Extracellular vesicle detection method based on magnetic bead capture and nano-enzyme labeling and application

Abstract

The invention discloses an extracellular vesicle detection method based on magnetic bead capture and nano-enzyme labeling, used Fe3O4 (at) TiO2 magnetic beads can be used for rapidly enriching small extracellular vesicles (sEVs), meanwhile, the integrity of surface proteins of the sEVs is reserved, and a high-quality sample is provided for subsequent detection and analysis; the used nano enzyme has good oxidase-like activity, no exogenous hydrogen peroxide (H2O2) is needed, errors caused by addition of exogenous substances are reduced, and the detection sensitivity and accuracy are improved. The invention breaks through the bottlenecks of insufficient sensitivity (the detection limit reaches 102 pieces / mu L) and obvious environmental interference in the traditional detection method, and can also provide a new idea and technical scheme for monitoring extracellular vesicle markers of diseases such as tumors and the like.

Description

Technical Field

[0001] The present invention relates to a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling and its application, belonging to the fields of nanomaterials and biomedical nanotechnology. Background Art

[0002] Extracellular vesicles (EVs) are phospholipid bilayer vesicles secreted by cells and participate in cell-to-cell communication. They can be divided into subtypes such as exosomes, microvesicles, and apoptotic bodies according to biogenesis and size. Among them, small extracellular vesicles (sEVs, 50-200 nm) originate from intracellular multi-vesicular structures, regulate various biological functions, and are particularly crucial in lymphoma. They can affect the disease process by mediating tumor progression, immune escape, and drug resistance. Lymphoma is a globally prevalent hematological malignancy, and its occurrence is closely related to the abnormal proliferation of mature B lymphocytes. CD20, as a specific antigen on the surface of B cells, plays a central role in cell activation, differentiation, and proliferation, and is an ideal target for immunotherapy of B cell lymphoma. Therefore, accurately detecting the expression level of CD20 on the surface of sEVs may provide important evidence for the dynamic monitoring of lymphoma treatment efficacy, the formulation of individualized medication plans, and prognosis assessment.

[0003] The detection of sEVs usually involves two core steps: separation and enrichment, and target analysis. In the field of separation technology, although the ultra-high-speed centrifugation method is regarded as the "gold standard", it relies on multiple gradient centrifugations (usually taking 6-12 hours) and has strict requirements for the performance of centrifugation equipment (such as a rotation speed ≥ 100,000×g), making it difficult to meet the clinical rapid detection needs. The separation method based on ultrafiltration is easy to operate, but sample loss of sEVs is likely to occur during the processing due to membrane adsorption and shear force (the recovery rate is less than 60%), especially for low-abundance samples, its applicability is poor. In recent years, Fe3O4@TiO2 magnetic beads can rapidly enrich plasma sEVs in a short time due to the efficient binding ability of TiO2 to the phosphate groups in the sEVs phospholipid layer, while retaining the integrity of their surface proteins.

[0004] In terms of detection methods, although surface-enhanced Raman scattering (SERS) and surface plasmon resonance (SPR) have single-molecule-level sensitivity, they rely on expensive and sophisticated instruments and complex optical systems, making it difficult to achieve point-of-care testing; chemiluminescence (CL) and electrochemistry (EC) methods have relatively high sensitivity, but are easily interfered by complex matrices in biological samples, resulting in an increased false positive rate. Colorimetric analysis has attracted much attention due to its advantages such as low cost and visible results to the naked eye. However, its single optical signal is easily affected by environmental temperature, light intensity, and the subjective judgment of operators, and the detection sensitivity is limited, making it difficult to meet the trace detection requirements. Existing technologies have tried to combine colorimetric analysis with fluorescence analysis, and the reliability can be improved through dual-signal cross-validation.

