Optical heterostructure aggregate, preparation method thereof, sensor, kit and detection method of extracellular vesicles

Abstract

The invention relates to a light heterostructure aggregate, a preparation method thereof, a sensor, a kit and a detection method of extracellular vesicles, and belongs to the technical field of functional materials and biosensing. The method solves the problem that the zinc oxide nanostructure which is controllable in structure and stable in combination is difficult to realize on the microsphere substrate in the prior art. According to the scheme, the method comprises the following steps: performing surface cleaning and hydroxyl activating treatment on hollow glass microspheres; dispersing the activated hollow glass microspheres in a mixed aqueous solution containing a zinc source and hexamethylenetetramine, and carrying out a stirring reaction at 60-95 DEG C for 2-8 h to make the zinc oxide nanostructure grow in situ on the surfaces of the hollow glass microspheres; after the reaction, quenching, washing and drying to obtain a composite material; the composite material is modified by adopting a silanization reagent, and a biological coupling functional group is introduced to obtain the photo-heterostructure aggregate. The aggregate prepared by the method is mainly used for constructing a high-sensitivity biosensor, and is particularly suitable for detecting biomarkers such as extracellular vesicles and the like.

Description

Technical Field

[0001] This invention belongs to the field of functional materials and biosensing technology, specifically relating to an optical heterostructure aggregate and its preparation method, sensor, reagent kit, and detection method for extracellular vesicles. Background Technology

[0002] In the field of photoheterogeneous aggregate fabrication, existing methods typically involve growing functional nanomaterials on the surface of microsphere substrates. Achieving a stable and controllable bond between the nanostructure and the substrate is a key step. Some current methods employ physical adsorption or simple chemical treatments to introduce nanostructures, but the interfacial bonding forces formed by these methods are often weak. During subsequent processing or applications, the nanostructures are prone to detaching from the substrate surface, leading to reduced material structural stability and performance reliability.

[0003] Furthermore, to achieve controllable growth of target nanostructures on substrate surfaces, precise control of the nucleation and growth processes is required. However, in practice, fluctuations in reaction parameters (such as temperature and precursor concentration) can easily lead to inhomogeneities in the morphology, size, and distribution of nanostructures. This structural inconsistency directly affects the optical or electrical properties of the material, making the performance of the final product difficult to predict and repeat.

[0004] Therefore, existing technologies still lack a preparation method that can simultaneously achieve excellent results in both interfacial bonding strength and structural morphology controllability. This limits the further development of such composite materials in applications requiring high stability and repeatability. Summary of the Invention

[0005] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0006] Another objective of this invention is to provide a method for preparing photoheterogeneous aggregates, which can form stable chemical bonding sites on the surface of hollow glass microspheres through hydroxyl activation treatment, control the morphology and bonding strength of zinc oxide nanostructures by using optimized range of hydrothermal reaction temperature and time parameters, and effectively introduce bio-coupled functional groups through subsequent silanization modification, thereby simultaneously achieving a strong bond between the nanostructure and the substrate, controllable morphology, and good biofunctionalization properties.

[0007] To achieve these objectives and other advantages of the present invention, a method for preparing an optical heterostructure aggregate is provided, comprising the following steps: 1) Surface cleaning and hydroxyl activation treatment of hollow glass microspheres; 2) The activated hollow glass microspheres were dispersed in a mixed aqueous solution containing a zinc source and hexamethylenetetramine. The zinc source was zinc acetate, zinc nitrate, or zinc chloride. The hydrothermal reaction was carried out at a temperature of 60-95℃ with stirring for 2-8 hours to allow zinc oxide nanostructures to grow in situ on the surface of the hollow glass microspheres. 3) After the reaction is complete, the mixture is transferred to an ice-water bath for quenching, centrifuged and the supernatant is discarded. The precipitate is resuspended and washed with ultrapure water until the supernatant is neutral. The precipitate is collected by gravity sedimentation and dried overnight in a vacuum drying oven to obtain the composite material GB@ZnO. 4) The GB@ZnO composite material was silanized by hexadecyltrimethoxysilane (HDS) and triethoxy(3-epoxypropyloxypropyl)silane (GPTES) to introduce bio-coupled functional groups and obtain photoheterogeneous aggregates.

