Bionic microsphere, non-diagnostic purpose extracellular vesicle detection method and extracellular vesicle detection kit

Abstract

The invention belongs to the technical field of biochemical detection, and particularly relates to a bionic microsphere, a non-diagnostic-purpose extracellular vesicle detection method and an extracellular vesicle detection kit, the bionic microsphere is prepared by modifying a homologous cell membrane CM on the surface of a silica microsphere MS; the bionic microsphere (CMS) is used for capturing specific types of extracellular vesicles (EVs) in a to-be-detected sample and an optical / electrochemical detection method, can specifically capture homologous EVs, and is combined with fluorescence, ultraviolet visible absorption, differential pulse voltammetry and electrochemical luminescence for detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical detection, and particularly relates to a biomimetic microsphere, a method for detecting extracellular vesicles for non-diagnostic purposes, and a kit for detecting extracellular vesicles. Background Art

[0002] Extracellular vesicles (EVs) are phospholipid bilayer vesicle bodies containing nucleic acids, lipids, and proteins secreted by most cells. Due to carrying various signal transduction-related cargos of the parental cells, tumor-related EVs play important roles in tumor formation, growth and metastasis, angiogenesis, and drug resistance. Therefore, the content changes of EVs and their cargos can reflect the situation of the parental cells and are also important disease markers in the process of cancer development. The content of EVs has been proven to be closely related to cancers such as prostate cancer, breast cancer, lung cancer, ovarian cancer, and bladder cancer. EVs also show many advantages as detection markers, such as good specificity and stability. Therefore, the detection of the content of tumor-related EVs is of great significance for the early diagnosis of tumors, drug guidance, prognosis monitoring, and the research on the occurrence, development, metastasis, and drug resistance mechanisms of tumors.

[0003] Although EVs are present in various biological fluids for non-invasive detection, the analysis of tumor-related EVs still faces challenges such as low content and being easily interfered by the complex environmental factors in body fluids. In recent years, a series of EVs detection methods have been developed, such as spectroscopy, electrochemical methods, enzyme-linked immunosorbent assay, etc. However, most of the methods are still based on the recognition and capture of EVs surface proteins by antibodies or aptamers, and this mode is significantly limited to the analysis of EVs with high expression of specific proteins. Although the introduction of multi-target recognition probes improves the accuracy of EVs detection, the system cost is further increased. A large number of studies have shown that the inherent homologous targeting of EVs can enter homologous cells through membrane fusion, receptor-mediated endocytosis, phagocytosis, pinocytosis, etc. Based on this, directly applying the homologous targeting of EVs to bind to the parental cell membrane can effectively avoid the dependence on antibodies or aptamers, simplify the capture process, achieve the purpose of high-specificity recognition, and reduce the detection cost at the same time. Summary of the Invention

[0004] Object of the Invention: To solve the above problems, an embodiment of the present invention provides a biomimetic microsphere for capturing homologous extracellular vesicles and an optical / electrochemical multiplex detection method.

[0005] Technical Solution:

[0006] A method for capturing and optically / electrochemically detecting specific types of extracellular vesicles (EVs) in a sample to be tested using a biomimetic microsphere (CMS). The biomimetic microsphere can specifically capture homologous EVs and is combined with fluorescence, ultraviolet-visible absorption, differential pulse voltammetry, and electrochemiluminescence methods for detection.

[0007] In a first aspect, the present invention provides a biomimetic microsphere CMS, as Figure 16 shown, the biomimetic microsphere is prepared by modifying the surface of a silica microsphere MS with a homologous cell membrane CM. The homologous cell membrane refers to a biological membrane extracted from cells of the same type as the target to be detected. The diameter of the silica microsphere is 10 micrometers;

[0008] For example, if the target cell to be detected is a tumor cell, then the tumor cell membrane is extracted and coated on the surface of the microsphere by ultrasonic method to prepare a biomimetic microsphere for the capture of EVs. Preferably, the step of modifying the surface of the silica microsphere with a homologous cell membrane includes:

[0009] The homologous cell membrane suspension and the silica microsphere are ultrasonically assembled at 50 W for 15 - 60 min to form CMS, and after centrifugation and precipitation, it is resuspended in physiological saline to obtain a CMS suspension. Among them, the mass ratio of the homologous cell membrane to the silica microsphere is (50 - 100):1;

[0010] Preferably, the ultrasonic time is 30 min;

[0011] Preferably, the centrifugation step is specifically: 3500 rpm, 4 °C, centrifugation for 5 min, discard the supernatant, and take the precipitate;

[0012] Preferably, after the precipitate is resuspended in physiological saline, the concentration range of the precipitate is 0.0001 - 0.001 g / L.