[0005] As an artificial nanomaterial that mimics the catalytic function of natural enzymes, nanozymes exhibit unique advantages in the field of biosensing. Compared with natural enzymes, nanozymes not only have excellent stability such as high temperature resistance and acid-base resistance, but also can be synthesized in large quantities, and their catalytic activity can be regulated by various means. These characteristics enable nanozymes to have broad application prospects in the field of biosensing. For example, nanozymes can be used as the core components of biosensors to detect metabolites such as glucose and uric acid in vivo, and their high stability and tunability can ensure the long-term stable operation of sensors in complex biological environments. In addition, nanozymes can be combined with fluorescent probes, electrochemical sensors, etc. to further improve the sensitivity and specificity of detection. By precisely regulating the composition and structure of nanozymes, specific recognition and detection of different biomolecules can be achieved, providing strong support for the early diagnosis and personalized treatment of diseases. Summary of the Invention

[0006] Object of the Invention: The object of the present invention is to provide a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling.

[0007] Technical Solution: The present invention provides a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling, including the following steps:

[0008] (1) Mix Fe3O4@TiO2 magnetic beads with a detection sample containing extracellular vesicles, incubate, wash, and prepare an Fe3O4@TiO2 magnetic bead / extracellular vesicle complex.

[0009] (2) Couple CuCo-ZIF / Pt nanozyme and an antibody for detecting extracellular vesicles, add BSA blocking solution to prepare a signal tag, and wash and resuspend it.

[0010] (3) Mix the complex formed by the binding of Fe3O4@TiO2 magnetic beads and extracellular vesicles with the signal tag and incubate. After the reaction is completed, wash with PBS, then add o-phenylenediamine (OPD) substrate solution. In the PBS buffer, the signal tag catalyzes the oxidation of OPD to generate DAP. Finally, use a UV spectrophotometer and a fluorescence analyzer to detect the absorbance value and fluorescence intensity value of DAP, and substitute the signal value into the corresponding standard curve to quantitatively analyze the concentration of extracellular vesicles in the sample.

[0011] Further, in step (1), the dosage of the Fe3O4@TiO2 magnetic beads is 0.6 - 1.8 mg, and the incubation time is 5 - 10 min.

[0012] Furthermore, the CuCo-ZIF / Pt described in step (2) is prepared by the following steps: first, Cu(NO3)2·3H2O, Co(NO3)2·6H2O and hexadecyltrimethylammonium bromide (CTAB) are dispersed in deionized water, and after being fully mixed, the above solution is quickly added to an aqueous solution containing 2-methylimidazole (2-MeIM), stirred at room temperature and dried under vacuum overnight to obtain CuCo-ZIF; the dried CuCo-ZIF is dispersed in a methanol solution, H2PtCl6 is added, stirred at room temperature, and then sodium borohydride (NaBH4) is added and stirred until the reaction is completed, and finally the mixture is centrifuged, washed, and vacuum dried overnight to obtain CuCo-ZIF / Pt.

[0013] Furthermore, the concentration of the antibody for detecting extracellular vesicles in step (2) is 10-50 μg / mL, and the concentration of the BSA blocking solution is 1-3%.

[0014] Furthermore, the concentration of the signal tag in step (3) is 1-3 mg / mL.

[0015] Furthermore, the method also includes the step of establishing a standard curve: in the colorimetric mode, the linear relationship between the extracellular vesicle concentration C and the signal value Y is: Y=0.401*logC-1.75; in the fluorescence mode, the linear relationship between the extracellular vesicle concentration C and the signal value Y is: Y=2037.21*logC-5818.84.

[0016] Furthermore, in step (3), the colorimetric mode detection of 2,3-diaminophenazine (DAP) produces a characteristic absorption peak at 420 nm; the fluorescence mode detection of DAP emits a 570 nm fluorescence signal under 420 nm excitation light.

[0017] The present invention also provides the application of the above-mentioned extracellular vesicle detection method based on magnetic bead capture and nanozyme labeling in detecting the extracellular vesicle content in plasma.