[0008] Preferably, step 1) specifically includes the following steps for surface cleaning and hydroxyl activation treatment of the hollow glass microspheres: 1.1) Immerse hollow glass microspheres in a piranha solution at a temperature of 20-25 °C for 50-70 min. The piranha solution is prepared by mixing 30% hydrogen peroxide and 98% concentrated sulfuric acid at a volume ratio of 1:3. 1.2) The treated hollow glass microspheres were washed with ultrapure water until the pH of the washing solution was 6.5-7.5; 1.3) Rinse with ultrapure water and dry in a vacuum environment at 35-40 °C for 10-14 h to obtain activated hollow glass microspheres.

[0009] Preferably, in step 1), the diameter of the hollow glass microspheres before activation is 1-100 μm.

[0010] Preferably, in step 2), the activated hollow glass microspheres are dispersed in a mixed aqueous solution containing a zinc source and hexamethylenetetramine. The specific process is as follows: Mix equal volumes of 0.025 M anhydrous zinc acetate aqueous solution and hexamethyltetramine in a beaker, add 100-300 mg of the activated hollow glass microspheres from step 1), and sonicate for 30-50 min to mix evenly.

[0011] Preferably, in step 4), the GB@ZnO composite material is silanized using hexadecyltrimethoxysilane (HDS) and triethoxy(3-epoxypropyloxypropyl)silane (GPTES), specifically including the following steps: 4.1) Dry the GB@ZnO composite material in a vacuum environment at 50-100℃ for 1-2 h; 4.2) Add 1% hexadecyltrimethoxysilane (HDS) to the GB@ZnO composite material and react with shaking at 100-150 rpm for 2 h at room temperature; 4.3) Transfer the reacted solution to a centrifuge tube and wash it three times with anhydrous ethanol to remove unreacted hexadecyltrimethoxysilane. 4.4) Add 5% (v / v) of triethoxy(3-epoxypropyloxypropyl)silane (GPTES) and incubate overnight in a 3D vortex mixer to form epoxy groups. Then wash the product 3-5 times with anhydrous ethanol. Subsequently, collect GB@ZnO by self-separation, dry it in an oven, and store it at 4 °C for later use.

[0012] Preferably, the zinc oxide nanostructure is a nanorod array with an axial length of 50-200 nm.

[0013] An optical heterostructure aggregate, characterized in that the optical heterostructure aggregate is prepared by the aforementioned method for preparing optical heterostructure aggregates.

[0014] A sensor comprising the aforementioned photoheterogeneous aggregate and a capture molecule immobilized on the photoheterogeneous aggregate; the capture molecule being an antibody, aptamer, or polypeptide that specifically binds to extracellular vesicle surface markers.

[0015] A kit comprising the aforementioned sensor, the kit being used for cancer diagnosis.

[0016] Preferably, the cancer is liver cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, colon cancer, rectal cancer, bladder cancer, ovarian cancer, pancreatic cancer, skin cancer, stomach cancer, thyroid cancer, head and neck cancer, esophageal cancer, uterine cancer, or brain cancer.

