A method for extracellular vesicle real-time enrichment and detection

Abstract

The present application relates to a kind of extracellular vesicle real-time enrichment and detection method;Belong to biological detection technical field;The present application first prepares silica colloidal crystal (SCC) film using vertical evaporation method;Then di-stearoyl phosphatidyl ethanolamine-polyethylene glycol-carboxyl (DSPE) is modified on silica colloidal crystal film, and the affinity characteristics of DSPE and phospholipid bilayer are used to separate and enrich extracellular vesicle in biological liquid;Again, by constructing reflection interference spectrum system, the optical thickness change caused in the process of separating extracellular vesicle is used to carry out real-time detection to extracellular vesicle;The present application can not only quickly and efficiently separate extracellular vesicle in a variety of biological liquids, but also can carry out real-time concentration and membrane protein expression amount analysis to separated extracellular vesicle;The present application provides a kind of fast low-cost integrated multifunctional analysis method for the extracellular vesicle separation of various body fluids.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for real-time enrichment and detection of extracellular vesicles, and belongs to the technical field of biological detection. BACKGROUND

[0002] Extracellular vesicles (EVs) are nanoscale particles derived from cells and possess a lipid bilayer structure, and their interiors are rich in diverse biological information molecules, including proteins, mRNAs, non-coding RNAs, DNA, and lipids. In recent years, the scientific community has shown increasing interest in EVs because of their significant therapeutic potential in various therapeutic fields, such as tissue repair and regeneration, infectious diseases, autoimmune diseases, and cancer. EVs are widely distributed in various body fluids, such as blood, urine, saliva, cerebrospinal fluid, and peritoneal fluid. As a direct reflection of the state of parent cells, their stable cell structure brings unprecedented convenience and possibility to clinical detection, indicating that EVs are expected to become an important part of liquid biopsy technology. Protein molecules located on the surface or inside EVs provide rich, stable, sensitive, and unique biological information. These proteins have been successfully developed as specific diagnostic and prognostic factors for various diseases, such as inflammation, neurodegenerative diseases, and cancer. Most importantly, when using membrane proteins as markers for detection, there is no need to destroy or further cut the EVs, thereby maintaining the physiological activity and integrity of EVs and their contents, which is undoubtedly an advantage in the detection field.

[0003] The small size and low density of EVs make it challenging to specifically isolate them from complex components of a wide range of cell fragments and biological fluids. Ultracentrifugation (UC) is currently the most commonly used method for isolating EVs in laboratories and is also the gold standard method; however, ultracentrifugation requires an expensive ultracentrifuge, and the extraction process requires multiple tedious purification steps, takes a long time, and the separation efficiency is generally less than 30%. In addition, there are methods such as immunoprecipitation, sedimentation polymerization, and ultrafiltration, but the separation efficiency and purity of these methods cannot meet the needs of scientific research, and moreover, these methods can damage the structure and function of extracellular vesicles, and the isolated extracellular vesicles are prone to clustering, which is not conducive to downstream analysis. At the same time, the enrichment and detection of EVs at the present stage are carried out separately, and EVs need to be separately packaged and detected during detection, which leads to the loss of EVs and results in a lower detection value than the enrichment value.

[0004] Therefore, there is an urgent need to develop a method that is simple, economical, and can quickly separate and enrich EVs from complex samples and perform real-time detection. SUMMARY

[0005] In view of some deficiencies in the prior art, the purpose of the present application provides a method for real-time enrichment and detection of extracellular vesicles, the method can be used for enriching extracellular vesicles and real-time detecting the concentration of extracellular vesicles; further, the method can be used for analyzing the expression amount of corresponding extracellular vesicle membrane proteins and the kinetics of antibody binding and dissociation.

[0006] To achieve the above technical purpose, the present application provides the following technical scheme:

[0007] The present application first provides a method for real-time enrichment and detection of extracellular vesicles, which comprises the following steps:

[0008] S1. A silica colloidal crystal film (SCC film) is prepared by a vertical deposition method;

[0009] The method for preparing the silica colloidal crystal film by the vertical deposition method comprises:

[0010] After centrifugation, the silica microspheres are repeatedly cleaned with anhydrous ethanol and centrifuged; the glass slide is soaked in the solution of E. gobbio and then washed with ultrapure water; the treated glass slide is vertically placed in a silica ethanol suspension (1%); after 7 days of ethanol evaporation, a large-area silica colloidal crystal film is formed on the surface of the glass.

[0011] Further, in step (3), the evaporation environment temperature is 25±5℃, and the humidity is 10±5%.

[0012] S2. A DSPE-modified silica colloidal crystal film is prepared;

[0013] The preparation method of the DSPE-modified silica colloidal crystal film comprises:

[0014] The SCC film is soaked in an ethanol solution of aminopropyl triethoxysilane to obtain an amino-modified SCC film; DSPE is dissolved in an ethanol solution to obtain solution one; N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are dissolved in a Tris-HCl buffer to obtain solution two; solution one and solution two are mixed and the pH value is adjusted to obtain a mixed solution; after the amino-modified SCC film is reacted with the mixed solution, it is washed with ultrapure water to obtain a DSPE-modified silica colloidal crystal film, which is stored in ultrapure water for standby use;

[0015] Further, the concentration of the ethanol solution of aminopropyl triethoxysilane is 0.1%-2% (volume / volume), and the soaking time of the SCC film in the ethanol solution of aminopropyl triethoxysilane is 1-48 hours;

[0016] Further, the DSPE solution is 1-100 mg: 1 mL, the N-hydroxysuccinimide, 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Tris-HCl buffer solution are 20-200 mg: 50-400 mg: 4 mL.

[0017] Further, the pH value of the solution is adjusted to 7.5 after mixing the solution one and the solution two; the reaction time is 4-12 h.