[0018] Furthermore, the plasma needs to be pre-treated, including low-speed centrifugation and filtration.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the Fe3O4@TiO2 magnetic beads used in the extracellular vesicle detection method based on magnetic bead capture and nanozyme labeling can quickly enrich sEVs while retaining the integrity of sEVs surface proteins, providing high-quality samples for subsequent detection and analysis; the nanozymes used have good oxidase-like activity, do not require exogenous H2O2 participation, reduce the error caused by the addition of exogenous substances, and improve the sensitivity and accuracy of detection. The present invention breaks through the lack of sensitivity of traditional detection methods (detection limit of 10 2The bottleneck of significant environmental interference with [number] / μL) can also provide new ideas and technical solutions for the monitoring of extracellular vesicle markers in diseases such as tumors. Description of the Drawings

[0020] Figure 1 It is a process principle flow chart, where: A, synthesis of signal tags; B, assembly and working principle of immunosensors.

[0021] Figure 2 It is a characterization diagram of the morphology and size of extracellular vesicles observed under a transmission electron microscope.

[0022] Figure 3 It is an experimental result diagram for optimizing the dosage of Fe3O4@TiO2 magnetic beads required in the sEVs separation process.

[0023] Figure 4 It is an experimental result diagram for optimizing the incubation time in the sEVs separation process.

[0024] Figure 5 It is a result diagram of the capture rate of sEVs by Fe3O4@TiO2 magnetic beads.

[0025] Figure 6 It is a characterization diagram of the morphology and size of CuCo-ZIF / Pt nanozyme observed under a transmission electron microscope.

[0026] Figure 7 It is a comparison diagram of the a nano values of CuCo-ZIF and CuCo-ZIF / Pt.

[0027] Figure 8 It is a comparison diagram of the colorimetric signals when PtNPs, CuCo-ZIF, and CuCo-ZIF / Pt are used as signal tags respectively.

[0028] Figure 9 It is a standard curve diagram for detecting CD20 on sEVs by a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling in the colorimetric analysis mode.

[0029] Figure 10 It is a standard curve diagram for detecting CD20 on sEVs by a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling in the fluorescence analysis mode.

[0030] Figure 11 It is an analysis result diagram of plasma sEVs from healthy people and lymphoma patients by a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling. Detailed Embodiments

[0031] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.

[0032] Example 1: Optimization of Conditions for Capturing Extracellular Vesicles by Fe3O4@TiO2 Magnetic Beads

[0033] 1. Reagents and Equipment

[0034] Fe3O4@TiO2 magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd. (concentration: 25 mg / mL), PBS buffer (pH 7.4), PKH26 dye, centrifuge, ultra-high-speed centrifuge, magnetic stand.

[0035] 2. Implementation Method

[0036] (1) Obtaining extracellular vesicles: The collected Raji cell culture medium was centrifuged successively at 4°C, and the specific steps were as follows: centrifuged at 300 g for 10 min to remove cells; centrifuged at 2000 g for 10 min to remove cell debris; centrifuged at 10,000 g for 30 min to further remove impurities. Then centrifuged at 100,000 g for 70 min at 4°C, the precipitate was collected, washed with PBS buffer, and centrifuged again at 100,000 g for 70 min at 4°C. Finally, the obtained sEVs were dispersed in 500 μL PBS buffer and stored at -80°C as sEVs standard for subsequent experiments.

[0037] (2) Process of capturing extracellular vesicles by Fe3O4@TiO2 magnetic beads: Prepare two parallel samples with a concentration of 9.4×10 6 per μL, take 10 μL of each of the above samples, and fluorescently label them with PKH26 dye. Treated sample I: directly detect the fluorescence signal intensity of the labeled sEVs, denoted as F0; Treated sample II: mix the labeled sEVs with 1.4 mg Fe3O4@TiO2 magnetic beads, incubate at 37°C for 7 min, and detect the fluorescence signal intensity of the supernatant after magnetic separation, denoted as F1. The capture efficiency is proportional to the subtraction of the fluorescence signals of sample I and sample II. The calculation formula for the capture efficiency is as follows:

[0038]

[0039] (3) Condition optimization: The effects of the amount of Fe3O4@TiO2 magnetic beads (0.2 - 1.8 mg) and incubation time (1 - 10 min) on the capture efficiency were also studied, as well as the advantages of this separation method compared with other separation methods.

