Metal-organic framework dense-encapsulated hollow glass microspheres and preparation and application thereof

Abstract

The application discloses a preparation method of metal organic framework densely wrapped hollow glass microspheres, which comprises the following steps: 1) adding the hollow glass microspheres into a piranha solution, stirring, removing the lower layer of debris after standing, washing with ultrapure water, drying to obtain product A; 2) adding polyethyleneimine solution into the product A, mixing and reacting, standing and separating, washing the upper layer of floating matter with ultrapure water, vacuum drying to obtain product B; 3) adding N, N-dimethylformamide into 2-amino terephthalic acid, benzoic acid and ZrCl4, ultrasonic dissolving, adding the product B, stirring and reacting, standing and separating, taking the upper layer of floating matter, washing with N, N-dimethylformamide and ethanol, vacuum drying, and the metal organic framework densely wrapped hollow microspheres prepared by the method can be used as a general type of capture probe, and can be used in the capture detection and analysis of target extracellular vesicles, phosphoproteome, phosphopeptide, nucleic acid and cells, and has a wide application prospect in liquid biopsy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical engineering and analysis. More particularly, the present application relates to a kind of metal organic framework dense package hollow glass microspheres and its preparation and application. BACKGROUND

[0002] Extracellular vesicles (EVs) are microvesicles released by cells into the surrounding biological fluids, whose lipid bilayer encapsulates various components of the original tumor, such as proteins, nucleic acids, metabolites, small molecules, organelles, etc. Therefore, EVs secreted in pathological microenvironment can carry intracellular molecular characteristics specific to a particular disease stage or injury, and are an important circulating biomarker for non-invasive cancer diagnosis and treatment monitoring. However, circulating tumor EVs are usually low in abundance, heterogeneous in size and molecular composition, and overexpressed proteins are not necessarily cancer-specific, which makes it difficult to effectively isolate and detect EVs. Therefore, it is necessary to prepare a material for rapid capture and detection of EVs to realize the phenotypic analysis of EVs. SUMMARY

[0003] An object of the present application is to solve at least the above problems and provide at least the advantages to be described later.

[0004] An object of the present application is to provide a preparation method of metal organic framework dense package hollow glass microspheres, which can realize rapid capture of EVs and provide a prerequisite for multi-element SERS analysis of EV surface protein markers.

[0005] To achieve these objects and other advantages of the present application, a preparation method of metal organic framework dense package hollow glass microspheres is provided, which comprises the following steps:

[0006] 1) Add hollow glass microspheres to a piranha solution, stir, remove the lower layer of debris after standing, wash with ultrapure water, and dry to obtain surface-activated hollow glass microspheres;

[0007] 2) Add polyethyleneimine solution to the surface-activated hollow glass microspheres, mix and react, separate after standing, wash the upper floating material with ultrapure water, and vacuum dry to obtain polyethyleneimine-coated hollow glass microspheres;

[0008] 3) Add N,N-dimethylformamide to 2-amino terephthalic acid, benzoic acid and ZrCl4, ultrasonically dissolve, add polyethyleneimine-coated hollow glass microspheres, stir and react, separate after standing, take the upper floating material, wash with N,N-dimethylformamide and ethanol, and vacuum dry to obtain metal organic framework dense package hollow glass microspheres with floating ability.

[0009] Preferably, in step 2), the mass ratio of the surface-activated hollow glass microspheres to the polyethyleneimine is 100:3-24, and the reaction is mixed by rotation at room temperature, and the reaction time is 0.5-12 h.

[0010] Preferably, in step 3), the mass ratio of the 2-amino terephthalic acid, the benzoic acid, the ZrCl4, the N,N-dimethylformamide, and the polyethyleneimine-coated hollow glass microspheres is 1-3:30-50:1-3:150-250:1-3, the reaction is stirred at 90-160℃, and the reaction time is 6-24 h.

[0011] A metal-organic framework densely coated hollow glass microsphere is prepared by the preparation method provided by the application.

[0012] A method for capturing extracellular vesicles, comprising the following steps:

[0013] i) extracting extracellular vesicles;

[0014] ii) taking the metal-organic framework densely coated hollow glass microspheres into a centrifuge tube, dispersing the microspheres with PBS buffer, adding the extracellular vesicles extracted in step i), mixing the reaction by vortexing at room temperature, after the reaction is completed, standing to make the microsphere compound completely float to the surface of the solution, removing the lower layer solution, and washing with PBS buffer, adding 1% BSA solution to block the active sites, separating and washing 3-5 times after 5-10 min, and obtaining the zirconium metal-organic framework densely coated hollow glass microspheres with the extracellular vesicles captured.