[0017] The present invention has at least the following beneficial effects: This invention achieves in-situ chemical bonding growth of zinc oxide nanostructures on the surface of hollow glass microspheres through optimized hydroxyl activation and hydrothermal growth processes, forming a stable core-shell composite unit that significantly improves the bonding strength and stability of the heterogeneous interface. Multiple composite units can self-aggregate in the liquid phase to form aggregates, and the optical properties of the hollow glass microspheres and the electromagnetic field enhancement effect of the zinc oxide nanostructures generate synergistic fluorescence enhancement, greatly improving the intensity and signal-to-noise ratio of the optical signal. Furthermore, customizable bio-coupling functional groups are introduced into the material surface through silanization modification, enabling it to easily couple with various capture molecules, laying the material foundation for constructing highly sensitive and specific biosensors, especially suitable for the detection of low-abundance biomarkers (such as extracellular vesicles) in complex biological samples.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the preparation and application principle of photoheterogeneous aggregates. Figure a shows the in-situ growth of zinc oxide (ZnO) nanorod arrays on the surface of hollow glass microspheres (GB), the formation of the core-shell structure through silanization modification and stabilizer coating; Figure b shows the process by which the aggregates self-aggregate in droplets by means of buoyancy (Fb) and specifically capture extracellular vesicles. Figure 2 Scanning electron microscope (SEM) images of GB@ZnO composite materials with different precursor concentrations; Figure 3 Chemical structure characterization diagrams of GB@ZnO composite materials and their derivatives; Figure 4 A comparison of the fluorescence properties of aggregates made of different materials; Figure 5 This is a diagram verifying the antibody modification effect; Figure 6 A fluorescence image of the sensor after capturing EVs; Figure 7 Scanning electron microscope (SEM) image of the GB@ZnO-EV complex; Figure 8 A standard curve of the sensor's sensitivity to detect extracellular vesicles (EVs); Figure 9 The image shows the sensor specificity verification results. Figure 10 The graph shows the verification results of the sensor's anti-interference performance. Figure 11 Figures showing the correlation between EV detection in clinical plasma samples are shown below; Figure a is a schematic diagram of the detection process, Figure b is a violin plot of fluorescence signal (P<0.0001), and Figure c is a heatmap of biomarker expression in the corresponding sample. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0021] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0022] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0023] Example 1 A method for preparing optical heterostructure aggregates, such as Figure 1 As shown, it includes the following steps: 1) Take 200 mg of hollow glass microspheres (GBs, 3M™ iM30K) with a diameter of about 18 μm and immerse them in 20 mL of freshly prepared piranha solution (H2O2:H2SO4=1:3, v / v). Treat them at room temperature for 1 h to thoroughly clean the surface and maximize the surface hydroxyl density. Wash them repeatedly with ultrapure water until the wash water is neutral. Dry them in a vacuum drying oven at 37 ℃ for 12 h to obtain activated GBs. Store them in a sealed container. 2) Mix 200 mL of 0.05 M zinc acetate aqueous solution and 200 mL of 0.05 M hexamethylenetetramine (HMTA) aqueous solution in a 500 mL beaker to form a precursor solution with a concentration of 0.025 M (1:1 zinc acetate aqueous solution and hexamethylenetetramine (HMTA) aqueous solution). Sonicate for 30 min to make it homogeneous. Add 200 mg of GBs activated in step 1). Place the beaker in an 80℃ constant temperature water bath and react for 5 h with magnetic stirring. 3) After the reaction was completed, the mixture was quickly transferred to an ice-water bath for 30 min to quench it, so as to terminate the reaction and prevent excessive growth of zinc oxide; the crude product was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the product was resuspended and washed with ultrapure water. This centrifugation-washing process was repeated 3 times until the supernatant was neutral; the final product was collected by gravity sedimentation and dried overnight in a vacuum drying oven at 60 ℃ to obtain GB@ZnO composite material; 4) Add 1% hexadecyltrimethoxysilane (HDS) to the GB@ZnO composite material and shake at 100-150 rpm for 2 h at room temperature. Transfer the reaction solution to a centrifuge tube, centrifuge at 4000 rpm for 5 min, and wash three times with anhydrous ethanol to remove unreacted hexadecyltrimethoxysilane. Add 5% triethoxy(3-epoxypropyloxypropyl)silane (GPTES) and continue to react in a 3D vortex mixer for 12 h to form epoxy groups. Then wash the product five times with anhydrous ethanol. Subsequently, collect the GB@ZnO by self-separation, dry it in an oven, and store it in a drying environment at 4℃ for later use.