[0040] 3. Implementation Results

[0041] As Figure 2As shown, the extracellular vesicles observed by transmission electron microscopy have a typical disc shape with a diameter of 50 - 200 nm. To obtain the best capture efficiency, the dosage of Fe3O4@TiO2 magnetic beads ( Figure 3 ) and the incubation time ( Figure 4 ) were determined by staining with PKH26 (a lipophilic dye). When the dosage of Fe3O4@TiO2 magnetic beads was 1.4 mg and the capture time of Fe3O4@TiO2 magnetic beads for sEVs was 7 min, the capture efficiency of Fe3O4@TiO2 magnetic beads for sEVs was 91.34% ( Figure 5 ). The capture efficiency of the Fe3O4@TiO2 magnetic beads used in the method of the present invention for extracellular vesicles is 80.83%, and the integrity of its surface markers (such as CD20) is retained, providing a high-quality sample basis for subsequent detection.

[0042] Example 2: Preparation and specific activity measurement of CuCo-ZIF / Pt nanozyme (a nano )

[0043] 1. Reagents and equipment

[0044] Cobalt nitrate hexahydrate [Co(NO3)2·6H2O], cetyltrimethylammonium bromide (CTAB), 2-methylimidazole (2-MeIM), sodium borohydride (NaBH4), chloroplatinic acid (H2PtCl6·6H2O), copper nitrate trihydrate [Cu(NO3)2·3H2O], methanol, o-phenylenediamine (OPD), stirrer, centrifuge, vacuum drying oven, microplate reader.

[0045] 2. Implementation method

[0046] (1) Preparation method of CuCo-ZIF / Pt: First, 96.6 mg of Cu(NO3)2·3H2O, 174.6 mg of Co(NO3)2·6H2O and 7.5 mg of CTAB were dispersed in 10 mL of ultrapure water to obtain a homogeneous solution. Then the above solution was quickly added to 70 mL of aqueous solution containing 4.54 g of 2-MeIM and stirred at room temperature for 1 h. It was dried overnight under vacuum to obtain CuCo-ZIF. Then, 30 mg of the dried CuCo-ZIF was dispersed in 10 mL of methanol solution. 0.48 mL of 1% H2PtCl6 was added and stirred at room temperature for 2 h, and then 5 mL (0.3 M) of NaBH4 was added and stirred for 30 min. Then, the mixture was centrifuged, washed 3 times with methanol, and dried overnight under vacuum at 60 °C.

[0047] (2) Measurement of nanozyme specific activity: CuCo-ZIF and CuCo-ZIF / Pt with different amounts (0.001, 0.005, 0.01, 0.02, 0.05 mg) were mixed with OPD (10 μL, 10 mg / mL) and PBS (200 μL, 0.1 M, pH 6.5), respectively. The absorbance at 420 nm was read after reacting at room temperature for 30 min.

[0048] 3. Implementation Results

[0049] like Figure 6 As shown in the figure, the CuCo-ZIF / Pt observed under transmission electron microscopy showed a typical cubic structure, and PtNPs were loaded on the surface of CuCo-ZIF. In the activity quantification experiment based on the nanozyme, 0.001 mg, 0.005 mg, 0.01 mg, 0.02 mg, and 0.05 mg of CuCo-ZIF and its composite CuCo-ZIF / Pt were weighed respectively, and their specific activity values ​​were measured under the same reaction conditions. Figure 7 It can be seen that the specific activity of CuCo-ZIF is 1.11 U·mg -1 The specific activity of CuCo-ZIF / Pt is 2.45 U·mg -1 The results showed that the loading of Pt NPs could significantly enhance the activity of CuCo-ZIF nanozymes, thereby increasing the sensitivity of the sensing device and improving its accuracy.