[0013] In a second aspect, the present invention provides a method for detecting extracellular vesicles for non-diagnostic purposes based on the biomimetic microsphere described in the first aspect. In addition to being able to be used for the early diagnosis of tumor cells, based on the same technical principle, the biomimetic microsphere can also be used for the determination of food nutrient content and the detection of microbial flora;

[0014] The method for detecting extracellular vesicles for non-diagnostic purposes includes a sample pretreatment step, a homologous target incubation step, and a signal detection step:

[0015] The sample pretreatment step includes: preliminarily purifying the sample to be tested to extract extracellular vesicles EVs in the sample as a test solution, then mixing the test solution with a TDA-Co-Ln solution to modify the EVs with TDA-Co-Ln, and then adding a signal molecule reagent for co-incubation to further modify the signal molecule to obtain a pretreated sample solution;

[0016] The homologous targeting incubation step includes: mixing and incubating the above-mentioned pretreated sample solution with the biomimetic microsphere CMS constructed according to the first aspect; preferably, the CMS suspension is co-incubated with the pretreated sample solution at 37 °C for 25 to 60 min;

[0017] The signal detection step includes: purifying the mixture after the above-mentioned homologous targeting incubation step to separate the CMS that has captured homologous EVs, and the content of homologous EVs can be determined by measuring the signals carried by the EVs in the CMS that has captured homologous EVs.

[0018] Technical principle: As Figure 17 shown, after the total extracellular vesicles in the sample to be tested are preliminarily purified, such as by centrifugation extraction, lanthanide ions (Ln 3+ ) are used to modify the highly expressed sialic acid SA on the surface of EVs to improve their homologous targeting ability to achieve enhanced homologous targeting recognition. Then, a probe with optical or electrochemical signals is labeled. The biomimetic microsphere is prepared by coating the cell membrane homologous to the detection target cell on a silica carrier to achieve homologous recognition and capture of the EVs secreted by the detection target cells in the sample. The CMS that has captured homologous EVs can be separated by simple centrifugation, and the signals of the probes carried by the EVs captured on the CMS are measured to respectively realize optical / electrochemical multiplex detection methods.

[0019] Dietary sialic acid is a type of neuraminic acid derivative, a keto sugar acid, which is widely present in various foods and has various beneficial effects on human health. Therefore, as a specific embodiment of the extracellular vesicle detection method of the present invention, the sample to be tested can be various foods containing glycan nutritional components; the constructed biomimetic microsphere CMS is to modify the cell membrane of cells with highly expressed dietary sialic acid on the surface of silica microspheres; by measuring the proportion of the content of extracellular vesicles of cells with highly expressed dietary sialic acid in the test solution through the extracellular vesicle detection method, the content level of the glycan nutritional components in the food can be judged.

[0020] Bacteria such as Escherichia coli and Tannerella forsythia can highly express sialic acid. Therefore, as another specific embodiment of the extracellular vesicle detection method of the present invention, the sample to be tested can be a drinking water source, the test solution is the bacterial extracellular vesicles secreted by bacteria in the water source extracted by centrifugation, and the constructed biomimetic microsphere CMS is to modify the cell membrane of the bacteria with highly expressed sialic acid, which is the object to be tested, on the surface of silica microspheres. By measuring the content of bacterial extracellular vesicles in the test solution through the extracellular vesicle detection method and constructing a standard curve, the monitoring of the microbial content in the drinking water source can be realized.

[0021] In a third aspect, the present invention provides the use of the above-mentioned bionic microspheres in the preparation of a kit for detecting tumor-related EVs in blood samples, that is, the detection kit includes the above-mentioned CMS suspension and also includes a sample pretreatment reagent; the sample pretreatment reagent is a TDA-Co-Ln solution and a signal molecule reagent; the signal molecule reagent is any one of a fluorescent signal molecule, an electrochemical signal molecule, or a biotinylated membrane material.

[0022] The TDA-Co-Ln solution is used to modify all EVs after extracting total EVs from blood samples; the signal molecule reagent is used for detecting signal labeling of EVs; the CMS suspension is used for specifically recognizing and capturing the labeled EVs, and the bionic microspheres are collected by centrifugation and the tumor-related EVs are detected based on the corresponding analysis method.