[0024] Morphological characterization: GB@ZnO synthesized at four different precursor concentrations (0.010 M, 0.025 M, 0.050 M, 0.100 M) was characterized using scanning electron microscopy (SEM). The results are as follows: Figure 2 As shown, the density and axial length of zinc oxide nanorods systematically increased with increasing precursor concentration from 0.010 M to 0.100 M. At a precursor concentration of 0.025 M, a uniform and moderately dense array of zinc oxide nanorods was successfully grown on the GB surface, with an average axial length of approximately 100 nm. This morphology is considered to be most favorable for subsequent bioconjugation and fluorescence enhancement.

[0025] Chemical structure characterization: The GB@ZnO interface was analyzed using X-ray photoelectron spectroscopy (XPS). The results are as follows: Figure 3 As shown, a Zn 2p characteristic peak exists near the binding energy of 1022 eV, confirming that a chemical bond is formed between ZnO and the glass microspheres, rather than physical adsorption.

[0026] Optical performance characterization: Equal amounts of SiO2 microspheres, SiO2@ZnO (prepared using the same method as GB@ZnO, except that SiO2 was used instead of GB), GBs, and GB@ZnO were incubated with FITC-BSA and then transferred to a PDMS hydrophobic substrate to form aggregates. Fluorescence imaging and quantification were then performed. Results are as follows: Figure 4 As shown, the fluorescence intensity of GB@ZnO aggregates was significantly higher than that of other control groups. After BET surface area normalization, the enhancement index (EI) of GB@ZnO was as high as 7.4, which was much higher than the sum of GB (EI≈3.0) and SiO2@ZnO (EI≈1.6) (4.6), confirming the synergistic enhancement effect. In addition, SiO2 microspheres are solid structures, and due to insufficient density matching with the system, they cannot achieve self-aggregation behavior in PDMS hydrophobic substrate droplets, and it is even more difficult to form aggregates at the droplet tip; while GBs are hollow structures, which can float and spontaneously aggregate at the droplet tip by means of density characteristics. This self-aggregation characteristic can significantly increase the local FITC-BSA concentration, thereby amplifying the fluorescence signal. Using SiO2 microspheres as a control, the unique advantages of GBs in "hollow structure-driven floating self-aggregation" were clarified.

[0027] Example 2 A method for constructing a sensor (using an anti-CD63 antibody as an example) includes the following steps: Take 0.06 mg of the epoxy-functionalized GB@ZnO composite material prepared in Example 1 and place it in an EP tube, then disperse it in phosphate buffer at pH 7.4 to make its concentration 2 mg / ml; Add 30 μL of GB@ZnO solution to an EP tube, then add 20 μL of 10 μg / mL anti-CD63 antibody solution. Gently shake at 37°C (100 rpm to 150 rpm) for 0.5 h to allow the antibody molecules to covalently couple with the epoxy groups on the material surface through their amino groups. After the reaction, discard the lower layer solution and add 300 μL of 5% BSA solution for blocking for 1 h. After blocking, wash three times with 200 μL of PBS to obtain the sensor.

[0028] Antibody modification effect verification: Fluorescence intensity was detected using fluorescent secondary antibody in two groups of GB@ZnO: one without anti-CD63 antibody (-anti-CD63 group) and the other with anti-CD63 antibody (+anti-CD63 group). Results are as follows: Figure 5 As shown, the fluorescence intensity of the +anti-CD63 group was significantly higher than that of the -anti-CD63 group, and the difference between the two groups was highly significant (P<0.0001). This proves that the anti-CD63 antibody has been successfully modified on the GB@ZnO surface, providing experimental evidence for subsequent antibody-specific recognition of EVs.

