged at 600g for 10 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was centrifuged at 14500g for 2 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was further centrifuged at 14500g for 1 h at 4°C, the supernatant was discarded, and the precipitate (i.e., EV) was collected; the obtained EV precipitate was resuspended in PBS and stored at -20°C for later use.

[0068] (3) Preparation of liposome probes

[0069] In a round-bottom flask, 15 mg of phosphatidylcholine was dissolved in a chloroform-methanol mixed solvent (chloroform: methanol volume ratio = 3:1) and mixed thoroughly until it was completely dissolved and the solution was clear and transparent; then, a rotary evaporator was used to evaporate the organic solvent at 12°C to form a liposome film.

[0070] At room temperature, place the flask in a vacuum for at least 2 hours to ensure drying; then, slowly add 3.5 ml PBS buffer and 1.5 ml mixed solution preheated to 37°C to the flask containing the liposome film, and then use a high-speed vortex oscillator to shake and mix at 37°C for 30 minutes to completely hydrate the film. Among them, the 1.5 ml mixed solution consists of: 250ul NADPH with a concentration of 3.36mM + 1ml L-arginine with a concentration of 5mM + 50ul nitric oxide fluorescent probe + 200ul sodium hydroxide.

[0071] The hydrated liposome solution was squeezed through a filter membrane with a pore size of 800 nm for 3 to 5 times, gently and slowly, to obtain a uniform liposome size; finally, the solution after extrusion was centrifuged at 5500g for 10 min at 4°C, the supernatant was discarded (to remove small particles), and the precipitate was collected; after the precipitate was dispersed with PBS buffer, it was centrifuged again at 1200g for 10 min at 4°C, and the supernatant was taken to obtain the desired liposome solution.

[0072] (4) Fusion of liposome probes and EVs

[0073] Mix 1 ml of the EV obtained in step (2) and 1 ul of the DIL dye, and place in a thermostatic oscillator at 37°C for 30 min. Next, centrifuge the resulting solution at 10,500 g for 10 min at 4°C, discard the supernatant, and collect the precipitate. Resuspend the precipitate with PBS, then take 25 ul of the resuspended solution and mix it with 25 ul of the liposome solution in step (3), and incubate in a thermostatic oscillator at 37°C for 90 min.

[0074] (5) Fluorescence detection

[0075] Imaging was performed using an inverted fluorescence microscope.

[0076] Example 3

[0077] A fluorescence detection method for a single extracellular vesicle in this embodiment includes the following steps:

[0078] (1) Polarization of macrophages

[0079] LPS (lipopolysaccharide) at a concentration of 500 ng / ml was added to the macrophages and incubated for 1 to 24 hours to obtain polarized macrophages.

[0080] (2) Preparation of EVs (extracellular vesicles)

[0081] The collected polarized macrophage culture fluid was centrifuged at 600g for 10 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was centrifuged at 15000g for 2 min at 4°C, the precipitate was discarded, and the supernatant was collected; the obtained supernatant was further centrifuged at 15000g for 1 h at 4°C, the supernatant was discarded, and the precipitate (i.e., EV) was collected; the obtained EV precipitate was resuspended in PBS and stored at -20°C for later use.

[0082] (3) Preparation of liposome probes

[0083] In a round-bottom flask, 20 mg of phosphatidylcholine was dissolved in a chloroform-methanol mixed solvent (chloroform: methanol volume ratio = 3:1) and mixed thoroughly until it was completely dissolved and the solution was clear and transparent; then, a rotary evaporator was used to evaporate the organic solvent at 45°C to form a liposome film.

[0084] At room temperature, place the flask in a vacuum for at least 2 hours to ensure drying; then, slowly add 3.5 ml PBS buffer and 1.5 ml mixed solution preheated to 37°C to the flask containing the liposome film, and then use a high-speed vortex oscillator to shake and mix at 37°C for 30 minutes to completely hydrate the film. Among them, the 1.5 ml mixed solution consists of: 250ul NADPH with a concentration of 3.36mM + 1ml L-arginine with a concentration of 5mM + 50ul nitric oxide fluorescent probe + 200ul sodium hydroxide.

[0085] The hydrated liposome solution was squeezed through a filter membrane with a pore size of 800 nm for 5 times, gently and slowly squeezing to obtain a uniform liposome size; finally, the solution after squeezing was centrifuged at 6000g for 10 min at 4°C, the supernatant was discarded (to remove small particles), and the precipitate was collected; after the precipitate was dispersed with PBS buffer, it was centrifuged again at 1500g for 10 min at 4°C, and the supernatant was taken to obtain the desired liposome solution.