[0015] Preferably, the extracellular vesicles are extracted from cell culture solution or plasma.

[0016] Preferably, the extracellular vesicles are derived from normal cells or cancer cells.

[0017] A method for analyzing extracellular vesicle surface protein markers, comprising the following steps:

[0018] I) mixing and incubating a Raman reporter molecule, 3,3'-dithiodipropionic acid di(N-hydroxy succinimidyl ester), and an AuAg solution at room temperature, removing free molecules by centrifugation, adding PBS solution to redisperse the precipitate, then adding an antibody, incubating at room temperature, centrifuging at low temperature after the incubation is completed, removing free antibodies, adding PBS buffer containing 0.1% BSA to redisperse the precipitate, and obtaining a SERS nanolabel;

[0019] II) The hollow glass microspheres densely coated with metal organic frameworks capturing extracellular vesicles of claim 5 are mixed with SERS nanotags, incubated, and allowed to float to the surface of the liquid, and the lower layer of free SERS nanotags is removed, and the incubated complex is washed with PBS buffer, dropped on a silicon wafer, and the Raman spectrum data is collected by a portable Raman spectrometer.

[0020] Preferably, the surface protein markers of extracellular vesicles include epidermal growth factor, four transmembrane proteins, human epidermal factor receptor 2, epithelial cell adhesion molecule, alpha-fetoprotein, carcinoembryonic antigen, mucin, programmed cell death ligand 1, prostate specific antigen, vascular endothelial growth factor.

[0021] Application of metal organic framework densely coated hollow glass microspheres as capture probes in target extracellular vesicles, phosphoproteome, phosphopeptide, nucleic acid and cell capture.

[0022] The present application at least includes the following beneficial effects:

[0023] First, the present application utilizes the high affinity of transition metal elements (Zr) to EVs, combined with hollow glass microspheres with self-driving, self-separation, and self-enrichment, to prepare a material that can quickly capture, separate and enrich EVs. Specifically, the present application can quickly capture EVs through the coordination between Zr on the zirconium metal organic framework (Zr-MOF) and the phosphate groups on the surface of EVs, and then use the buoyancy of the hollow glass microspheres to achieve the separation and enrichment of EVs. Compared to bioaffinity magnetic beads and microfluidic technology, Zr-MOF has higher affinity to EVs, and does not require expensive antibodies and external forces, which can shorten the separation time, reduce costs and reduce energy consumption. 4+

[0024] Second, the present application co-modifies different Raman reporter molecules and antibodies on the surface of anisotropic gold-silver alloy nanoboxes (AuAg) with good Raman enhancement effect, to prepare a series of SERS nanotags that can specifically recognize and detect target EV surface protein markers, achieving ultra-sensitive and multi-element analysis of target EVs, and solving the problem of multi-element analysis of EVs.

[0025] Third, the present application uses floating B@MOF to quickly capture target EVs, and then uses the specific recognition of antibodies on the surface of SERS nanotags to the surface proteins of target EVs, to achieve the rapid capture and multi-element SERS analysis of EVs. The present application is a rapid, sensitive, simplified, energy-saving and multi-element EV detection method, which has great application potential in clinical analysis, diagnosis and treatment.

[0026] ​Fourthly, the floating B@MOF has many sites for coordination with phospholipid groups, and has the ability of rapid capture and separation of both cell-derived and plasma-derived EVs. The floating B@MOF has simple preparation steps, cheap raw materials, and good stability, and can realize separation of target EVs without external force by using its own buoyancy.

[0027] Fifthly, the floating B@MOF prepared by the application can be used as a general capture probe for capture and detection of target extracellular vesicles, phosphoproteome, phosphopeptide, nucleic acid and cells, and has wide application prospects in liquid biopsy.