[0029] The capture unit of this invention can be flexibly constructed by changing the immobilized capture molecule and can be matched with various detection probes to achieve specific recognition and signal output of different markers on the surface of extracellular vesicles. For example, one or a combination of the following typical strategies can be used during construction: Firstly, using anti-CD9 antibodies (or aptamers) as capture molecules can achieve broad-spectrum enrichment of all EVs expressing CD9. During testing, Cy3-labeled GPC3 aptamers and Cy5-labeled EpCAM aptamers can be used together as detection probes to simultaneously distinguish between GPC3-positive and EpCAM-positive EV subpopulations in a single test.

[0030] Secondly, if high-specificity enrichment is required, anti-CD81 aptamers can be used as capture molecules to specifically capture CD81-positive EVs. The corresponding detection probes can be FITC-labeled anti-HER2 antibodies or Cy5-labeled anti-PSA nucleic acid probes, which are used to analyze the expression of HER2 or PSA disease markers in this subgroup.

[0031] Third, a sensor with anti-CD63 peptide as the capture molecule can be constructed, and fluorescently labeled antibodies against multiple disease biomarkers such as GPC3, EpCAM, and HER2 can be used as detection probes to achieve multi-parameter parallel phenotypic analysis of CD63-positive EVs.

[0032] The above schemes indicate that the capture molecules can be antibodies, aptamers, or peptides that specifically bind to universal surface markers of EVs such as CD9, CD63, and CD81; while the detection probes can be fluorescently labeled antibodies, aptamers, or nucleic acid probes targeting disease-related markers such as GPC3, EpCAM, HER2, and PSA.

[0033] Example 3 A method for detecting extracellular vesicles 1. EV Sample Preparation: After each cell type reached confluence greater than 80%, the culture medium was replaced with DMEM containing 1% P / S (HepG2, THLE2). Cells were cultured at 37 °C and 5% CO2 for 48 h. After collecting the culture medium, intact cells and cell debris were removed by centrifugation at 3000 g for 15 min at 4 °C. The supernatant was filtered through a syringe-driven filter (0.45 μm) and then concentrated using an Amicon Ultra-15 centrifuge filter (MWCO 10 kDa). The concentrated solute was collected and mixed with ExoQuick-TC exosome precipitation buffer (concentrated solute to ExoQuick-TC exosome precipitation buffer volume ratio 5:1). The mixture was incubated overnight at 4 °C, followed by centrifugation at 1500 g for 30 min at 4 °C to remove the supernatant for EV collection. Finally, the EVs were resuspended in PBS buffer and immediately analyzed or stored at −80 °C for later use.

[0034] 2. Capture reaction: Take the sensor prepared in Example 2 (i.e. GB@ZnO conjugated with anti-CD63 antibody), wash with PBS, add 10 μL of EV sample, add PBS to the total volume of 100 μL, and incubate with gentle shaking at room temperature for 20 min.

[0035] 3. Washing: After incubation, add 200 μL of PBS and mix gently. The conjugate will separate on its own and be washed three times with PBS to remove non-specific binding impurities.

[0036] 4. DiI Fluorescence Staining Detection: Prepare a 1:200 dilution of DiI working solution (prepare fresh before use). Add 200 μL of DiI working solution to the washed complex and incubate at 37 ℃ in the dark for 15 min. Then wash twice with PBS to remove free dye. Add the complex to a fluorescence microscope slide, cover with a coverslip, and observe and acquire fluorescence images. A blank control group without added EVs was also prepared using the same procedure.

[0037] 5. SEM morphology observation: The washed GB@ZnO-EV composite was dropped onto the conductive adhesive, allowed to air dry naturally, and then sputtered with gold. The composite was then observed and images were acquired under a scanning electron microscope (SEM).

[0038] like Figure 6 As shown, the experimental group incubated with EV and GB@ZnO exhibited strong fluorescence signals, while the blank control group without EV showed almost no fluorescence. Since DiI dye can specifically insert into the phospholipid bilayer of EV, this result directly indicates that GB@ZnO modified with anti-CD63 antibody successfully captured a large amount of EV. Figure 6 In the diagram, BF represents the bright field, and DF represents the dark field.