[0018] S3. Assemble the OPLI (ordered porous layer interferometry) system:

[0019] Assemble the reaction pool with the DSPE modified silica colloidal crystal thin film as the substrate, and assemble the OPLI (ordered porous layer interferometry) system with the inverted microscope and the fiber spectrometer;

[0020] Further, the assembling of the OPLI system includes the following steps:

[0021] Drill holes on the silica gel pad, cover it on the DSPE modified silica colloidal crystal thin film glass slide to form a reaction pool; press the upper end with another glass slide to form a closed reaction pool; fix the reaction pool on the inverted microscope and adjust the focal length to the silica colloidal crystal layer; couple the fiber through the spectroscope and connect the spectrometer; connect the conduit to the reaction pool and connect the conduit to the peristaltic pump to make the reaction liquid flow into the hole in the middle of the silica gel pad; repeat the operation to connect another conduit to make the reacted liquid flow out.

[0022] S4. Isolation and enrichment of extracellular vesicles:

[0023] Pass the PBS solution into the reaction pool of the OPLI system, and after the baseline is stable, pass the sample solution of the extracellular vesicles to be separated into the reaction pool, and when the spectrometer shows that the baseline is stable, perform spectral line fitting on the selected reflection interference spectrum band, and calculate the optical thickness 1 of the silica colloidal crystal thin film by using the extreme value method, so as to complete the isolation and enrichment of the extracellular vesicles.

[0024] Further, the sample solution of the extracellular vesicles to be separated includes cell culture medium, urine, milk, cerebrospinal fluid or blood.

[0025] S5. Dissociation of the enriched extracellular vesicles:

[0026] After the enrichment is completed, pass the detergent to wash away the impurities adsorbed on the SCC thin film; finally, pass the elution solution to dissociate the extracellular vesicles enriched by the SCC thin film.

[0027] Further, the washing agent comprises Triton X-100 or ethyl phenyl polyethylene glycol, and the elution solution comprises triethanolamine.

[0028] S6. Establishing a linear relationship to analyze the concentration of extracellular vesicles:

[0029] The circulation is performed on the sample solution of the extracellular vesicles to be separated at different dilution ratios, and steps S4 and S5 are repeated, a linear relationship is established with the optical thickness 1 as the ordinate and the dilution ratio as the abscissa, and the concentration of extracellular vesicles is analyzed.

[0030] S7. Repeating steps S1-S5, except that in step S5, the elution solution is not introduced, but the extracellular vesicle membrane protein antibody and the PBS solution are introduced; the spectrum line fitting is performed on the selected reflection interference spectrum wave band, the optical thickness 2 and the optical thickness 3 of the silicon dioxide colloidal crystal film are calculated by the extremum method fitting, the binding and dissociation curves of the membrane protein and the antibody are obtained, and the expression amount of the corresponding extracellular vesicle membrane protein and the kinetics of the binding and dissociation of the antibody are analyzed.

[0031] Further, the optical thickness in step S4 and step S7 is fitted according to the formula , wherein λ is the real-time wavelength of the interference peak in the interval of 430-1100 nm, that is, the selected reflection interference spectrum wave band, k' is a relative order, k' = 0, 1, 2, 3, 4,..., nd is the optical thickness; the extracellular vesicle membrane protein antibody comprises the antibody corresponding to the membrane protein CD9, CD63 or CD81.

[0032] The working principle of the technical scheme of the present application is described as follows:

[0033] The DSPE on the modified silicon dioxide colloidal crystal film can specifically bind to the phospholipid bilayer of EVs, so as to separate the EVs in the sample. The OPLI system is based on the reflection interference phenomenon caused by the reflection of white light on the upper and lower boundaries of the SCC film layer, and the wavelength of the interference peak is converted into the change (ΔOT) of the optical thickness by the extremum tracking method. When the observation ΔOT changes from rising to stable, it indicates that the separation of EVs is completed, and then the elution solution is introduced to elute the EVs fixed on the SCC film to realize the separation. The corresponding antibody is directly introduced without the elution process, and the antibody can bind to the surface membrane protein of the EVs fixed on the SCC film. By fitting the first-order kinetic model of the obtained real-time optical thickness change curve, the antibody binding rate constant, dissociation rate constant and equilibrium constant and other information are calculated, so that the method provided by the present application can analyze the binding and dissociation constants of the EV membrane protein and the antibody or the drug, and can be further used for drug research and development or target protein screening.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] (1) The method of the present application enriches EVs by using a silica colloidal crystal film modified with DSPE, and the whole process does not require the use of large instruments, and the operation is simple, time-consuming is short, and the whole EVs separation process can be completed in about 1 hour.

[0036] (2) The method of the present application uses a silica colloidal crystal film modified with DSPE as a substrate to assemble a reaction pool, and together with an inverted microscope and a fiber spectrum, an OPLI system is formed, which can obtain real-time curves of the combination of EVs and the silica colloidal crystal film. The real-time curves can observe the start and end of the EVs capture process, and the capture process can be recorded in real time through ΔOT, and the EVs separation process can be visually observed. The scheme of the present application can calculate the specific concentration of EVs while separating EVs, predict the quality of EVs in the sample, and achieve separation and detection at the same time; it does not need to separate and monitor the sample again, reduces the sample loss, simplifies the operation process, realizes the separation and concentration detection of EVs in one step, and the efficiency and concentration of the separated and captured EVs are high.

[0037] (3) The method of the present application can also fix the separated EVs on the silica colloidal crystal film, analyze the expression amount of the membrane proteins of EVs from different sources, screen antibodies for the surface membrane proteins, and detect the kinetics; at the same time, the expression amount of the surface membrane protein markers of EVs of the corresponding disease can be analyzed to realize the liquid biopsy function; the method is based on the porous and pore size characteristics of the silica colloidal crystal film, the signal acquisition area is not affected by the micron-level cells, and the OPLI system uses an inverted microscope integrated with an optical system, so the flow of the sample above the film will not interfere with the system monitoring, and therefore the method can be applied to more complex clinical samples, such as cerebrospinal fluid, emulsion and blood, etc. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a scanning electron microscope image of the silica colloidal crystal film prepared in Example 1, wherein Figure A is a front view of the silica colloidal crystal film, and the scale is 1 μm; Figure B is a cross-sectional view of the silica colloidal crystal film, and the scale is 200 nm.