[0028] Other advantages, objects and features of the application will be apparent from the following description, and will be understood by those skilled in the art through a study of the application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a preparation flow chart of the floating B@MOF of the application and a principle diagram of capture of EVs; wherein a is a preparation flow chart; b is a principle diagram of capture of EVs;

[0030] Figure 2 It is a TEM and SEM characterization diagram of B@PEI and floating B@MOF of the application;

[0031] Figure 3 It is a TEM characterization diagram of target EVs;

[0032] Figure 4 It is a fluorescence diagram of capture of EVs of different cells by the floating B@MOF of the application; wherein BF is a bright field diagram, and DF is a dark field diagram; PBS is a control, that is, an equal amount of PBS solution is used instead of EV solution; HepG2 is a human hepatoma cell; L02 is a normal liver cell; MCF-7, MDA-MB-231 and SK-BR-3 are human breast cancer cells; A-431 is a human epidermoid carcinoma cell; A549 is a human lung cancer cell; HeLa is a human cervical cancer cell; and RAW264.7 is a mouse macrophage;

[0033] Figure 5 It is a characterization diagram of capture kinetics of EVs by the floating B@MOF of the application;

[0034] Figure 6 It is a preparation flow chart of SERS nanolabels and a Raman spectrum diagram thereof;

[0035] Figure 7 It is a multivariate Raman confocal imaging and a relative Raman intensity columnar diagram of L02 and HepG2 EVs captured by the floating B@MOF of the application, and the scale is 5 microns;

[0036] Figure 8A phenotype analysis chart of the floating B@MOF after capturing the cell-derived EVs of the present application;

[0037] Figure 9 A multi-element SERS analysis chart of the floating B@MOF after capturing the plasma-derived EVs of the present application. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with the accompanying drawings, so that those skilled in the art can implement the present application according to the description and drawings.

[0039] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0040] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0041] As shown in the following embodiments, the present application provides a preparation method of metal organic framework densely wrapped hollow glass microspheres, which comprises the following steps: Figures 1-9

[0042] 1) Add the hollow glass microspheres to the aiptasia solution, stir, remove the lower debris after standing, wash with ultrapure water, and dry to obtain surface-activated hollow glass microspheres;

[0043] 2) Add polyethyleneimine solution to the surface-activated hollow glass microspheres, mix and react, separate after standing, wash the upper floating material with ultrapure water, and vacuum dry to obtain polyethyleneimine-wrapped hollow glass microspheres;

[0044] 3) Add N,N-dimethylformamide to 2-amino terephthalic acid, benzoic acid and ZrCl4, ultrasonically dissolve, add polyethyleneimine-wrapped hollow glass microspheres, stir and react, separate after standing after the reaction is completed, take the upper floating material, and wash with N,N-dimethylformamide and ethanol, and vacuum dry to obtain metal organic framework densely wrapped hollow glass microspheres with floating ability.

[0045] In another technical solution, in step 2), the mass ratio of the surface-activated hollow glass microspheres to polyethyleneimine is 100:3-24, and the reaction is mixed by rotation at room temperature for 0.5-12h.

[0046] ​In another technical solution, in step 3), the mass ratio of 2-amino terephthalic acid, benzoic acid, ZrCl4, N,N-dimethylformamide and hollow glass microspheres wrapped by polyethyleneimine is 1-3:30-50:1-3:150-250:1-3, the stirring reaction is at 90-160℃, and the reaction time is 6-24h.

[0047] A metal organic framework densely wrapped hollow glass microsphere is prepared by the preparation method provided by the application.

[0048] A method for capturing extracellular vesicles, comprising the following steps:

[0049] i) extracting extracellular vesicles;

[0050] ii) taking the metal organic framework densely wrapped hollow glass microspheres into a centrifuge tube, dispersing the microspheres with PBS buffer, adding the extracellular vesicles extracted in step i), vortex mixing at room temperature, after the reaction is completed, standing to make the microsphere compound completely float to the surface of the solution, removing the lower solution, and washing with PBS buffer, adding 1% BSA solution to block the active sites, separating and washing 3-5 times after 5-10 minutes, to obtain zirconium metal organic framework densely wrapped hollow glass microspheres with captured extracellular vesicles.

[0051] In another technical solution, the extracellular vesicles are extracted from cell culture solution or plasma.

[0052] In another technical solution, the extracellular vesicles are derived from normal cells or cancer cells. The normal cells include normal liver cells, normal mammary epithelial cells, normal lung cells, normal brain cells, nerve cells, immune cells, etc. The cancer cells can be cancer cells of liver cancer, breast cancer, skin cancer, lung cancer, cervical cancer, colon cancer, rectal cancer, bladder cancer, pancreatic cancer, gastric cancer, thyroid cancer, prostate cancer, head and neck cancer, esophageal cancer or brain cancer, etc. The immune cells can be monocyte macrophages, T cells, B cells, etc.