[0039] SEM morphology results are as follows Figure 7 As shown, a large number of spherical / quasi-spherical membrane structure particles (consistent with the typical morphology of EV) are visible on the GB@ZnO surface. SEM images further confirm the abundant EV membrane structure on the GB@ZnO surface.

[0040] In summary, the detection method of this embodiment achieves efficient EV capture through antibody-specific binding. Combined with DiI fluorescence staining and SEM morphology results, the capture effect of EV can be accurately demonstrated, providing reliable technical support for subsequent EV analysis.

[0041] Example 4 This embodiment aims to verify the sensitivity, specificity, and anti-interference of the sensor (GB@ZnO@Anti-CD63) constructed in Embodiment 2 of the present invention in detecting extracellular vesicles.

[0042] 1. Sensitivity Verification Using precisely counted HepG2 cell-derived EVs obtained through nanoparticle tracking analysis, they were serially diluted with PBS buffer to prepare particle concentrations of 1×10⁻⁶. 8 1×10 7 1×10 6 1×10 5 1×10 4 And samples with 0 particles / mL.

[0043] Strictly following the detection method described in Example 3, EVs samples of various concentrations were mixed with the sensor suspension, allowed to stand to form aggregates, and then 0.6 μM of Cy5-labeled anti-EpCAM aptamer was added as a signal probe. After incubation in the dark for 30 min, fluorescence imaging was performed.

[0044] The mean fluorescence intensity (MFI) of each sample aggregation region was quantified using image analysis software, with three parallel experiments set up for each concentration.

[0045] Results and Analysis: The results are as follows Figure 8As shown, the detected fluorescence signal intensity systematically decreases with decreasing EV concentration. A standard curve is plotted with the logarithm of EV concentration on the x-axis and the corresponding average fluorescence intensity on the y-axis. At 1×10⁻⁶... 4 particles / mL to 1×10 8 Within the concentration range of particles / mL, the fluorescence signal showed a good linear relationship with the EV concentration (R²>0.99).

[0046] Based on the method recommended by the International Union of Pure and Applied Chemistry, and using the concentration corresponding to three times the standard deviation of the blank control signal value, the detection limit of this sensor for GPC3 is as low as 9.5 × 10⁻⁶. 3 Particles / mL, as low as 5.5 × 10⁻⁶ when EpCAM is the target. 3 Particles / mL, compared to ELISA (10 8 The particle count (particles / mL) is four orders of magnitude lower. This indicates that the detection method has great potential for rapidly identifying subtle changes in EV phenotype.

[0047] 2. Specificity verification Experimental group: EVs derived from HepG2 cells.

[0048] Negative control: EVs derived from normal hepatocytes of THLE-2.

[0049] All EV samples were uniformly adjusted to the same particle concentration.

[0050] Following the detection procedure of Example 3, all samples were processed in parallel and subjected to fluorescence imaging analysis of the Cy5 (EpCAM) channel.

[0051] Results and Analysis: The results are as follows Figure 9 As shown, the sensor only produces a strong, specific fluorescence signal for the experimental group (HepG2 EVs).

[0052] For the negative control (THLE-2 EVs), although the sensor captured the EVs via anti-CD63, the signal probe could not bind effectively because the EVs did not express or expressed low levels of the target biomarker EpCAM. Therefore, the resulting fluorescence signal intensity was extremely low, showing no statistically significant difference compared to the blank control group (p>0.05). This demonstrates the specificity of the sensor for identifying disease-related biomarkers.

[0053] 3. Anti-interference performance verification Experimental Design: To further investigate the sensor's anti-interference capability, HepG2 exosomes were added to PBS buffer and 10% FBS solution (without exosomes) to achieve a concentration of 10. 610 7 10 8 particles / mL. Results as follows Figure 10 As shown, there was no significant difference in relative fluorescence intensity between PBS buffer and 10% FBS, indicating that it has good anti-interference performance.