[0086] (4) Fusion of liposome probes and EVs

[0087] Mix 1 ml of the EV obtained in step (2) and 1 ul of the DIL dye, and place in a thermostatic oscillator at 37°C for 30 min. Next, centrifuge the resulting solution at 11,000 g for 10 min at 4°C, discard the supernatant, and collect the precipitate. Resuspend the precipitate with PBS, then take 25 ul of the resuspended solution and mix it with 25 ul of the liposome solution in step (3), and incubate in a thermostatic oscillator at 37°C for 90 min.

[0088] (5) Fluorescence detection

[0089] Imaging was performed using an inverted fluorescence microscope.

[0090] In the following experimental examples, RAW 264.7 macrophages were used as an example for culture, polarization treatment and EV extraction. RAW 264.7 is a mouse macrophage cell line, which is cultured and expanded in RPMI 1640 medium containing 1.5% penicillin-streptomycin and 10% fetal bovine serum, and cultured in a 37°C cell culture incubator containing 5% CO2. It should be noted that it is known that when the RAW 264.7 cells are polarized, the macrophages are polarized from M0 to M1 (more iNOS) and / or M2 (more iNOS).

[0091] Experimental Example 1

[0092] This experimental example is used to verify the reliability and accuracy of the method of the present invention.

[0093] In order to test whether the method of the present invention can reliably and accurately detect the expression of specific proteins on EVs, a control experiment was carried out in this experimental example (comparing M1 and M2 macrophage-derived EVs).

[0094] First, referring to the method of implementation 3, the expression of the marker iNOS on EVs isolated from M1 and M2 macrophages was studied. The schematic diagram of the detection principle is shown in Figure 1 As shown, the test results are Figure 2 shown.

[0095] From the above results, it can be seen that after the fusion of "M1 macrophage-derived EV (M1-EV)" and "liposome probe", a strong fluorescence signal will be generated. However, after the fusion of "M2 macrophage-derived EV (M2-EV)" and "liposome probe", its fluorescence intensity is weaker than that of M1-EV. Figure 1 , 2 EV and liposome probe fusion emit yellow light, which can also eliminate the factor of background fluorescence interference.

[0096] The results of this experiment showed that by detecting the marker iNOS, EVs derived from M1 and M2 macrophages can be effectively distinguished when analyzing the fluorescence of single vesicle particles.

[0097] Experimental Example 2

[0098] This experimental example is used to verify the specificity of the method of the present invention.

[0099] In order to evaluate the specificity of the method of the present invention, the "EV containing iNOS (M1-EV)" and "free iNOS" were mixed with the liposome probe respectively according to the method of Example 3. Figure 3 shown.

[0100] Depend on Figure 3 The results show that after the mixed solution of liposome probe and free iNOS was incubated at 37°C for a period of time, the detected fluorescence signal was extremely weak, and its intensity did not change significantly compared with the fluorescence intensity of the liposome probe alone. However, when the liposome probe and the "EV containing iNOS" were fused, they showed strong fluorescence. Therefore, this detection method will not be affected by free iNOS.

[0101] This result means that the detection method of the present invention can specifically detect iNOS protein on EV, and the membrane fusion detection method avoids some non-specific detection problems, thereby ensuring that the fluorescent signal of each vesicle is derived from iNOS in EV, so that other substances in the solution will not affect the effect of membrane fusion detection. The fusion detection of two vesicles ensures the purity and specificity of the detection.

[0102] Experimental Example 3

[0103] This experimental example is used to verify the effect of changes in LPS concentration on the detection results of the present invention.

[0104] Lipopolysaccharide (LPS) is a component of the outer cell wall of Gram-negative bacteria. Under normal conditions, LPS has a wide range of biological activities. It can not only activate immune cells including T lymphocytes, B lymphocytes, macrophages, and natural killer cells, but also promote the production of cytokines, regulating the immune system from multiple aspects. In addition, LPS can stimulate immune cells and induce inflammation by increasing the production of cytokines. In the body, LPS can transmit cell signals, activate mononuclear macrophages, endothelial cells, epithelial cells, etc., causing them to synthesize and release a variety of cytokines and inflammatory mediators, thereby causing the body to produce a series of reactions. Given the advantages of LPS, such as easy control, good model reproducibility, and easy identification and measurement of systemic effects, it is the gold standard reagent for establishing an acute inflammation model.