[0053] A method for analyzing extracellular vesicle surface protein markers, comprising the following steps:

[0054] I) mixing and incubating a Raman reporter molecule, 3,3'-dithiodipropionic acid di(N-hydroxy succinimidyl ester) and an AuAg solution at room temperature, centrifuging to remove free molecules, adding PBS solution to redisperse the precipitate, then adding an antibody, incubating at room temperature, centrifuging at low temperature after the incubation is completed, removing free antibodies, adding PBS buffer containing 0.1% BSA to redisperse the precipitate, to obtain a SERS nanolabel;

[0055] II) The hollow glass microspheres densely wrapped with metal organic frameworks capturing extracellular vesicles in claim 5 are mixed with SERS nanotags and incubated, and the incubated complex is allowed to float to the liquid surface, the lower layer of free SERS nanotags is removed, and the incubated complex is washed with PBS buffer, dropped on a silicon wafer, and Raman spectrum data is collected by a portable Raman spectrometer.

[0056] In the technical solution, the Raman reporter molecule can be 4-mercaptopyridine, 4-nitrothiophenol, 4-ethynylbenzoic acid, 4-mercaptobenzonitrile, 4-mercaptobenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid), or other Raman reporter molecules; and the antibody can be EGFR, CD63, HER2, EpCAM, or other antibodies.

[0057] In another technical solution, the surface protein markers of extracellular vesicles include epidermal growth factor, four transmembrane proteins, human epidermal factor receptor 2, epithelial cell adhesion molecule, alpha-fetal protein, carcinoembryonic antigen, mucin, programmed cell death ligand 1, prostate-specific antigen, and vascular endothelial growth factor.

[0058] Application of metal organic framework densely wrapped hollow glass microspheres as capture probes in target extracellular vesicles, phosphoproteome, phosphopeptide, nucleic acid, and cell capture.

[0059] The present application uses Zr 4+ Through coordination between the phosphoric groups on the surface of target extracellular vesicles, phosphoproteome, phosphopeptide, nucleic acid, and cells and the Zr

[0060] <Experiment I> Preparation experiment of metal organic framework densely wrapped hollow glass microspheres

[0061] The preparation method of the metal organic framework densely wrapped hollow glass microspheres comprises the following steps:

[0062] 1) Add the hollow glass microspheres to piranha solution (H2O2 / H2SO4=1:3), stir for 1 h at a rotation speed of 1000 rpm, separate and remove the lower layer of debris using a separatory funnel after standing, wash with ultrapure water, and dry at 80°C to obtain surface-activated hollow glass microspheres;

[0063] 2) Add 30 mL of 2 mg / mL polyethyleneimine (PEI) aqueous solution to 1 g of surface-activated hollow glass microspheres, rotate and mix at room temperature for 2 h at 10 rpm, let stand and separate, wash the upper floating matter with ultrapure water, and vacuum dry at 37 °C to obtain polyethyleneimine-coated hollow glass microspheres (B@PEI);

[0064] 3) Add 10 mL of N,N-dimethylformamide (DMF) to 110 mg of 2-aminoterephthalic acid, 1.9 g of benzoic acid and 120 mg of ZrCl4. Sonicate to completely dissolve the solids. Add 100 mg of polyethyleneimine-coated hollow glass microspheres. Stir at 120 °C for 24 h at a stirring speed of 1000 rpm. After the reaction is complete, allow to stand and separate. Take the upper floating material and wash it with N,N-dimethylformamide and ethanol. Vacuum dry to obtain hollow glass microspheres (floating B@MOF) with dense metal-organic frameworks that have buoyancy.

[0065] like Figure 2 As shown, the surface of the hollow glass microspheres is coated with irregular PEI, which provides abundant metal chelation sites, which is conducive to the growth of Zr-MOF. Ultimately, a high density of Zr-MOF is formed on the surface of the bubble, proving the successful preparation of floating B@MOF.