[0039] Figure 2 It is a reflection interference spectrum of the silica colloidal crystal film prepared in Example 1.

[0040] Figure 3 It is a reflection interference spectrum of the silica colloidal crystal film prepared in Example 1 before and after modification with DSPE.

[0041] Figure 4 It is a real-time optical thickness change graph during the separation of extracellular vesicles in the supernatant of the cell culture solution in Example 1.

[0042] Figure 5 Figure of the results of the Western blotting experiment of the CD9 protein of the EVs completely separated and eluted from the cell culture supernatant of the method of the present application and the UC method.

[0043] Figure 6 Figure of the transmission electron microscope photograph of the EVs completely separated and eluted in Example 2, with a scale of 200 nm.

[0044] Figure 7 Figure of the real-time optical thickness change during the separation of the extracellular vesicles in the urine in Example 2.

[0045] Figure 8 Figure of the results of the Western blotting experiment of the CD9 protein of the EVs eluted from the urine of the method of the present application and the UC method.

[0046] Figure 9 Figure of the transmission electron microscope photograph of the EVs completely separated and eluted in Example 2, with a scale of 200 nm.

[0047] Figure 10 Figure of the real-time optical thickness change during the separation of the extracellular vesicles in the supernatant of the MSC culture solution diluted at different ratios in Example 3.

[0048] Figure 11 Figure of the linear relationship of the different ratios of dilution as the abscissa and the optical thickness change as the ordinate in Example 3.

[0049] Figure 12 Figure of the real-time optical thickness curve of the real-time separation of the EVs in the supernatant of the MSC culture solution and the real-time curve of the binding of the CD9 antibody to the membrane protein in Example 4.

[0050] Figure 13 Figure of the real-time curve of the optical thickness change of the binding and dissociation of the EVs membrane protein to the CD9, CD63 and CD81 antibodies in the supernatant of the MSC culture solution in Example 4.

[0051] Figure 14 Figure of the optical thickness change caused by the circulation of the ανβ3 antibody into the solution and the binding to the immobilized EVs in the urine sample of the prostate cancer patient in Example 5.

[0052] Figure 15 Figure of the optical thickness change caused by the circulation of the Trop-2 antibody into the solution and the binding to the immobilized EVs in the urine sample of the prostate cancer patient in Example 5.

[0053] Figure 16 Figure of the optical thickness change caused by the circulation of the tumor marker antibodies ανβ3 and Trop-2 solution into the solution after the immobilization of the EVs in the urine samples of the prostate cancer patient and the healthy population in Example 5. DETAILED DESCRIPTION

[0054] In order for those skilled in the art to better understand the technical solutions of the present application, the preferred embodiments of the present application are described in detail below, but the following embodiments do not limit the protection scope of the present application.

[0055] In the embodiments of the present application, those not described in detail are completed by using conventional experimental methods, and the processes involved in the embodiments not described in detail are understood and easily realized by those skilled in the art according to product instructions or basic knowledge in the art, and thus are not described in detail.

[0056] In the following embodiments, the water-dispersed silica microspheres with a diameter of 190 nm are purchased from Japan DnKem Co., Ltd.; DSPE-PEG2000-COOH is purchased from Xi'an Ruixi Biological Co., Ltd.; the fiber-optic spectrometer is a PG2000 PRO type spectrometer; and other unspecified items are all ordinary commercially available products.

[0057] The piranha solution described in the embodiments is obtained by a conventional method.

[0058] Embodiment 1: Isolation and enrichment of EVs in MSC (mesenchymal stem cell) culture supernatant

[0059] S1. Preparation of silica colloidal crystal film (SCC film)

[0060] (1) All glass slides are immersed in the piranha solution overnight in advance, and then washed with ultrapure water.

[0061] (2) The 190 nm diameter silica microspheres are centrifuged, and then repeatedly washed and centrifuged with anhydrous ethanol to form a 1% silica ethanol suspension.

[0062] (3) The glass slides soaked with piranha are vertically placed in the silica ethanol suspension, and after 7 days, a silica colloidal crystal film is formed. The temperature of the placement environment is 25±5℃, and the humidity is 10±2%.

[0063] The silica colloidal crystal film prepared in step S1 is scanned by a scanning electron microscope, and the results are shown in Figure 1 , wherein Figure 1 is a scanning electron microscope image of the silica colloidal crystal film, wherein Figure 1 A is a front view of the silica colloidal crystal film, Figure 1 B is a cross-sectional view of the silica colloidal crystal film. As can be seen from Figure 1 A, the silica microspheres are closely arranged under the scanning electron microscope photo, and there is no random arrangement structure. As can be seen from Figure 1BIt can be seen from the cross-sectional electron micrograph that the silica microspheres are closely packed and form a typical colloidal crystal structure, indicating that the silica colloidal crystal thin film is successfully prepared by the method described in step S1.

[0064] The reflection interference of the silica colloidal crystal thin film obtained in step S1 is detected, and the results are shown in FIG. 3. Figure 2 As can be seen from the figure, the silica colloidal crystal thin film has good interference fringes, and the interference peak is obvious, which is convenient for subsequent optical thickness calculation of EVs separation and detection.

[0065] S2. DSPE is modified on the silica colloidal crystal thin film

[0066] (1) The silica colloidal crystal thin film is soaked in a 0.1% APTES (aminopropyl triethoxysilane) ethanol solution (v / v) overnight for amino modification.

[0067] (2) 1 mg of DSPE is dissolved in 1 mL of a 30% ethanol solution (v / v) to obtain solution one; 20 mg of NHS (N-hydroxysuccinimide) and 50 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) are dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution two. The two solutions are mixed and the pH value is adjusted to 7.5. The amino-modified silica colloidal crystal thin film is reacted with the mixed solution for 6 h to obtain the DSPE-modified silica colloidal crystal thin film.

[0068] (3) The DSPE-modified silica colloidal crystal thin film obtained in step (2) is washed with ultrapure water for 3 times and can be used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for standby use.