[0023] As a preferred embodiment of the detection kit described in the third aspect of the present invention, that is, a kit for detecting tumor-related EVs in blood samples based on fluorescence method is constructed, as Figure 18 shown, the signal molecule reagent in the sample pretreatment reagent is the green fluorescent dye PKH67;

[0024] Preferably, the reagents and sample solutions in the kit for detecting cancer cell extracellular vesicles based on fluorescence method are configured according to the volume ratio as follows:

[0025]

[0026] The use steps of the kit for detecting cancer cell extracellular vesicles based on fluorescence method are:

[0027] According to the above volume ratio, in the obtained sample EVs solution, add the above TDA-Co-Ln solution to enhance homologous targeting, then add the above fluorescent dye PKH67, incubate at room temperature for 5 - 30 min, make up the system with physiological saline, add 50% PEG-8000 and incubate at 4°C for 90 - 120 min and then centrifuge, discard the supernatant, obtain EVs with fluorescent signals, and then capture with the above CMS suspension for 25 - 60 min. After the capture is completed, centrifuge and discard the supernatant, and then detect the signal of the CMS capturing EVs by a fluorescence spectrophotometer and calculate the content of EVs in the blood sample.

[0028] As another preferred embodiment of the detection kit described in the third aspect of the present invention, that is, a kit for detecting tumor-related EVs in blood samples based on ultraviolet-visible absorption spectrometry is constructed, as Figure 19 shown, the signal molecule reagent in the sample pretreatment reagent is phospholipid-PEG-biotin, streptavidin-horseradish peroxidase, and 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution.

[0029] Preferably, the reagents and sample solution in the cancer cell extracellular vesicle detection kit based on ultraviolet-visible absorption spectrometry are configured according to the volume ratio as follows:

[0030]

[0031]

[0032] The usage steps of the cancer cell extracellular vesicle detection kit based on ultraviolet-visible absorption spectrometry are as follows:

[0033] According to the above volume ratio, in the obtained sample EVs solution, add the above TDA-Co-Ln solution to enhance homologous targeting; then add DSPE-PEG-Bio and incubate at 37 °C for 15 - 60 min; then add the above SA-HRP and incubate at 37 °C for 30 min, then transfer to a 30 kDa ultrafiltration tube and centrifuge at 12000 g at 4 °C for 15 min, and resuspend the precipitate in the tube; further capture with the above CMS suspension for 25 - 60 min. After the capture, centrifuge and discard the supernatant, add the above 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution and react at 37 °C for 15 min. After adding the above stop solution, measure the absorbance at 450 nm and calculate the content of EVs in the blood sample.

[0034] As another preferred embodiment of the detection kit described in the third aspect of the present invention, that is, to construct a detection kit for tumor-related EVs in blood samples based on differential pulse voltammetry, as Figure 20 shown, the signal molecule reagent in the sample pretreatment reagent is methylene blue.

[0035] Preferably, the reagents and sample solution in the cancer cell extracellular vesicle detection kit based on differential pulse voltammetry are configured according to the volume ratio as follows:

[0036]

[0037] The usage steps of the cancer cell extracellular vesicle detection kit based on differential pulse voltammetry are as follows:

[0038] According to the above volume ratio, in the obtained sample EVs solution, add the above TDA-Co-Ln solution to enhance homologous targeting, then add the above methylene blue MB solution of the electro-signal molecule, incubate at room temperature for 5 - 30 min, then make up the system with physiological saline, add 50% PEG-8000 and incubate at 4 °C for 90 - 120 min, then centrifuge and discard the supernatant to obtain EVs with electrochemical signals, and then capture with the above CMS suspension for 25 - 60 min. After the capture, centrifuge and discard the supernatant, and then detect the signal of the CMS capturing EVs through an electrochemical workstation and calculate the content of EVs in the blood sample.

[0039] As another preferred embodiment of the detection kit described in the third aspect of the present invention, that is, to construct a detection kit for tumor-related EVs in blood samples based on electrochemiluminescence method, such as Figure 21 shown, the signal molecule reagent in the sample pretreatment reagent is phospholipid-PEG-biotin cascade streptavidin-horseradish peroxidase and luminol.

[0040] Preferably, each reagent and the sample solution in the cancer cell extracellular vesicle detection kit based on electrochemiluminescence method are configured according to the volume ratio as follows:

[0041]

[0042] The use steps of the cancer cell extracellular vesicle detection kit based on differential pulse voltammetry are as follows: according to the above volume fraction ratio, in the obtained sample EVs solution, take the above EVs solution and add the above TDA-Co-Ln solution to enhance the homologous targeting; then add the above phospholipid-PEG-biotin (DSPE-PEG-Bio) and incubate at 37 °C for 15-60 min; then add the above streptavidin-horseradish peroxidase (SA-HRP) and incubate at 37 °C for 30 min, then transfer to a 30 kDa ultrafiltration tube and centrifuge at 12,000 g and 4 °C for 15 min, and resuspend the precipitate in the tube; further capture with the above CMS suspension for 25-60 min, after the capture is completed, centrifuge to discard the supernatant, add the above luminol and 1% H 2 O 2 Measure the electrochemiluminescence intensity and calculate the content of EVs in the blood sample.