[0054] Systematic interference experiments have demonstrated that the sensor based on the GB@ZnO photoheterogeneous aggregate not only possesses excellent anti-interference performance but also achieves accurate detection even in the presence of high concentrations of interfering substances. These characteristics make it highly suitable for applications such as primary healthcare institutions and rapid on-site detection, providing strong support for the stable and reliable detection of extracellular vesicles.

[0055] Example 5: Detection and Disease Differentiation of EV Surface Proteins in Clinical Plasma Samples Based on GB@ZnO@Anti-CD63 Experimental objective: To verify the enrichment ability of GB@ZnO@Anti-CD63 on extracellular vesicles (EVs) in clinical plasma samples, and the clinical feasibility of distinguishing between healthy controls (NC) and liver cancer (HCC) samples by detecting EV surface proteins.

[0056] 1. Experimental Materials - Sample Source: Fresh whole blood samples (10 healthy controls (NC) and 10 patients with hepatocellular carcinoma (HCC)); Reagents and materials: GB@ZnO@Anti-CD63, GPC3 / EpCAM aptamer (labeled with fluorescent group), sterile PBS buffer.

[0057] 2. Experimental Procedure 1. Plasma sample pretreatment: Take 5 mL of fresh whole blood and first centrifuge at 3000 rpm for 5 min to separate the plasma; then centrifuge at 2000 g for 10 min and 10000 g for 30 min to remove cell debris and apoptotic bodies; then filter the plasma through a 0.45 μm pore size membrane filter to remove large molecular impurities; the processed plasma samples should be tested immediately or aliquoted and frozen at −80℃, avoiding repeated freeze-thaw cycles throughout the process.

[0058] 2. Enrichment of plasma vesicles with GB@ZnO@Anti-CD63: Take 10 μL of pretreated plasma sample and incubate it with GB@ZnO composite material at room temperature for 20 min. The EV capture capability of GB@ZnO is used to enrich the target vesicles.

[0059] 3. Fluorescence detection of EV surface proteins: Fluorescent aptamers corresponding to GPC3 and EpCAM were added to the incubation system. After incubation for 30 min, the fluorescence signal of the system was detected by a fluorescence analyzer. The protein expression level was characterized by the relative fluorescence intensity value. Finally, the data were normalized to obtain the "Normalization" value.

[0060] 3. Experimental Results and Analysis: The operation procedure of this embodiment is as follows: Figure 11 a. Fluorescence detection results as follows Figure 11 b. The "Normalization" values ​​for GPC3 and EpCAM in HCC samples were significantly higher than those in NC samples (P=0.0001), indicating that the expression levels of these two proteins on the EV surface of HCC patients were much higher than in healthy individuals. Expression profile differences ( Figure 11 c): The heatmap results further validated that the expression levels of GPC3 and EpCAM in the 10 HCC samples were significantly higher than those in the 10 NC samples, effectively distinguishing healthy controls from liver cancer samples. This example demonstrates that the plasma EV surface protein detection method based on GB@ZnO can reliably distinguish between healthy individuals and liver cancer patients, possessing potential for clinical diagnostic applications.

[0061] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A method for preparing an optical heterostructure aggregate, characterized in that, Includes the following steps: 1) Surface cleaning and hydroxyl activation treatment of hollow glass microspheres; 2) The activated hollow glass microspheres were dispersed in a mixed aqueous solution containing a zinc source and hexamethylenetetramine. The zinc source was zinc acetate, zinc nitrate, or zinc chloride. The hydrothermal reaction was carried out at a temperature of 60-95 °C with stirring for 2-8 h to allow zinc oxide nanostructures to grow in situ on the surface of the hollow glass microspheres. 3) After the reaction is complete, the mixture is transferred to an ice-water bath for quenching, centrifuged and the supernatant is discarded. The precipitate is resuspended and washed with ultrapure water until the supernatant is neutral. The precipitate is collected by gravity sedimentation and dried overnight in a vacuum drying oven to obtain the composite material GB@ZnO. 4) The GB@ZnO composite material was silanized by hexadecyltrimethoxysilane and triethoxy(3-epoxypropyloxypropyl)silane to introduce bio-coupled functional groups and obtain photoheterogeneous aggregates.