[0069] The DSPE-modified silica colloidal crystal thin film obtained in step S2 is subjected to reflection interference spectrum detection, and the results are shown in FIG. 4. Figure 3 Figure 3 is the reflection interference spectrum of the same position on the silica colloidal crystal thin film before and after DSPE modification. As can be seen from the figure, after the DSPE modification, the reflection interference spectrum of the silica colloidal crystal thin film as a whole moves to the right, proving that the DSPE is successfully modified on the silica colloidal crystal thin film.

[0070] S3. Build OPLI system

[0071] (1) Drill holes on the silica gel pad, cover it on the DSPE-modified silica colloidal crystal thin film glass slide to form a reaction pool.

[0072] (2) Use another glass slide to press the upper end to form a closed reaction pool.

[0073] ​(3) Fix the closed reaction cell on the inverted microscope and adjust the focal length to the silica colloidal crystal layer.

[0074] (4) Coupling the optical fiber and connecting the spectrometer through the beam splitter.

[0075] (5) Connect the conduit to the reaction cell and connect the conduit to the peristaltic pump to enable the reaction liquid to flow into the hole in the middle of the silica gel pad. Repeat the operation to connect another conduit to enable the reaction liquid to flow out.

[0076] S4. Isolation and enrichment of EVs and elution of silica colloidal crystal film enriched EVs

[0077] Using the peristaltic pump, 0.1M PBS solution is introduced into the closed reaction cell through the conduit in step S3(5) at a flow rate of 0.4mL / min. When the baseline of the spectrometer is stable, the selected reflection interference spectrum band is fitted by the extremum method, and the real-time optical thickness of the silica colloidal crystal film is calculated. When the optical thickness of the silica colloidal crystal film is stable, 1mL of MSC culture supernatant is introduced into the closed reaction cell through the conduit in step S3(5) using the peristaltic pump, and the selected reflection interference spectrum band is fitted by the extremum method. The optical thickness of the silica colloidal crystal film is calculated (the obtained thickness of the silica colloidal crystal film is optimal in the range of 4000-6000nm, but the specific initial thickness of the silica colloidal crystal film does not affect the thickness increment).

[0078] The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase. After the curve rises smoothly, PBS solution is introduced again to stabilize the baseline. Then, 0.02% Triton X-100 or NP40 is introduced as a detergent to wash away the excess protein and other impurities in the culture supernatant adsorbed by the silica colloidal crystal film. PBS solution is introduced again to stabilize the baseline. Finally, 1mL of triethanolamine (TEA) solution is introduced to elute the silica colloidal crystal film enriched EVs and collect them in a centrifuge tube for subsequent experiments. The entire process of EV separation is recorded by OPLI.

[0079] Results are as follows Figure 4As shown, after the baseline was stable, the 200s circulation of MSC culture supernatant was introduced, and the optical thickness was obviously increased. The curve rose rapidly during 200-1800s, and then tended to be stable. At 2000s, the separation was basically completed. After the Triton X-100 or NP40 flushing, the optical thickness slightly decreased at 2200s to 2500s, and then quickly stabilized. After the PBS solution was introduced, the optical thickness was still very stable, indicating that the DSPE could be stably combined with the EVs. After the TEA solution was introduced, the optical thickness was significantly decreased, and basically returned to the initial value, proving that the EVs adsorbed on the silica colloidal crystal film were completely eluted.

[0080] The calculated optical thickness of the silica colloidal crystal film was subjected to spectral line fitting by using the extreme value method, and the specific formula was as follows: The optical thickness was fitted, wherein λ was the real-time wavelength of the interference peak in the interval of 430-1100nm, k' was the relative order, k'=0, 1, 2, 3, 4···, and nd was the optical thickness.

[0081] After the optical thickness was completely stable, the EVs eluted were subjected to Western blotting experiment, and the expression amount of the EVs specific protein CD9 was analyzed. The results were as shown in Figure 5 As shown in the gel map, CD9 was obviously expressed, indicating that the method (OPLI) of the application could separate and enrich the EVs in the MSC culture supernatant.

[0082] After the optical thickness was completely stable, the EVs eluted were subjected to transmission electron microscopy observation. As shown in Figure 6 Many circular structures could be obviously observed, and the size was consistent with the particle size (30-150nm) range of the EVs.

[0083] Example 2: Separation and enrichment of EVs in urine

[0084] S1. Preparation of silica colloidal crystal film (SCC film)

[0085] (1) All glass slides were immersed in piranha solution overnight in advance, and then washed with ultrapure water.

[0086] (2) The 190nm diameter silica microspheres were centrifuged, and then repeatedly washed and centrifuged with anhydrous ethanol to form a silica ethanol suspension with a mass concentration of 1%.

[0087] (3) The glass slides soaked with piranha were vertically placed in the silica ethanol suspension, and the silica colloidal crystal film could be formed after 7 days. The temperature of the placement environment was 25±5℃, and the humidity was 10±2%.

[0088] S2. Modification of DSPE on silica colloidal crystal thin film

[0089] (1) The silica colloidal crystal thin film was immersed in 2% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amino modification.

[0090] (2) 100 mg DSPE was dissolved in 1 mL 30% ethanol solution (v / v) to obtain solution one; 200 mg NHS (N-hydroxysuccinimide) and 400 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL Tris-HCl buffer (pH = 6.5) to obtain solution two. Solution one and solution two were mixed and the pH value was adjusted to 7.5. The amino-modified silica colloidal crystal thin film was reacted with the mixed solution for 6 h to obtain the DSPE-modified silica colloidal crystal thin film.

[0091] (3) The DSPE-modified silica colloidal crystal thin film obtained in step (2) was washed with ultrapure water for 3 times and could be used for subsequent analysis or experiment. When not in use, it could be stored in ultrapure water for standby use.

[0092] S3. Construction of OPLI system

[0093] (1) A hole was drilled on the silica gel pad, which was covered on the DSPE-modified silica colloidal crystal thin film glass slide to form a reaction cell.

[0094] (2) Another glass slide was used to press the upper end to form a closed reaction cell.

[0095] (3) The closed reaction cell obtained in step (2) was fixed on an inverted microscope, and the focal length was adjusted to the silica colloidal crystal layer.