[0050] Example 3: Comparison of colorimetric signals when Pt NPs, CuCo-ZIF and CuCo-ZIF / Pt nanozymes are used as signal labels

[0051] 1. Reagents and Equipment

[0052] PBS buffer (pH 7.4), anti-CD20 monoclonal antibody (human-mouse chimeric monoclonal antibody) was purchased from Xinxinda Biopharmaceutical (Suzhou) Co., Ltd.

[0053] 2. Implementation methods

[0054] (1) Preparation method of signal tags: First, disperse 2.6 mg of PtNPs, CuCo-ZIF or CuCo-ZIF / Pt nanomaterials in 500 μL of PBS buffer (pH 7.4) respectively. Then, add 500 μL of CD20 antibody solution with a concentration of 52 μg / mL to the above suspension, and continuously stir at a speed of 500 rpm for 12 h under the condition of constant temperature at 4 °C to complete antibody conjugation. Add 300 μL (1.0 wt%) BSA blocking solution, and stir at 500 rpm at 4 °C for 2 h to block the non-specific adsorption sites on the material surface. Adopt a centrifugal purification process (5000 rpm, 5 min, 4 °C), discard the supernatant, resuspend and wash twice with PBS to remove unbound biomolecules. Finally, resuspend the obtained composite probe in 500 μL of PBS buffer and store it at 4 °C for later use.

[0055] (2) Comparison of colorimetric signals generated by three signal tags: Mix 1.4 mg of Fe3O4@TiO2 magnetic beads with 10 μL of a standard solution of sEVs with a concentration of 9.4×106 particles / μL, and incubate in a constant temperature oscillator at 37 °C for 7 min to complete the capture of sEVs; then transfer the mixture to a magnetic separation device to achieve solid-liquid separation, and discard the supernatant; add PBS and wash twice to remove unbound impurities; add 40 μL of signal tags (i.e., the three signal tags prepared above) to the Fe3O4@TiO2 magnetic bead / sEVs complex, and incubate in a constant temperature oscillator at 37 °C for 30 min to form a Fe3O4@TiO2 magnetic bead / sEVs / signal tag complex. Add PBS and wash twice to remove unbound signal tags, then add 8 μL of OPD substrate solution (concentration 0.1 M), and react in the dark at 37 °C for 30 min; finally, detect the intensity of the colorimetric signal by an ultraviolet-visible spectrophotometer.

[0056] 3. Experimental results

[0057] The experimental data of colorimetric signals show that (as Figure 8As shown in the figure, when CuCo-ZIF / Pt nanozyme is used as the signal tag, the intensity of the characteristic absorption peak it generates is significantly enhanced compared to the CuCo-ZIF system. After analysis, it is considered that the surface of the CuCo-ZIF / Pt nanomaterial generates specific charge distribution characteristics due to the loading of PtNPs, and forms a stable directional binding with the Fc fragment of the CD20 antibody through electrostatic adsorption. In contrast, the charge distribution on the surface of the unmodified platinum CuCo-ZIF material is not conducive to the effective immobilization of antibody molecules, resulting in a serious decrease in the antibody binding rate, thus significantly reducing the signal response intensity. In addition, when separate PtNPs are used as the signal tag, although it also has a certain signal response ability, due to the lack of the synergistic effect brought by its binding to CuCo-ZIF, its signal intensity and stability are not as good as those when CuCo-ZIF / Pt nanozyme is used as the signal tag, which further highlights the advantages of CuCo-ZIF / Pt nanozyme as the signal tag in colorimetric signal detection.

[0058] Example 4: The analytical performance of the biosensor was judged by a method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling.

[0059] 1. Reagents and equipment

[0060] Fe3O4@TiO2 magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd., PBS buffer (pH 7.4), extracellular vesicles, ultraviolet spectrophotometer, fluorescence analyzer, magnetic stand.