2. The method for preparing the optical heterostructure aggregate according to claim 1, characterized in that, Step 1) involves surface cleaning and hydroxyl activation treatment of the hollow glass microspheres, specifically including the following steps: 1.1) Immerse hollow glass microspheres in a piranha solution at a temperature of 20-25 °C for 50-70 min. The piranha solution is prepared by mixing 30% hydrogen peroxide and 98% concentrated sulfuric acid at a volume ratio of 1:

3. 1.2) The treated hollow glass microspheres were washed with ultrapure water until the pH of the washing solution was 6.5-7.5; 1.3) The washed hollow glass microspheres are dried in a vacuum environment at 35-40℃ for 10-14 h to obtain activated hollow glass microspheres.

3. The method for preparing the optical heterostructure aggregate according to claim 1, characterized in that, In step 1), the diameter of the hollow glass microspheres before activation is 1-100 μm.

4. The method for preparing the optical heterostructure aggregate according to claim 1, characterized in that, In step 2), the activated hollow glass microspheres are dispersed in a mixed aqueous solution containing a zinc source and hexamethylenetetramine. The specific process is as follows: Mix equal volumes of 0.025 M anhydrous zinc acetate aqueous solution and hexamethyltetramine in a beaker, add 100-300 mg of the hollow glass microspheres activated in step 1), and sonicate for 30-50 min to mix evenly.

5. The method for preparing the optical heterostructure aggregate according to claim 1, characterized in that, In step 4), the GB@ZnO composite material is silanized using hexadecyltrimethoxysilane and triethoxy(3-epoxypropyloxypropyl)silane, specifically including the following steps: 4.1) Dry the GB@ZnO composite material in a vacuum environment at 50-100 ℃ for 1-2 h; 4.2) Add 0.5%-1.5% (v / v) of hexadecyltrimethoxysilane to the GB@ZnO composite material and react with shaking at 100-150 rpm for 2 h at room temperature; 4.3) Transfer the reacted solution to a centrifuge tube and wash it three times with anhydrous ethanol to remove unreacted hexadecyltrimethoxysilane. 4.4) Add 5% (v / v) of triethoxy(3-epoxypropyloxypropyl)silane, and incubate overnight in a 3D vortex mixer to form epoxy groups. Then wash the product 3-5 times with anhydrous ethanol. Subsequently, collect GB@ZnO by self-separation, dry it in an oven, and store it at 4 °C for later use.

6. The method for preparing the optical heterostructure aggregate according to claim 1, characterized in that, The zinc oxide nanostructure is an array of nanorods with an axial length of 50-200 nm.

7. An optical heterostructure aggregate, characterized in that, The optical heterostructure aggregate was prepared by the method for preparing optical heterostructure aggregates according to any one of claims 1-6.

8. A sensor, characterized in that, It includes the photoheterogeneous aggregate as described in claim 7 and the capture molecule immobilized on the photoheterogeneous aggregate; the capture molecule is an antibody, aptamer or polypeptide that specifically binds to extracellular vesicle surface markers.

9. A reagent kit, characterized in that, It includes the sensor described in claim 8.

10. A method for detecting extracellular vesicles, characterized in that, Includes the following steps: S1. The sensor of claim 8, a PBS solution or plasma sample containing extracellular vesicles, and at least one signal probe are mixed to form a mixture, wherein the signal probe is capable of specifically binding to disease-related biomarkers on the surface of target extracellular vesicles and is fluorescently labeled; S2. Place the mixture on the substrate and let it stand, so that the photoheterogeneous aggregate floats to the gas-liquid interface or the top of the droplet under the action of buoyancy, and self-aggregates to form aggregates, thereby completing the capture and labeling of the target extracellular vesicles. S3. Perform fluorescence detection on the aggregates, and perform qualitative and / or quantitative analysis of the target extracellular vesicles based on the fluorescence signal.

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