[0096] (4) The spectroscope, coupling fiber and spectrometer were connected.

[0097] (5) The catheter was connected to the reaction cell, and the catheter was connected to the peristaltic pump to make the reaction liquid flow into the hole in the middle of the silica gel pad. Another catheter was connected to make the liquid after reaction flow out.

[0098] S4. Using a peristaltic pump, 0.1M PBS solution was introduced into the closed reaction cell through the conduit (5) in step S3 at a flow rate of 0.4 mL / min. After the baseline of the spectrometer was stable, the selected reflection interference spectrum band was subjected to spectral line fitting, and the real-time optical thickness of the silica colloidal crystal film was obtained by calculation. After the optical thickness of the silica colloidal crystal film was stable, 1 mL of urine was introduced into the closed reaction cell through the conduit (5) in step S3 using a peristaltic pump, and the selected reflection interference spectrum band was subjected to spectral line fitting. The optical thickness of the silica colloidal crystal film was obtained by calculation using the extremum method.

[0099] The DSPE molecules on the surface of the silica colloidal crystal film can specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase. After the curve rises steadily, PBS solution is introduced again to stabilize the baseline. Then, Triton X-100 or NP40 with a volume concentration of 0.02% is introduced as a detergent to wash away the excess proteins and other impurities in the culture supernatant adsorbed on the silica colloidal crystal film. PBS solution is introduced again to stabilize the baseline. Finally, 1 mL of triethanolamine (TEA) solution is introduced to elute the EVs enriched on the silica colloidal crystal film and collect them in a centrifuge tube for subsequent experiments. The entire process of EV separation is recorded by OPLI.

[0100] The results are shown in Figure 7 After the baseline is stable, the optical thickness increases significantly after 200 s of urine introduction. The curve rises rapidly during the period of 200-1800 s, and then tends to be stable. The separation is basically completed at 2000 s. After washing with Triton X-100 or NP40, the optical thickness decreases slightly from 2200 s to 2500 s and stabilizes quickly. After introducing PBS solution again, the optical thickness remains very stable, indicating that DSPE can stably bind to EVs. After introducing TEA solution, the optical thickness decreases significantly and basically returns to the initial value, indicating that the EVs adsorbed on the silica colloidal crystal film are completely eluted.

[0101] The calculated optical thickness of the silica colloidal crystal film is subjected to spectral line fitting using the extremum method, and the formula for fitting the optical thickness is as follows: where λ is the real-time wavelength of the interference peak in the range of 430-1100 nm, i.e., the selected reflection interference spectrum band, k' is the relative order, k' = 0, 1, 2, 3, 4,..., and nd is the optical thickness.

[0102] After the optical thickness rises completely and stably, the eluted EVs are subjected to Western blotting to analyze the expression of EV-specific protein CD9. The results are shown in Figure 8As shown in the gel part, CD9 has obvious expression, indicating that the method (OPLI) described in the application can separate and enrich EVs in urine.

[0103] The EVs eluted after the optical thickness rose completely smoothly were observed by transmission electron microscopy. As shown in FIG. 3, many circular structures can be observed obviously, and the size is consistent with the particle size range of EVs. Figure 9

[0104] Example 3: Verification of effectiveness of real-time concentration detection of EVs

[0105] S1. Preparation of silica colloidal crystal film (SCC film)

[0106] (1) All glass slides were immersed in piranha solution overnight and then rinsed with ultrapure water.

[0107] (2) The 190 nm diameter silica microspheres were centrifuged and repeatedly washed with anhydrous ethanol, centrifuged, and formed into a 1% silica ethanol suspension.

[0108] (3) The piranha-soaked glass slides were vertically placed in the silica ethanol suspension, and after 7 days, a silica colloidal crystal film was formed. The temperature of the placement environment was 25±5℃, and the humidity was 10±2%.

[0109] S2. Modification of DSPE on the silica colloidal crystal film

[0110] (1) The silica colloidal crystal film was soaked in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amino functionalization.

[0111] (2) 10 mg of DSPE was dissolved in 1 mL of a 30% ethanol solution (v / v) to obtain solution one; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution two. Solution one and solution two were mixed and the pH value was adjusted to 7.5. The amino-functionalized silica colloidal crystal film was reacted with the mixed solution for 6 h to obtain a DSPE-modified silica colloidal crystal film.

[0112] (3) The DSPE-modified silica colloidal crystal film obtained in step (2) was washed with ultrapure water for 3 times and then used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for standby.

[0113] S3. Construction of OPLI system

[0114] ​(1) Drill holes in the silicone pad and cover it on a glass slide with silica gel crystal film modified with DSPE to form a reaction cell.

[0115] (2) Press the top with another glass slide to form a closed reaction pool.

[0116] (3) Fix the closed reaction cell obtained in step (2) on an inverted microscope and adjust the focus to the silica gel crystal layer.

[0117] (4) Couple the optical fiber and connect it to the spectrometer through the beam splitter.

[0118] (5) Connect the conduit to the reaction tank and connect the conduit to the peristaltic pump so that the reaction liquid can flow into the hole in the middle of the silicone pad. Repeat the operation to connect another conduit so that the liquid after the reaction can flow out.

[0119] S4. Using a peristaltic pump, 0.1MPBS solution is introduced into the closed reaction chamber at a flow rate of 0.4mL / min through the conduit in step S3 (5). After the spectrometer baseline stabilizes, spectral line fitting is performed on the selected reflection interference spectral band using the extreme value method. The optical thickness of the silica crystalline film in real time is calculated. After the optical thickness of the silica crystalline film stabilizes, 1mL of MSC culture supernatant is circulated through the conduit in step S3 (5) using a peristaltic pump. Spectral line fitting is performed on the selected reflection interference spectral band using the extreme value method. The optical thickness of the silica crystalline film is calculated. DSPE molecules on the surface of the silica crystalline film specifically bind to the phospholipid bilayer of EVs, causing an increase in optical thickness.