[0066] <Experiment 2> Extraction of Cell-Derived EVs

[0067] Once the cell density reached 80%, the complete culture medium was changed to serum-free medium, and the cells were incubated in an incubator for another 48 hours. Afterward, the supernatant was collected and centrifuged at 4°C (3000g, 15min) to remove intact cells and cell debris. The supernatant was then filtered through a 0.45μm membrane, and the filtrate was transferred to a 15mL ultrafiltration tube and centrifuged at 4°C (5000g, 15min) to concentrate the medium. The concentrated medium was collected in centrifuge tubes, and ExoQuick-TC was added. TM Reagents (concentrated culture medium and ExoQuick-TC) TM The reagent volume ratio is 5:1. After mixing evenly, the mixture is incubated overnight at 4°C. Finally, the mixture is centrifuged at 4°C (1500g, 30min), the supernatant is removed, and the collected EV precipitate is dispersed with PBS buffer and stored at -80°C for later use.

[0068] The morphology of EVs was characterized using TEM, such as... Figure 3 As shown, a typical butterfly-shaped structure can be observed, indicating that cell-derived EVs were successfully extracted.

[0069] <Experiment 3> Floating B@MOF Capture of Cell-Derived EVs Experiment

[0070] Take 0.3 mg of floating B@MOF into a 1.5 mL centrifuge tube, add 90 μL of PBS buffer dispersion, then add 10 μL of EV solution from different sources (10 8 particles / mL), vortex mix at room temperature; after the reaction is completed, let the B@MOF@EV complex float completely to the surface of the solution, aspirate the lower solution, and wash the B@MOF@EV complex with PBS buffer 3 times, add 100 μL of BSA (1%) to block the active sites, separate and wash 3 times after 5 min, use DiI solution to stain the EV, separate and wash 3 times after 20 min, and perform fluorescence imaging on the B@MOF@EV complex.

[0071] The results, as shown in Figure 4 , show that the surface of the floating B@MOF has obvious fluorescence, proving that the EV from different cell sources is successfully captured by the B@MOF.

[0072] <Experiment Four> Speed and stability test of floating B@MOF capturing cell-derived EV

[0073] Take 0.3 mg of floating B@MOF into a 1.5 mL centrifuge tube, add 90 μL of PBS buffer dispersion, then add 10 μL of EV solution extracted from liver cancer cells (HepG2) (10 8 particles / mL), vortex mix at room temperature for 0, 5, 10, 20, 30, 60, and 90 min, respectively, after the reaction is completed, let the B@MOF@EV complex float completely to the surface of the solution, aspirate the lower solution, and wash the B@MOF@EV complex with PBS buffer 3 times, add 100 μL of BSA (1%) to block the active sites, separate and wash 3 times after 5 min, then add DiI solution to stain the HepG2 EV, separate and wash 3 times after 20 min, and finally perform fluorescence imaging on the complex.

[0074] As shown in Figure 5 , after 5 min of incubation, the surface of the B@MOF@EV complex has obvious fluorescence and is stable, indicating that the EV can be quickly captured by the floating B@MOF.

[0075] <Experiment Five> Preparation test of SERS nanolabels

[0076] According to the procedure shown in Figure 6 , a plurality of SERS nanolabels are prepared.

[0077] Take 1 μL Raman reporter molecules (4-mercapto pyridine (MPY), 4-nitro thiophenol (NTP), 4-ethynyl benzoic acid (EBA), 4-mercapto benzonitrile (MBN), 10 mM) and 1 μL 3,3'-dithiodipropionic acid di(N-hydroxy succinimidyl ester) (DSP, 10 mM) respectively mixed with 100 μL AuAg solution at room temperature for 5 h to assemble the molecular layer on the surface of AuAg, centrifuged (8000 rpm, 5 min) to remove free molecules, and 100 μL PBS (0.1 mM) was added to re-disperse the precipitate, then 1 μL antibody (anti-EGFR, ant-CD63, anti-HER2 and anti-EpCAM, 500 μg / mL) was added respectively, incubated at room temperature for 2 h, then centrifuged (8000 rpm, 5 min) at 4°C to remove free antibodies, 100 μL PBS buffer (containing 0.1% BSA) was added to re-disperse the precipitate, and finally the prepared SERS nanolabels (MPY-EGFR, NTP-CD63, EBA-HER2 and MBN-EpCAM) were stored in a 4°C refrigerator for subsequent experiments.