[0105] In this experiment, the sample without LPS was set as the experimental control group, and two different concentrations of LPS (10μg / mL and 100μg / mL) were selected and incubated with RAW 264.7 macrophages of the same density for 24 hours. After the culture, the cell supernatant was collected and purified by centrifugation to obtain EVs (including M1 and M2 EVs). Subsequently, equal amounts of liposome probes were added to the three groups of EV samples to carry out fusion experiments.

[0106] The fusion experiment results are as follows Figure 4 As shown. Figure 4 The results show that the fluorescence of the "EV sample not stimulated by LPS" image is weak. With the addition of LPS and the increase in concentration, the proportion of M1-type EVs increases, the total iNOS content increases, the fluorescence increases, and the number of fluorescent bright spots in the fluorescence image also increases.

[0107] The above results strongly prove that the method of detecting NO by membrane fusion detection to characterize iNOS has good sensitivity and strong detection performance.

[0108] Experimental Example 4

[0109] This experimental example is used to verify the influence of the time variation of drug polarization on the detection results of the present invention.

[0110] In this study, the sensitivity of the detection system was evaluated by measuring iNOS in EVs.

[0111] First, RAW264.7 macrophages were plated at 8 × 10 per well. 4 The cells were then inoculated at a density of 10 cells / mL. Then, the cells were treated with polarization regulators. First, 25 ng / mL of IL-4 was incubated with macrophages RAW264.7 for 24 hours to ensure that most of the cultured cells were M2 type. Since iNOS only exists in M1 macrophages, it can be an ideal detection indicator. After 24 hours, the IL-4 solution was removed, and to prevent IL-4 from continuing to play a role in subsequent experiments, we used phosphate buffered saline (PBS) to rinse the adherent cells three times.

[0112] After that, 100 ng / mL of LPS was immediately used to continue culturing with the cells for 0 h, 8 h, and 24 h. After the culture was completed, the cell culture medium was collected, and the EVs were purified and concentrated by ultracentrifugation. Subsequently, the EV precipitate was collected and incubated with 10 μM Dil dye for 20 min, and then the dye was removed by centrifugation, and then the membrane probe prepared in advance was added to carry out the membrane fusion experiment. Finally, an inverted fluorescence microscope was used for detection, and the data were analyzed using Image J software.

[0113] When the fused solution was dropped onto a glass slide and observed under an inverted fluorescence microscope, it was shown that EVs were distributed as single spots in the image, and the locations of the spots were different under different excitation lights. At the same time, we observed that orange-red and green fluorescence could be observed simultaneously in some EV populations. Dil and NO fluorescent probes coexisted on the same EV particle, which appeared as yellow spots (mixed from orange-red and green). Figure 5 As shown in A.

[0114] First, the fluorescence images of the two fluorescence channels are converted into grayscale images respectively. After the EV area is clearly defined, the fluorescence intensity inside and outside the area can be calculated, and the overlapping area can be determined based on the comparison of the two images. The overlapping area of ​​the two images, that is, the overlapping area of ​​the Dil and NO probes, is identified as EVs containing iNOS. The processed merged image shows the state of a fused fluorescence image. We found that with the extension of LPS stimulation time of macrophages, the iNOS content in its EVs also gradually increased. This phenomenon can be clearly observed from the co-localization image of a single EV, and the fluorescence intensity is positively correlated with the concentration of iNOS expressed by the EV. The signal can be detected even in a changing situation, which shows that our detection system has extremely high sensitivity and can detect changes in iNOS in EVs, such as Figure 5 As shown in C.

[0115] At the same time, as the polarization time stimulated by LPS increases, the NO fluorescence gradually increases, such as Figure 5 As shown in B in Figure 1, this means that "the proportion of M1 EVs is gradually increasing."

[0116] Then, the co-localized EVs can be further quantitatively analyzed by processing the individual fluorescence images and the merged images with Image J to calculate the proportion of EV subpopulations that simultaneously express the two fluorescences in the entire EV population. In other words, in a field of view, the proportion of "EVs with iNOS" in "total EVs".