[0120] S5. Dilute 1 mL of MSC culture supernatant with PBS solution in a series of concentrations (dilution ratios include 0.1, 0.25, 0.5, 0.75, and 1), repeating step S4. Figure 10 As shown, the supernatant of undiluted MSC culture medium (i.e., at a dilution ratio of 1) exhibits the fastest curve rise and ultimately results in the highest change in optical thickness. With increasing culture medium dilution ratio, the curve rises more slowly, and the resulting change in optical thickness decreases accordingly.

[0121] The optical thickness of the calculated silica bulk crystal film was fitted to the spectrum using the extreme value method, according to the formula... Fit the optical thickness, where λ is the real-time wavelength of the interference peak in the wavelength range of 430-1100nm, i.e. the selected reflection interference spectrum band, k′ is the relative order, k′=0, 1, 2, 3, 4···,nd is the optical thickness.

[0122] Figure 11is taken as the horizontal coordinate and the change of optical thickness is taken as the vertical coordinate. From the figure, it can be seen that the linear relationship between the dilution concentration of the supernatant of the culture solution and the change of optical thickness is very good, R Figure 11 = 0.991, which indicates that the detection method provided by the present application can effectively enrich EVs and realize real-time detection of the concentration. 2

[0123] Example 4: Kinetic analysis of EVs membrane proteins

[0124] S1. Preparation of silica colloidal crystal film (SCC film)

[0125] (1) All glass slides were immersed in piranha solution overnight and then washed with ultrapure water.

[0126] (2) The 190 nm diameter silica microspheres were centrifuged and repeatedly washed with anhydrous ethanol, centrifuged, and formed into a 1% silica ethanol suspension.

[0127] (3) The glass slides soaked with piranha were vertically placed in the silica ethanol suspension, and after 7 days, the silica colloidal crystal film was formed. The temperature of the placement environment was 25±5℃, and the humidity was 10±2%.

[0128] S2. Modification of DSPE on the silica colloidal crystal film

[0129] (1) The silica colloidal crystal film was soaked in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amino functionalization.

[0130] (2) 10 mg of DSPE was dissolved in 1 mL of a 30% ethanol solution (v / v) to obtain solution one; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH = 6.5) to obtain solution two. Solution one and solution two were mixed and the pH value was adjusted to 7.5. The amino-functionalized silica colloidal crystal film was reacted with the mixed solution for 6 h to obtain the DSPE-modified silica colloidal crystal film.

[0131] (3) The DSPE-modified silica colloidal crystal film obtained in step (2) was washed with ultrapure water for 3 times and then used for subsequent analysis or experiments. When not in use, it can be stored in ultrapure water for standby.

[0132] S3. Construction of OPLI system

[0133] ​(1) Drill a hole on a glass slide and cover it on the modified DSPE silica colloidal crystal film glass slide to form a reaction cell.

[0134] (2) Press the upper end with another glass slide to form a closed reaction cell.

[0135] (3) Fix the reaction cell on an inverted microscope and adjust the focal length to the silica colloidal crystal layer.

[0136] (4) Coupling optical fiber through a spectroscope and connecting a spectrometer.

[0137] (5) Connect a conduit to the reaction cell and connect the conduit to a peristaltic pump to enable the reaction liquid to flow into the hole in the middle of the silica gel pad. Repeat the operation to connect another conduit to enable the reaction liquid to flow out.

[0138] S4. Using the peristaltic pump to pass 0.1M PBS solution through the conduit in step S3 (5) at a flow rate of 0.4mL / min to the closed reaction cell, and when the baseline of the spectrometer is stable, the selected reflection interference spectrum band is fitted by the extreme value method, and the real-time optical thickness of the silica colloidal crystal film is calculated. When the optical thickness of the silica colloidal crystal film is stable, 1mL of MSC culture supernatant is circulated through the conduit in step S3 (5) using a peristaltic pump, and the selected reflection interference spectrum band is fitted by the extreme value method, and the optical thickness of the silica colloidal crystal film is calculated. The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase.

[0139] S5. Pass 0.1M PBS solution, and when the baseline is stable, pass the antibody solution of CD9. After the binding is stable, pass 0.1M PBS solution again, as shown in Figure 12 It can be seen from Figure 12 that, taking the binding of CD9 antibody as an example, after the EVs are fixed on the silica colloidal crystal film, the optical thickness gradually increases after the antibody solution is circulated, which is caused by the specific binding of the CD9 antibody to the EV membrane protein CD9 fixed on the silica colloidal crystal film. After passing PBS, the optical thickness will slowly decrease, and there is a slight dissociation process between CD9 and its antibody.

[0140] The optical thickness of the silica colloidal crystal film is calculated by fitting the spectrum by the extreme value method, and the fitted optical thickness is calculated according to the formula , where λ is the real-time wavelength of the interference peak in the interval of 430-1100nm, i.e. the selected reflection interference spectrum band, k' is the relative order, k'=0, 1, 2, 3, 4···, nd is the optical thickness.

[0141] The steps 1-6 were repeated with the antibody solution of membrane protein CD63, CD81, respectively, and the dissociation curves of the binding of EVs membrane protein and antibody were recorded. The real-time optical thickness changes of the binding and dissociation of EVs membrane protein and the antibody of membrane protein CD9, CD63 and CD81 in the liquid supernatant of MSC culture solution were shown as Figure 13 Figure 13 It can be seen that the optical thickness changes caused by the binding of membrane protein CD9, CD63 and CD81 with the corresponding antibody respectively are not very significant, and there is a slight dissociation phenomenon after the PBS is passed, which proves that the binding between them is very firm. The above results show that the method described in the application can be used to detect the firmness of the binding of EVs membrane protein and antibody, and can be used for drug screening.

[0142] Example 5: Prostate cancer liquid biopsy assisted diagnosis of EVs

[0143] S1. Preparation of silica colloidal crystal film (SCC film)

[0144] (1) All glass slides were immersed in piranha solution overnight in advance, and then washed with ultrapure water.

[0145] (2) The 190 nm diameter silica microspheres were centrifuged and repeatedly washed with anhydrous ethanol, centrifuged to form a silica ethanol suspension with a mass concentration of 1%.