[0078] The Raman spectra of the four SERS nanolabels were collected using a portable Raman spectrometer. The results are shown in Figure 6 As shown, the characteristic Raman peaks (MPY-EGFR: 1005 cm -1 , NTP-CD63: 1350 cm -1 , EBA-HER2: 2026 cm -1 , MBN-EpCAM: 2230 cm -1 ) of each SERS nanolabel can be observed and are not overlapped, so that multi-element SERS analysis can be achieved.

[0079] <Experiment six> Characterization experiment of floating B@MOF for the feasibility of EV multi-element SERS analysis

[0080] In order to verify the feasibility of multi-element analysis, the floating B@MOF (B@MOF@EV complex) capturing EVs was incubated with MPY-EGFR, NTP-CD63, EBA-HER2, MBN-EpCAM respectively for 45 min, then the complex was allowed to float to the liquid surface, the free SERS nanolabels in the lower layer were removed, and washed with PBS buffer for three times, finally the complex was dropped on a silicon wafer, and the single bubble was imaged by a confocal Raman spectrometer or the Raman spectrum was collected by a portable Raman spectrometer.

[0081] As shown in Figure 7As shown, the Raman imaging and relative Raman intensity of single bubble in blank group (PBS) (replaced with PBS instead of EV), L02 EV (normal liver cell source) group and HepG2 EV (human hepatoma cell source) group have significant difference, which proves the feasibility of floating B@MOF for EV multi-element SERS analysis.

[0082] Experiment Seven: Floating B@MOF for phenotype analysis of EVs from different cell sources

[0083] According to Figure 8 As shown in a, after the EVs from different cell sources were captured by floating B@MOF, the SERS nanotags surface antibodies can recognize the EV surface protein markers, and then SERS analysis is carried out.

[0084] In order to verify that floating B@MOF can be used for phenotype analysis of EVs from different cell sources, after floating B@MOF captured EVs from different cell sources (human hepatoma cells (HepG2), human normal liver cells (L02), human breast cancer cells (MCF-7, MDA-MB-231, SK-BR-3), human epidermal carcinoma cells (A-431), human lung cancer cells (A549), human cervical cancer cells (HeLa)), they were incubated with MPY-EGFR, NTP-CD63, EBA-HER2, MBN-EpCAM respectively for 45 min.

[0085] Then, the complex was allowed to float to the liquid surface, the free SERS nanotags in the lower layer were removed, and the complex was washed with PBS buffer for three times. Finally, the complex was dropped on a silicon wafer, and the Raman spectrum was collected by a portable Raman spectrometer.

[0086] As Figure 8 As shown in b, 8c, CD63 is highly expressed in EVs from different cell sources, while the expression of other surface protein markers has differences. For example, EGFR is highly expressed in A-431 cell-derived EVs, HER2 is highly expressed in SK-BR-3 cell-derived EVs, and EpCAM is highly expressed in HepG2, MCF-7 and A-431 cell-derived EVs. Therefore, floating B@MOF can be used for phenotype analysis of EVs from different cell sources.

[0087] Experiment Eight: Floating B@MOF for multi-element SERS analysis of EVs from different disease plasma sources

[0088] To verify the floating B@MOF could be used for the multiplex SERS analysis of plasma-derived EVs from healthy people (HD, number of people n=10) and different diseases (liver cancer (LiC, number of people n=10), breast cancer (BC, number of people n=10), lung cancer (LuC, number of people n=10)), the floating B@MOF after capturing plasma-derived EVs was incubated with MPY-EGFR, NTP-CD63, EBA-HER2, MBN-EpCAM respectively for 45 min. Then the complex was allowed to float to the liquid surface, and the free SERS nanotags in the lower layer were removed, and washed with PBS buffer for three times. Finally, the complex was dropped on a silicon wafer, and the Raman spectrum was collected with a portable Raman spectrometer.

[0089] As Figure 9 a, 9b, the expression levels of EGFR, CD63, HER2 and EpCAM were heterogeneous in 40 clinical samples, but the expression levels of plasma-derived EV surface proteins in cancer patients were mostly higher than those in healthy people. Figure 9 c Receiver operating characteristic (ROC) analysis also proved that the protein significance (p<0.01) of plasma-derived EV surface in cancer patients was higher than that of plasma-derived EV surface in healthy people. In addition, by comparing the area under the curve (AUC, Figure 9 d-f), it was found that the four protein markers had high accuracy (AUC>0.85) in distinguishing cancer patients from healthy people. Therefore, these results show that the floating B@MOF can be used for the multiplex SERS analysis of plasma-derived EVs from different diseases, and can achieve accurate diagnosis of cancer.