[0043] The detection kit described in the third aspect of the present invention is used to realize the detection of tumor-related EVs in blood samples. Different from the traditional extracellular vesicle detection method, the biomimetic microspheres (CMS) prepared by coating tumor cell membranes with silica microspheres as carriers retain the inherent homologous targeting recognition ability of cell membranes, can be used for the specific recognition of EVs, and can be collected by centrifugation after capture to achieve the rapid enrichment of tumor-related EVs.

[0044] The TDA-Co-Ln complex formed by 2,2'-thiodiacetic acid (TDA) as a ligand and cobalt ions and lanthanide ions can specifically modify Ln 3+ onto the surface of EVs, thereby promoting the binding of EVs to the homologous cell membranes on the surface of biomimetic microspheres and further improving the homologous capture efficiency.

[0045] On the basis of modifying the metal complex TDA-Co-Ln to enhance the homologous targeting effect, signal molecules such as the fluorescent signal PKH67, the electrochemical signal methylene blue, and biotinylated membrane molecules are labeled on all extracellular vesicles. Then, the captured bionic microspheres are collected by centrifugation to achieve the specific enrichment of tumor-related extracellular vesicles. Further, the signals carried by the bionic microspheres are detected, and through the optimization of the modification and capture conditions and the targeting and recovery rate tests, a highly specific and highly sensitive detection of tumor-related EVs in blood is established, realizing the fluorescence, ultraviolet-visible absorption spectroscopy, differential pulse voltammetry, and electrochemiluminescence analysis of tumor-related extracellular vesicles.

[0046] Since the cell membrane coated on the CMS can be replaced, the electrochemiluminescence platform combined with 5 kinds of cell membranes coated on the CMS can also be used for the detection of different tumor-related EVs in blood to achieve the diagnosis of cancer types. In addition, based on the fluorescence microplate reader, the tumor-related EVs modified with fluorescent signals captured by the CMS can also be used for batch screening of blood samples.

[0047] Therefore, the present invention not only provides an efficient and accurate method for capturing extracellular vesicles and an optical / electrochemical multiplex detection strategy, but also the preparation process of the bionic microspheres is simple, time-consuming, easy to separate, and low-cost. Using the homologous targeting recognition of the bionic microspheres and extracellular vesicles, no modification of antibodies or aptamers is required, and the detection cost is low. The method of using the homologous targeting bionic microspheres for capturing extracellular vesicles in blood samples and optical / electrochemical multiplex detection has great application potential and broad market prospects. Description of the Drawings

[0048] Figure 1 It is the fluorescence micrograph of the silica microspheres before and after membrane modification in Example 1;

[0049] Figure 2 It is the scanning electron microscope image of the silica microspheres before and after membrane modification in Example 1;

[0050] Figure 3 It is the content of the silica microspheres modified with the membrane in Example 1;

[0051] Figure 4 It is the nanoparticle tracking analysis of extracellular vesicles in Example 2;

[0052] Figure 5 It is the modification of extracellular vesicles with Ln in Example 3 3+ The particle size and zeta potential result graphs before and after;

[0053] Figure 6 It is the statistical analysis of a single bionic microsphere capturing extracellular vesicles at the saturated concentration in Example 4;

[0054] Figure 7For the comparison of the effects before and after the capture and modification of Ln by the bionic microspheres in Example 5 3+ Before and after;

[0055] Figure 8 For the comparison of the effects before and after the capture and modification of Ln by the bionic microspheres in Example 6 3+ Before and after;

[0056] Figure 9 For the comparison of the effects before and after the capture and modification of Ln by the bionic microspheres in Example 7 3+ Before and after;

[0057] Figure 10 For the capture and detection of extracellular vesicles by the bionic microspheres in Example 8 using fluorescence analysis;

[0058] Figure 11 For the capture and detection of extracellular vesicles by the bionic microspheres in Example 8 using ultraviolet-visible absorption analysis;

[0059] Figure 12 For the capture and detection of extracellular vesicles by the bionic microspheres in Example 8 using differential pulse voltammetry;

[0060] Figure 13 For the capture and detection of extracellular vesicles by the bionic microspheres in Example 8 using electrochemiluminescence analysis;

[0061] Figure 14 For the results of the detection of 5 cancer blood samples using the electrochemiluminescence platform combining 5 CMSs in Example 9;