[0061] 2. Implementation method

[0062] (1) Construction of biosensor: Mix 1.4 mg of Fe3O4@TiO2 magnetic beads (concentration: 25 mg / mL) with 10 μL of standard solutions of sEVs at different concentrations, incubate in a constant temperature shaker at 37 °C for 7 min to complete the capture of sEVs; then transfer the mixture to a magnetic separation device to achieve solid-liquid separation, discard the supernatant; add PBS and wash twice to remove unbound impurities; add 40 μL of signal tag (i.e., the CD20 antibody / CuCo-ZIF / Pt nanozyme composite probe prepared in Example 3) to the Fe3O4@TiO2 magnetic bead / sEVs complex, incubate in a constant temperature shaker at 37 °C for 30 min to form the Fe3O4@TiO2 magnetic bead / sEVs / signal tag complex, add PBS and wash twice to remove unbound signal tags, then add 8 μL of OPD substrate solution (concentration: 0.1 M), react in the dark at 37 °C for 30 min; finally, detect the colorimetric signal and fluorescence signal intensity by ultraviolet-visible spectrophotometer and fluorescence analyzer, and quantify the concentration of extracellular vesicles in the sample in combination with the standard curve. In the colorimetric mode, DAP produces a characteristic absorption peak at 420 nm; in the fluorescence mode, DAP emits a fluorescence signal at 570 nm under the excitation light of 420 nm.

[0063] (2) Evaluation of analytical performance: Evaluate the performance of the biosensor under optimal conditions. As the concentration of sEVs increases, CuCo-ZIF / Pt exhibits good peroxidase-like activity, which can oxidize OPD to generate DAP, resulting in a gradual increase in absorbance and fluorescence intensity. Read the signals by ultraviolet spectrophotometer and fluorescence analyzer, and plot the standard curve.

[0064] 3. Implementation results

[0065] (1) Colorimetric mode: As Figure 9 shown, in the colorimetric mode, when the concentration of sEVs is 4.7×10 4 -9.4×10 6 particles / μL, the absorbance value shows a good linear relationship with the concentration of sEVs.

[0066] (2) Fluorescence mode: As Figure 10 shown, in the fluorescence mode, when the concentration of sEVs is 9.4×10 2 -9.4×10 6 particles / μL, the fluorescence intensity is positively correlated with the concentration of sEVs.

[0067] Example 5: Verification of the detection ability of an extracellular vesicle detection method based on magnetic bead capture and nanozyme labeling in actual samples.

[0068] 1. Reagents and equipment

[0069] The Fe3O4@TiO2 magnetic beads were purchased from Nanjing Dongna Biotechnology Co., Ltd., PBS buffer (pH 7.4), plasma samples, ultraviolet spectrophotometer, fluorescence analyzer, and magnetic stand.

[0070] 2. Implementation method

[0071] (1) Treatment of plasma samples: Plasma samples were collected from healthy donors and lymphoma patients in Zhongda Hospital Affiliated to Southeast University. First, cells and cell debris were removed by low-speed centrifugation, and then large extracellular vesicles were filtered out using a 0.22 μm filter.

[0072] (2) Capture and detection of sEVs: 1.4 mg of Fe3O4@TiO2 magnetic beads (concentration 25 mg / mL) were mixed with the filtered plasma sample (20 μL) and incubated in a 37°C constant temperature oscillator for 7 min to complete the capture of sEVs in the plasma; then the mixture was transferred to a magnetic separation device to achieve solid-liquid separation, and the supernatant was discarded; then PBS was added and washed 2 times to remove unbound impurities; 40 μL of signal tag (i.e., the CD20 antibody / CuCo-ZIF / Pt nanozyme composite probe prepared in Example 3) was added to the Fe3O4@TiO2 magnetic bead / sEVs complex and incubated in a 37°C constant temperature oscillator for 30 min to form a Fe3O4@TiO2 magnetic bead / sEVs / signal tag complex, and PBS was added and washed 2 times to remove unbound signal tags, then 8 μL of OPD substrate solution (concentration 0.1 M) was added and reacted in the dark at 37°C for 30 min; finally, the colorimetric signal and fluorescence signal intensity were detected by an ultraviolet-visible spectrophotometer and a fluorescence analyzer, and the concentration of extracellular vesicles in the sample was quantified by combining with the standard curve in Example 4.