[0090] Although embodiments of the present application have been disclosed as above, it is not limited only to the use listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily implemented by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A method for preparing hollow glass microspheres densely encapsulated by a metal-organic framework, characterized in that, Includes the following steps: 1) Add hollow glass microspheres to the piranha solution, stir, let stand, remove the lower layer of fragments, wash with ultrapure water, and dry to obtain surface-activated hollow glass microspheres; 2) Add polyethyleneimine solution to surface-activated hollow glass microspheres, mix and react, let stand and separate, wash the upper floating matter with ultrapure water, and vacuum dry to obtain hollow glass microspheres coated with polyethyleneimine. The surface-activated hollow glass microspheres and polyethyleneimine were mixed in a mass ratio of 100:3-24 and reacted by rotation at room temperature for 0.5-12 h. 3) Add N,N-dimethylformamide to 2-aminoterephthalic acid, benzoic acid and ZrCl4, dissolve by sonication, add hollow glass microspheres coated with polyethyleneimine, stir the reaction, let stand and separate after the reaction is complete, take the upper floating matter, wash with N,N-dimethylformamide and ethanol, and vacuum dry to obtain hollow glass microspheres densely coated with metal-organic framework with floating ability; The mass ratio of hollow glass microspheres encapsulated with 2-aminoterephthalic acid, benzoic acid, ZrCl4, N,N-dimethylformamide, and polyethyleneimine was 1-3:30-50:1-3:150-250:1-3. The stirring temperature was 90-160℃, and the reaction time was 6-24h.

2. Hollow glass microspheres densely encapsulated by a metal-organic framework, characterized in that, It is prepared by the method of claim 1.

3. A method for capturing extracellular vesicles for non-disease diagnosis, characterized in that, Includes the following steps: i) Extract extracellular vesicles; ii) Take the metal-organic framework densely wrapped hollow glass microspheres as described in claim 1 into a centrifuge tube, add PBS buffer to disperse the microspheres, add the extracellular vesicles extracted in step i), vortex mix at room temperature, after the reaction is completed, let stand to allow the microsphere complex to completely float to the surface of the solution, remove the lower layer of solution, wash with PBS buffer, add BSA solution to block the active sites, separate and wash 3-5 times after 5-10 min to obtain the metal-organic framework densely wrapped hollow glass microspheres with extracellular vesicles captured.

4. The method for capturing extracellular vesicles for non-disease diagnosis according to claim 3, characterized in that, Extracellular vesicles are extracted from cell culture medium or plasma.

5. The method for capturing extracellular vesicles for non-disease diagnosis according to claim 3, characterized in that, Extracellular vesicles originate from normal cells or cancer cells.

6. A method for analyzing extracellular vesicle surface protein markers for non-disease diagnosis, characterized in that, Includes the following steps: I) Mix Raman reporter molecule, 3,3'-dithiodipropionate di(N-hydroxysuccinimide) and AuAg solution and incubate at room temperature. Centrifuge to remove free molecules, add PBS solution to redisperse the precipitate, then add antibody and incubate at room temperature. After incubation, centrifuge at low temperature to remove free antibody, add PBS buffer containing 0.1% BSA to redisperse the precipitate to obtain SERS nanotags; II) The hollow glass microspheres densely wrapped with metal-organic frameworks containing extracellular vesicles as described in claim 3 were mixed with SERS nanotags and incubated. The incubation complex was allowed to float to the surface of the liquid, the lower free SERS nanotags were removed, and the mixture was washed with PBS buffer. The incubation complex was then dropped onto a silicon wafer, and Raman spectral data were collected using a portable Raman spectrometer.

7. The method for analyzing extracellular vesicle surface protein markers for non-disease diagnosis according to claim 6, characterized in that, Extracellular vesicle surface protein markers include epidermal growth factor, four-transmembrane protein, human epidermal growth factor receptor 2, epithelial cell adhesion molecule, alpha-fetoprotein, mucin, and vascular endothelial growth factor.

8. The application of the metal-organic framework-densely-encapsulated hollow glass microspheres as described in claim 2 as a capture probe in the capture of target extracellular vesicles, phosphoproteomics, phosphopeptides, nucleic acids, and cells for non-disease diagnosis.

Images