[0062] Figure 15 For the batch detection of extracellular vesicles in blood samples using bionic microspheres in Example 10;

[0063] Figure 16 For the preparation process of the bionic microspheres described in the first aspect of the present invention;

[0064] Figure 17 For the schematic diagram of the steps of the detection method described in the second aspect of the present invention;

[0065] Figure 18 For the schematic diagram of the detection principle of the detection kit for tumor-related EVs in blood samples based on fluorescence method described in the present invention;

[0066] Figure 19 For the schematic diagram of the detection principle in the detection kit for tumor-related EVs in blood samples based on ultraviolet-visible absorption spectrometry described in the present invention;

[0067] Figure 20 For the application schematic diagram in the detection kit for tumor-related EVs in blood samples based on differential pulse voltammetry described in the present invention;

[0068] Figure 21 This is a schematic diagram of the application in the kit for detecting tumor-related EVs in blood samples based on the electrochemiluminescence method of the present invention. Detailed implementation mode

[0069] The technical solution of the present invention will be described in detail below. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0070] Sources of main raw materials and reagents:

[0071] Silica microspheres were purchased from Jiangsu Zhichuan Technology Co., Ltd., with a model of 10 microns; the breast cancer cell line MDA-MB-436 was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the cells used for extracting extracellular vesicles were the MDA-MB-436 cell line, purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; PEG-8000 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0072] Example 1: Preparation of CMS

[0073] Extraction of cell membranes: Discard the DMEM medium from the obtained breast cancer cells MDA-MB-436, wash with PBS, digest with 1 mL of trypsin for 1 minute and 45 seconds, then add 3 mL of 10% DMEM medium to terminate digestion. Gently pipette the cells down and aliquot them into EP tubes; centrifuge at 3000 rpm, 4°C for 5 minutes, discard the supernatant, and resuspend the precipitate with 100 μL of 0.01 M Tris-Mg. Then perform cell disruption treatment. The operation of cell disruption treatment here is: sonicate for 5 seconds, pause for 10 seconds, for 5 minutes, with a power of 20 W; centrifuge at 11200 rpm, 4°C for 15 minutes, discard the supernatant containing cell contents, resuspend the precipitate in 300 μL of 0.01 M Tris-Mg buffer, and add a sucrose solution with a final concentration of 0.25 M; centrifuge at 2000 g, 4°C for 10 minutes to further remove organelle precipitates, take the supernatant; centrifuge at 3000 g, 4°C for 30 minutes, discard the supernatant, and resuspend the precipitate in 100 μL of physiological saline.

[0074] Preparation of CMS: Add 20 μL of the above-prepared cell membrane suspension (0.15 mg / mL) to silica microspheres with a final concentration of 0.2 mM, sonicate at 50 W for 30 minutes, then centrifuge at 3500 rpm, 4°C for 5 minutes, discard the supernatant, and resuspend the precipitate in 200 μL of physiological saline.

[0075] As Figure 1As shown, it is the fluorescence micrograph before and after the modification of the silica microspheres with the membrane (the cell membrane was pre-modified with DiI red fluorescence signal). It can be seen that the cell membrane was evenly coated on the surface of the microspheres, showing red fluorescence;

[0076] As Figure 2 Shown is the scanning electron micrograph before and after the modification of the silica microspheres with the membrane. It can be seen that a transparent film-like substance was wrapped on the surface of the microspheres, and the surface fine texture structure became rough;

[0077] As Figure 3 Shown is the standard curve and calculation result for the content calculation of the silica microsphere-modified membrane. Among them, Figure a is the standard curve of the membrane protein BCA, which is the standard curve of the protein content measured by the BCA kit and the ultraviolet absorption intensity at 450 nm; Figure b is the calculation result of the content of the membrane modified on the surface of the silica microspheres, which is the result calculated by comparing the ultraviolet absorption intensity at 450 nm after adding the BCA working solution with the standard curve according to different contents of the cell membrane coated on the surface of the silica microspheres;

[0078] Example 2: Extraction of the detection sample, i.e., extracellular vesicles

[0079] Collect the DMEM medium with the growth density of the breast cancer cell line MDA-MB-436 exceeding 80%. Centrifuge at 3000 g and 4 °C for 15 min, and take the supernatant; centrifuge at 10000 g and 4 °C for 30 min, and take the supernatant; filter with a 0.22 μM filter membrane; add PEG-8000 with a final concentration of 10% and let it stand overnight at 4 °C; centrifuge at 6500 g and 4 °C for 20 min, and discard the supernatant. Add PEG-8000 with a final concentration of 10% to the precipitate again and let it stand at 4 °C for 2 h, centrifuge at 6500 g for 20 min, discard the supernatant, and repeat the secondary extraction with PEG-8000. The final precipitate is resuspended in 100 μL of normal saline.