[0146] (3) The glass slides soaked with piranha were vertically placed in the silica ethanol suspension, and the silica colloidal crystal film was formed after 7 days. The temperature of the placement environment was 25±5℃, and the humidity was 10±2%.

[0147] S2. Modification of DSPE on the silica colloidal crystal film

[0148] (1) The silica colloidal crystal film was soaked in a 1% APTES (aminopropyltriethoxysilane) ethanol solution (v / v) overnight for amino functionalization.

[0149] (2) 10 mg of DSPE was dissolved in 1 mL of 30% ethanol solution (v / v) to obtain solution one; 50 mg of NHS (N-hydroxysuccinimide) and 100 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were dissolved in 4 mL of Tris-HCl buffer (pH=6.5) to obtain solution two. The two solutions were mixed and the pH value was adjusted to 7.5. The amino-functionalized silica colloidal crystal film was reacted with the mixed solution for 6 h to obtain the DSPE-modified silica colloidal crystal film.

[0150] ​(3) The silica colloidal crystal film modified with DSPE obtained in step (2) is washed with ultrapure water for 3 times, and can be used for subsequent analysis or experiment. If not used, it can be stored in ultrapure water for standby use.

[0151] S3. Building OPLI system

[0152] (1) Drill holes on the silica gel pad, cover it on the glass slide of the silica colloidal crystal film modified with DSPE, and form a reaction pool.

[0153] (2) Press the upper end with another glass slide to form a closed reaction pool.

[0154] (3) Fix the closed reaction pool obtained in step (2) on an inverted microscope, and adjust the focal length to the silica colloidal crystal layer.

[0155] (4) Coupling optical fiber and connecting spectrometer through a spectroscope.

[0156] (5) Connect the conduit with the reaction pool, and connect the conduit to the peristaltic pump, so that the reaction liquid can flow into the hole in the middle of the silica gel pad. Repeat the operation to connect another conduit, so that the liquid after reaction can flow out.

[0157] S4. Use the peristaltic pump to pass 0.1M PBS solution into the closed reaction pool through the conduit in step S3 (5) at a flow rate of 0.4 mL / min. When the baseline of the spectrometer is stable, perform spectral line fitting on the selected reflection interference spectrum band. The spectral line fitting method is the extreme value method. After calculation, the real-time optical thickness of the silica colloidal crystal film is obtained. When the optical thickness of the silica colloidal crystal film is stable, use the peristaltic pump to pass 1 mL of urine of prostate cancer patient through the conduit in step S3 (5) in a cycle. Perform spectral line fitting on the selected reflection interference spectrum band. The spectral line fitting method is the extreme value method. After calculation, the optical thickness of the silica colloidal crystal film is obtained. The DSPE molecules on the surface of the silica colloidal crystal film will specifically bind to the phospholipid bilayer of EVs, causing the optical thickness to increase.

[0158] S5. Pass 0.1M PBS solution, and when the baseline is stable, pass CD9 antibody solution. After the binding is stable, pass αvβ3 antibody solution as the cancer marker, as shown in Figure 14 . As can be seen from Figure 14 , taking αvβ3 cancer marker as an example, after the EVs in the urine of prostate cancer patient are fixed on the silica colloidal crystal film, the optical thickness gradually increases after the antibody solution is circulated and passed. This is caused by the specific binding of the antibody to the high expression of αvβ3 membrane protein of prostate cancer patient EVs fixed on the silica colloidal crystal film.

[0159] S6. The antibody solution of αvβ3 and Trop-2 cancer markers is introduced, and steps 1-6 are repeated, and the optical thickness change of the binding of EVs in the urine samples of healthy people and prostate cancer patients with the antibody is recorded.

[0160] The optical thickness of the silica colloidal crystal film is calculated by using the extremum method for spectral line fitting, according to the formula The optical thickness is fitted, wherein λ is the real-time wavelength of the interference peak in the interval of 430-1100 nm, that is, the selected reflection interference spectrum band, k' is the relative order, k' = 0, 1, 2, 3, 4,..., and nd is the optical thickness.

[0161] The optical thickness changes caused by the combination of the EVs in the urine samples of the prostate cancer patients with the αvβ3 and Trop-2 antibodies circulating into the solution are shown in Figure 14 and Figure 15 It can be seen from Figure 14 and Figure 15 that during the fixation of the EVs in the urine samples of the prostate cancer patients on the silica colloidal crystal film, the optical thickness rapidly rises, and then the PBS is washed. After the tumor marker antibodies αvβ3 and Trop-2 are pumped into the solution, the optical thickness also significantly increases. Figure 16 is the optical thickness change value caused by the combination of the EVs in the urine samples of the prostate cancer patients and healthy people with the tumor marker antibodies αvβ3 and Trop-2 circulating into the solution, and the optical thickness change value caused by the prostate cancer patients is significantly higher than that of the healthy people, P<0.001; it is indicated that the method can be used for assisting the liquid biopsy of prostate cancer.

[0162] Comparative Example 1: Separation of EVs in MSC culture supernatant by ultracentrifugation method

[0163] Ultracentrifugation (UC) method:

[0164] 1 mL of MSC culture solution was taken. First, 2500g centrifugation for 10 min, and the supernatant was taken; then, 10000g centrifugation at 4℃ for 45 min, and the supernatant was taken; then, 120000g centrifugation at 4℃ for 70 min, and the precipitate was taken; finally, PBS was added to the precipitate, the precipitate was dispersed, and 120000g centrifugation at 4℃ for 70 min was performed, and the precipitate was taken. The precipitate was resuspended with 1 mL of PBS.

[0165] By analyzing the expression amount of the specific protein CD9 of the EVs separated by the method (OPLI) and the ultracentrifugation (UC) method, the separation efficiency of the two methods is compared, and the results are shown in Figure 5As shown in the histogram part, the capture intensity of EVs isolated from MSC culture supernatant by the method of the present application is set to 1, and the capture intensity of EVs directly isolated from the supernatant by ultracentrifugation method is only 0.22; indicating that the efficiency of the method of the present application for isolating EVs from MSC culture supernatant is significantly higher than that of the ultracentrifugation method for isolating EVs from MSC culture supernatant.