[0117] In summary, the detection technology of the present invention uses the "lipid bilayer membrane fusion" of lipid complexes and EV vesicles to detect and analyze the internal information of the vesicles, demonstrating its ability to visualize single EVs and synchronously analyze and detect information within EVs, which can avoid the problems of membrane structure damage, vesicle content leakage, and difficult operation caused by existing lysis solution methods. At the same time, since the probe and the target detection object are transmitted through fusion, nonspecific adsorption is reduced and the interference of the sample environment is also reduced, which helps to improve the quality of the inverted image during detection and the accuracy of the results. The detection technology of the present invention can provide reliable liquid biopsy examinations for early screening and detection.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescence detection method for a single extracellular vesicle, characterized in that: The steps include: (1) Add lipopolysaccharide (LPS) to macrophages and co-culture them to obtain polarized macrophages; (2) extracting extracellular vesicles (EVs) from the polarized macrophages in step (1) using differential centrifugation; (3) Dissolve phosphatidylcholine in a chloroform-methanol mixed solvent and perform rotary evaporation to form a liposome film; add 3.5 ml PBS buffer and 1.5 ml mixed solution to the flask containing the liposome film, shake and mix to hydrate the film; the composition of the mixed solution is: 250 ul NADPH with a concentration of 3.36 mM + 1 ml L-arginine with a concentration of 5 mM + 50 ul nitric oxide fluorescent probe + 200 ul sodium hydroxide; the hydrated liposome solution is squeezed through a filter membrane, centrifuged, and the precipitate is collected; the precipitate is dispersed with PBS, centrifuged again, and the supernatant is taken to obtain the desired liposome solution; (4) Add DIL dye to the EV obtained in step (2), incubate with shaking, then centrifuge, collect the precipitate, and resuspend it in PBS; then, take the resuspended solution and mix it with the liposome solution in step (3), and incubate with shaking; (5) Imaging was performed using an inverted fluorescence microscope.

2. The fluorescence detection method for a single extracellular vesicle according to claim 1, characterized in that: In the step (1), LPS at a concentration of 200-500 ng / ml is added to macrophages and incubated for 1-24 hours to obtain polarized macrophages.

3. The fluorescence detection method for a single extracellular vesicle according to claim 1, characterized in that: In step (2), the specific process of extracting EVs by differential centrifugation is as follows: collect the polarized macrophage culture fluid, centrifuge it at 600g at 4°C for 10 minutes, discard the precipitate, and collect the supernatant; centrifuge the obtained supernatant at 14000-15000g at 4°C for 2 minutes, discard the precipitate, and collect the supernatant; continue to centrifuge the obtained supernatant at 14000-15000g at 4°C for 1 hour, discard the supernatant, collect the precipitate, and obtain EVs.

4. The fluorescence detection method for a single extracellular vesicle according to claim 1, characterized in that: In the step (3), the specific preparation process of the liposome film is as follows: in a round-bottom flask, 10-20 mg of phosphatidylcholine is dissolved in a chloroform-methanol mixed solvent, and mixed thoroughly until it is completely dissolved and the solution is clear and transparent; then, a rotary evaporator is used to rotary evaporate at 40-45° C. until the organic solvent is removed to form a liposome film.

5. The fluorescence detection method for a single extracellular vesicle according to claim 4, characterized in that: In the chloroform-methanol mixed solvent, the volume ratio of chloroform to methanol is 3:

1.

6. The fluorescence detection method for a single extracellular vesicle according to claim 1, characterized in that: In the step (3), the specific preparation process of the liposome solution is as follows: placing the flask in a vacuum at room temperature for at least 2 hours; then, adding the PBS buffer and the mixed drug solution to the flask containing the liposome film, and then using a high-speed vortex oscillator to shake and mix at 37°C for 30 minutes to completely hydrate the film; then, the hydrated liposome solution is squeezed through a filter membrane with a pore size of 800 nm for 3 to 5 times to obtain a uniform liposome size; finally, the solution after squeezing is first centrifuged at 5000~6000g at 4°C for 10 minutes, the supernatant is discarded, and the precipitate is collected; after the precipitate is dispersed with PBS buffer, it is centrifuged again at 1000~1500g at 4°C for 10 minutes, and the supernatant is collected to obtain the desired liposome solution.

7. The fluorescence detection method for a single extracellular vesicle according to claim 1, characterized in that: In step (4), the specific fusion process of the plastid probe and EV is as follows: EV and DIL dye are mixed and placed in a thermostatic oscillator at 37°C for 30 minutes; then, the obtained solution is centrifuged at 10000-11000g for 10 minutes at 4°C, the supernatant is discarded, and the precipitate is collected; the precipitate is resuspended with PBS, and then the resuspended solution is mixed with the liposome solution and incubated in a thermostatic oscillator at 37°C for 90 minutes.

8. The fluorescence detection method for single extracellular vesicles according to claim 7, characterized in that: When dyeing the EV, 1 ml of EV was mixed with 1 ul of DIL dye; when fusing EV with liposomes, 25 ul of the resuspension was mixed with 25 ul of liposome solution.

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