[0080] As Figure 4 Shown is the nanoparticle tracking analysis of the extracellular vesicles (EVs). The concentration in the figure refers to the standardized concentration of the extracellular vesicles extracted in Example 2.

[0081] Example 3: Enhanced modification of the targeting of the sample

[0082] Enhanced modification of the targeting of extracellular vesicles: After fully mixing TDA-Co-Ln with the above-extracted EVs, centrifuge at 6500 g and 4 °C for 20 min, discard the supernatant, and resuspend the precipitate in 100 μL of normal saline. Measure its particle size and potential changes. The results are as Figure 5 shown, and the test results verify the success of the enhanced targeting modification.

[0083] Example 4: Detection of extracellular vesicles captured by CMS by fluorescence method

[0084] Take 10 μL of the above EVs solution (1.26×10 8 EVs / mL), add 8 μL of TDA-Co-Eu / La / Dy (0.1 mg / mL) to enhance homologous targeting, then add 10 μL of the green fluorescent dye PKH67 diluted 250 times. After incubating at room temperature for 30 min, make up the system to 100 μL with normal saline. Add PEG-8000 with a final concentration of 10% and incubate at 4 °C for 120 min, then centrifuge. Discard the supernatant to obtain EVs with enhanced homologous targeting. Then capture with 4 μL of CMS suspension for 30 min. After the capture, centrifuge at 3500 rpm and 4 °C for 5 min, discard the supernatant, and then detect the signal of CMS capturing EVs by a fluorescence spectrophotometer.

[0085] Figure 6 For the statistical analysis of the capture of extracellular vesicles at the saturated concentration by a single biomimetic microsphere, where Figure a is the fluorescence microscopic images of a single biomimetic microsphere capturing EVs and EVs-Ln: EVs-Eu, EVs-La, EVs-Dy; the cell membrane is pre-modified with DiI red fluorescence signal, and then extracellular vesicles modified with PKH67 green fluorescence signal are captured; Figure b is the fluorescence intensity quantification diagram of the fluorescence image;

[0086] Example 5: Detection of biotinylated extracellular vesicles captured by CMS by ultraviolet-visible absorption spectrometry

[0087] Take 10 μL of the above EVs solution (1.26×10 8 EVs / mL), add 8 μL of TDA-Co-Eu / La / Dy (0.1 mg / mL) to enhance homologous targeting; then add 100 μM of phospholipid-PEG-biotin (DSPE-PEG-Bio) and incubate at 37 °C for 15 - 60 min; then add 10 μL of streptavidin-horseradish peroxidase (SA-HRP) and incubate at 37 °C for 30 min, then transfer to a 30 kDa ultrafiltration tube and centrifuge at 12000 g and 4 °C for 15 min to resuspend the precipitate in the tube; further capture with 4 μL of CMS suspension for 30 min. After the capture, centrifuge and discard the supernatant, add 90 μL of 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution and react at 37 °C for 15 min, and then measure the absorbance at 450 nm after adding 40 μL of the termination solution. As Figure 7 For the comparison of the effects of biomimetic microspheres capturing and modifying Ln 3+ before and after.

[0088] Example 6: Detection of extracellular vesicles captured by CMS by electrochemical method

[0089] Take 10 μL of the above EVs solution (1.26×10 8EVs / mL), add 8 μL of TDA-Co-Eu / La / Dy (0.1 mg / mL) to enhance homologous targeting, then add 10 μL of the electro-signal molecule MB (250 μM). After incubating at room temperature for 30 min, make up the system to 100 μL with normal saline, add PEG-8000 with a final concentration of 10% and incubate at 4 °C for 120 min, then centrifuge, discard the supernatant to obtain EVs with enhanced homologous targeting. Then capture with 4 μL of CMS suspension for 30 min. After the capture, centrifuge at 3500 rpm and 4 °C for 5 min, discard the supernatant, and then detect the signal of CMS capturing EVs through an electrochemical workstation. As Figure 8 shown is the comparison of the effects before and after the differential pulse voltammetry analysis of the biomimetic microspheres capturing the modified Ln 3+ before and after.