[0073] 3. Implementation results

[0074] This biosensor was used to analyze clinical plasma samples. As Figure 11 shown, both colorimetric and fluorescence results showed that the concentration of sEVs in the plasma of the patient group (4 - 8) was significantly higher than that of the healthy group (1 - 3). This indicates that this biosensor has certain application potential in distinguishing lymphoma patients from healthy individuals.

Claims

1. A method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling, characterized in that: The following methods are included: (1) mixing Fe3O4@TiO2 magnetic beads with a test sample containing extracellular vesicles, incubating, and washing to prepare a Fe3O4@TiO2 magnetic bead / extracellular vesicle complex; (2) coupling the CuCo-ZIF / Pt nanozyme with an antibody for detecting extracellular vesicles, adding BSA blocking solution to obtain a signal tag, and then washing and resuspending the tag; (3) The complex formed by the binding of Fe3O4@TiO2 magnetic beads and extracellular vesicles was mixed and incubated with the signal tag. After the reaction was completed, it was washed with PBS, and then the OPD substrate solution was added. The signal tag catalyzed the oxidation of OPD to generate DAP in the PBS buffer. Finally, the absorbance and fluorescence intensity of DAP were detected using an ultraviolet spectrophotometer and a fluorescence analyzer. The signal value was substituted into the corresponding standard curve to quantitatively analyze the concentration of extracellular vesicles in the sample.

2. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: The amount of Fe3O4@TiO2 magnetic beads used in step (1) is 0.6-1.8 mg, and the incubation time is 5-10 min.

3. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: The CuCo-ZIF / Pt described in step (2) is prepared by the following steps: first, Cu(NO3)2·3H2O, Co(NO3)2·6H2O and hexadecyltrimethylammonium bromide are dispersed in deionized water, and after being fully mixed, the above solution is quickly added to an aqueous solution containing 2-methylimidazole, and after stirring at room temperature, it is dried under vacuum overnight to obtain CuCo-ZIF; the dried CuCo-ZIF is dispersed in a methanol solution, H2PtCl6 is added, stirred at room temperature, and then sodium borohydride is added and stirred until the reaction is completed. Finally, the mixture is centrifuged, washed, and vacuum dried overnight to obtain CuCo-ZIF / Pt.

4. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: The concentration of the antibody for detecting extracellular vesicles in step (2) is 10-50 μg / mL, and the concentration of the BSA blocking solution is 1-3%.

5. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: The concentration of the signal tag described in step (3) is 1-3 mg / mL.

6. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: The method also includes the step of establishing a standard curve: in the colorimetric mode, the linear relationship between the extracellular vesicle concentration C and the signal value Y is: Y=0.401*logC-1.75; in the fluorescence mode, the linear relationship between the extracellular vesicle concentration C and the signal value Y is: Y=2037.21*logC-5818.

84.

7. The method for detecting extracellular vesicles based on magnetic bead capture and nanozyme labeling according to claim 1, characterized in that: In step (3), the colorimetric mode detection of DAP produces a characteristic absorption peak at 420 nm; the fluorescence mode detection of DAP emits a 570 nm fluorescence signal under 420 nm excitation light.

8. Use of the extracellular vesicle detection method based on magnetic bead capture and nanozyme labeling according to any one of claims 1 to 7 in detecting the extracellular vesicle content in plasma.

9. The use according to claim 8, characterized in that: The plasma also requires pretreatment, including low-speed centrifugation and filtration.

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