[0166] Comparative Example 2: Ultracentrifugation method for isolating EVs from urine

[0167] Ultracentrifugation (UC) method:

[0168] Take 1 mL of MSC culture medium. First, centrifuge at 2500g for 10 min, take the supernatant; then, centrifuge at 10000g at 4°C for 45 min, take the supernatant; then, centrifuge at 120000g at 4°C for 70 min, take the precipitate; finally, add PBS to the precipitate, disperse the precipitate, and then centrifuge at 120000g at 4°C for 70 min, take the precipitate. Resuspend the precipitate with 1 mL of PBS.

[0169] By analyzing the expression amount of specific protein CD9 of EVs isolated by the method of the present application (OPLI) and ultracentrifugation (UC) method, the separation efficiency of the two methods was verified, and the results are as follows Figure 8 As shown in the histogram part, the capture intensity of EVs isolated from MSC culture supernatant by the method of the present application is set to 1, and the capture intensity of EVs directly isolated from the supernatant by ultracentrifugation method is only 0.22; indicating that the efficiency of the method of the present application for isolating EVs from MSC culture supernatant is significantly higher than that of the ultracentrifugation method for isolating EVs from MSC culture supernatant.

Claims

1. A method for extracellular vesicle real-time enrichment and detection, characterized by, The method comprises the following steps: S1. Preparing a silica colloidal crystal film, i.e. an SCC film, by using a vertical deposition method; S2. Preparing a DSPE-modified silica colloidal crystal film; The preparation method of the DSPE-modified silica colloidal crystal film comprises: immersing the SCC film in an ethanol solution of aminopropyltriethoxysilane to obtain an aminated SCC film; dissolving DSPE in an ethanol solution to obtain solution one; dissolving N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in a Tris-HCl buffer to obtain solution two; mixing solution one and solution two and adjusting the pH value to obtain a mixed solution; reacting the aminated SCC film with the mixed solution and then washing with ultrapure water to obtain the DSPE-modified silica colloidal crystal film, which is stored in ultrapure water for standby use; S3. Assembling an OPLI (ordered porous layer interferometry) system: Assembling a reaction cell with the DSPE-modified silica colloidal crystal film as a substrate and combining the reaction cell with an inverted microscope and a fiber-optic spectrometer to form the OPLI system; S4. Separating and enriching extracellular vesicles: circulating a PBS solution into the reaction cell of the OPLI system, stabilizing the baseline, then circulating a sample solution of extracellular vesicles to be separated into the reaction cell, waiting for the spectrometer to show that the baseline is stable, performing spectral line fitting on a selected reflection interference spectrum band, and using an extreme value method to fit and calculate the optical thickness 1 of the silica colloidal crystal film, thereby completing the separation and enrichment of the extracellular vesicles; S5. Dissociating the enriched extracellular vesicles: after the enrichment is completed, circulating a detergent into the reaction cell to wash away impurities adsorbed on the SCC film, and finally circulating an elution solution into the reaction cell to dissociate the extracellular vesicles enriched by the SCC film; S6. Establishing a linear relationship and analyzing the concentration of extracellular vesicles: circulating sample solutions of extracellular vesicles to be separated at different dilution ratios into the reaction cell, repeating steps S4 and S5, taking the optical thickness 1 as the vertical coordinate and taking the dilution ratio as the horizontal coordinate to establish a linear relationship, and analyzing the concentration of the extracellular vesicles; S7. Repeating steps S1-S5, except that in step S5, instead of circulating the elution solution, a solution of extracellular vesicle membrane protein antibodies and a PBS solution are circulated into the reaction cell; performing spectral line fitting on a selected reflection interference spectrum band, using an extreme value method to fit and calculate the optical thickness 2 and the optical thickness 3 of the silica colloidal crystal film, and analyzing the expression amount of the corresponding extracellular vesicle membrane proteins and the kinetics of the binding and dissociation of the proteins with the antibodies.

2. The method of claim 1, wherein, The concentration of the ethanol solution of aminopropyltriethoxysilane is 0.1%-2% (volume / volume), and the immersion time of the SCC film in the ethanol solution of aminopropyltriethoxysilane is 1-48 hours.

3. The method of claim 1, wherein, The amount of DSPE used in the ethanol solution is 1-100 mg: 1 mL.

4. The method of claim 1, wherein, The N-hydroxy succinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and Tris-HCl buffer are used in an amount of 20-200 mg: 50-400 mg: 4 mL.

5. The method of claim 1, wherein, The pH value of the mixture of the solution one and the solution two is adjusted to 7.5, and the reaction time is 4-12 h.

6. The method of claim 1, wherein, The assembling of the OPLI system in step S3 comprises the following steps: Drill holes on a silica gel pad, cover it on the modified DSPE silica colloidal crystal film glass slide to form a reaction pool, press the upper end with another glass slide to form a closed reaction pool, fix the reaction pool on an inverted microscope and adjust the focal length to the silica colloidal crystal layer, couple the optical fiber through the beam splitter and connect the spectrometer, connect the conduit with the reaction pool and connect the conduit to the peristaltic pump to make the reaction liquid flow into the hole in the middle of the silica gel pad, repeat the operation to connect another conduit to make the reacted liquid flow out.

7. The method of claim 1, wherein, The sample solution of the extracellular vesicles to be separated in step S4 comprises cell culture fluid, urine, milk, cerebrospinal fluid or blood.

8. The method of claim 1, wherein, The detergent in step S5 comprises Triton X-100 or ethyl phenyl polyethylene glycol, and the elution solution comprises triethanolamine.

9. The method of claim 1, wherein, The optical thicknesses in step S4 and step S7 are according to the formula are fitted, where is the real time wavelength of the interference peak in the interval of 430-1100 nm for the wavelength, i.e. the selected reflection interference spectrum wavelength band, is the relative order, = 0, 1, 2, 3, 4, ···, nd is the optical thickness.

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