[0090] Example 7: Detection of biotinylated extracellular vesicles captured by CMS by electrochemiluminescence method

[0091] Take 10 μL of the above EVs solution (1.26×10 8 EVs / mL), add 8 μL of TDA-Co-Eu / La / Dy (0.1 mg / mL) to enhance homologous targeting; then add 100 μM of phospholipid-PEG-biotin (DSPE-PEG-Bio) and incubate at 37 °C for 30 min; then add 10 μL of streptavidin-horseradish peroxidase (SA-HRP) and incubate at 37 °C for 30 min, then transfer to a 30 kDa ultrafiltration tube and centrifuge at 12000 g and 4 °C for 15 min, and resuspend the precipitate in the tube; further capture with 4 μL of CMS suspension for 30 min. After the capture, centrifuge and discard the supernatant, add 50 μL of 10 mM luminol and 15 μL of 1% H 2 O 2 to measure the electrochemiluminescence intensity. As Figure 9 shown is the comparison of the effects before and after the electrochemiluminescence analysis of the biomimetic microspheres capturing the modified Ln 3+ before and after.

[0092] Example 8: Detection of extracellular vesicles from different cell sources by four methods respectively

[0093] The four detection methods in Examples 4 to 7 all show that the effect of the biomimetic microspheres capturing the modified Eu 3+ extracellular vesicles is better than that of the other two lanthanide elements. Here, in Example 8, TDA-Co-Eu is selected as the targeting-enhancing modifier of the sample for the experiment. Using the four detection methods in Examples 4 to 7, CMS is used to capture and detect four EVs (NEVs and TEVs) containing healthy and tumor cell sources respectively. The EVs from the four cell sources are MB-MDA-436, MCF-7, MB-MDA-10A, and SK-OV-3 respectively.

[0094] Figure 10 For the capture and detection of extracellular vesicles by fluorescence analysis biomimetic microspheres, where Figure a is the fluorescence spectrum diagram with the gradually increasing concentration of extracellular vesicles; Figure b is the standard curve for the detection of extracellular vesicles; Figure c is the sample addition recovery rate for the detection of extracellular vesicles by the fluorescence analysis method; Figure d is the selectivity for the detection of extracellular vesicles by fluorescence analysis;

[0095] Figure 11 For the capture and detection of extracellular vesicles by ultraviolet-visible absorption analysis biomimetic microspheres, Figure a is the standard curve for the detection of extracellular vesicles; Figure b is the sample addition recovery rate for the detection of extracellular vesicles by the fluorescence analysis method; Figures c and d are the selectivity for the detection of extracellular vesicles by fluorescence analysis.

[0096] Figure 12 For the capture and detection of extracellular vesicles by differential pulse voltammetry analysis biomimetic microspheres, where Figure a is the electrochemical signal diagram with the gradually increasing concentration of extracellular vesicles; Figure b is the standard curve for the detection of extracellular vesicles; Figure c is the sample addition recovery rate for the detection of extracellular vesicles by the differential pulse voltammetry analysis method; Figure d is the selectivity for the detection of extracellular vesicles by differential pulse voltammetry analysis;

[0097] Figure 13 For the capture and detection of extracellular vesicles by electrochemiluminescence analysis biomimetic microspheres, where Figure a is the electrochemiluminescence signal diagram with the gradually increasing concentration of extracellular vesicles; Figure b is the standard curve for the detection of extracellular vesicles; Figure c is the sample addition recovery rate for the detection of extracellular vesicles by the electrochemiluminescence analysis method; Figure d is the selectivity for the detection of extracellular vesicles by electrochemiluminescence analysis.

[0098] Example 9: Detection of 5 types of cancer blood samples using an ECL platform combining 5 types of CMS

[0099] The cell membranes of hepatocellular carcinoma HepG2 cells, breast cancer MB-MDA-436 cells, colorectal cancer HCT-116 cells, ovarian cancer SK-OV-3 cells, and cervical cancer Hela cells were respectively extracted for constructing 5 types of CMS. EVs were extracted from the plasma of healthy and cancer mice (including hepatocellular carcinoma, breast cancer, colorectal cancer, ovarian cancer, cervical cancer). The EVs were modified with TDA-Co-Eu and biotinylated membrane system, and then homologously captured with 5 types of CMS. After the capture, the luminol luminescence system was combined and the electrochemiluminescence intensity of each sample was measured.

[0100] Figure 14 Results of using the electrochemiluminescence platform combining 5 types of CMS for the detection of 5 types of cancer blood samples.

[0101] Example 10: Batch detection of mouse serum samples using a fluorescence microplate reader

[0102] Construction of CMS coated with SK-OV-3 cell membrane of ovarian cancer (CMS OV-3 ) for the detection of mouse serum. EVs were extracted from the sera of 10 healthy mice and 20 ovarian cancer mice, modified with TDA-Co-Eu and PKH67, and then homologous capture was performed using CMS OV-3 . After the capture, centrifugation was carried out. The CMS was suspended in physiological saline and transferred to a 96-well plate for batch detection using a fluorescence microplate reader. Figure 15 It is a bionic microsphere for batch detection of extracellular vesicles in blood samples.

Claims

1. A bionic microsphere, characterized in that: The biomimetic microspheres are prepared by modifying the surface of silica microspheres MS with homologous cell membranes CM, wherein the homologous cell membranes refer to biological membranes extracted from cells of the same type as the target to be tested, and the diameter of the silica microspheres is 10 microns.

2. A bionic microsphere according to claim 1, characterized in that: If the target cells to be detected are tumor cells, the tumor cell membranes are extracted and coated on the surface of the microspheres by ultrasound to prepare bionic microspheres for capturing EVs. The step of modifying the homologous cell membranes on the surface of the silica microspheres includes: self-assembling the homologous cell membrane suspension and the silica microspheres under ultrasound at 50W for 15 to 60 minutes to form CMS, and preparing the CMS suspension by centrifugation, precipitation and resuspending in physiological saline, wherein the mass ratio of the homologous cell membrane to the silica microspheres is (50 to 100):

1.

3. A method for detecting extracellular vesicles for non-diagnostic purposes, characterized in that: The non-diagnostic extracellular vesicle detection method comprises a sample pretreatment step, a homologous targeted incubation step and a signal detection step: The sample pretreatment step includes: preliminarily purifying the sample to be tested to extract extracellular vesicles EVs in the sample as a test solution, then mixing the test solution with a TDA-Co-Ln solution to modify the EVs with TDA-Co-Ln, then adding a signal molecule reagent to co-incubate to further modify the signal molecule, and obtaining a pretreated sample solution; The homologous targeted incubation step comprises: mixing and incubating the pretreated sample solution with the biomimetic microspheres (CMS) according to claim 1; The signal detection step includes: purifying the mixed solution after the above-mentioned homologous targeting incubation step, isolating the CMS that has captured the homologous EVs, and determining the content of the homologous EVs by measuring the signal carried by the EVs in the CMS that has captured the homologous EVs.

4. The detection method according to claim 3, characterized in that The sample to be tested is a food containing polysaccharide nutrients. The constructed biomimetic microsphere CMS is a cell membrane of cells that highly express dietary sialic acid modified on the surface of the silica microsphere; by measuring the proportion of extracellular vesicles that highly express dietary sialic acid in the test solution, the content of polysaccharide nutrients in the food can be judged.

5. The detection method according to claim 3, characterized in that: The sample to be tested is a drinking water source, the test liquid is the bacterial extracellular vesicles secreted by bacteria in the water source extracted by centrifugation, and the constructed bionic microsphere CMS is a silica microsphere surface modified with the cell membrane of the test object, i.e. bacteria that highly express sialic acid. By measuring the content of bacterial extracellular vesicles in the test liquid, a standard curve is constructed to monitor the content of microorganisms in the drinking water source.

6. The use of the bionic microspheres according to claim 1 in the preparation of a tumor-related EVs detection kit in a blood sample, that is, the detection kit includes the above-mentioned CMS suspension and also includes a sample pretreatment reagent; the sample pretreatment reagent is a TDA-Co-Ln solution and a signal molecule reagent; the signal molecule reagent is any one of a fluorescent signal molecule, an electrochemical signal molecule or a biotinylated membrane material; the TDA-Co-Ln solution is used to modify all EVs after extracting the total EVs in the blood sample; the signal molecule reagent is used to mark the detection signal of EVs; the CMS suspension is used to specifically identify and capture the marked EVs, collect the bionic microspheres by centrifugation, and detect tumor-related EVs based on the corresponding analysis method.

7. The use according to claim 6, characterized in that The detection kit is a tumor-related EVs detection kit in blood samples based on a fluorescence method, and the signal molecule reagent in the sample pretreatment reagent is a green fluorescent dye PKH67.

8. The use according to claim 6, characterized in that The detection kit is a tumor-related EVs detection kit in blood samples based on ultraviolet-visible absorption spectroscopy, and the signal molecule reagents in the sample pretreatment reagents are phospholipid-PEG-biotin, streptavidin-horseradish peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB) color development solution.

9. The use according to claim 6, characterized in that The detection kit is a tumor-related EVs detection kit in blood samples based on differential pulse voltammetry, and the signal molecule reagent in the sample pretreatment reagent is methylene blue.

10. The use according to claim 6, characterized in that The signal molecule reagents in the sample pretreatment reagents of the detection kit are phospholipid-PEG-biotin, streptavidin-horseradish peroxidase and luminol.

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