Tumor exosome detection method based on hierarchical iron-based heterojunction nano-enzyme

Abstract

The invention relates to a tumor exosome detection method based on hierarchical iron-based heterojunction nano-enzyme. The core of the system is a composite nano-enzyme material with a layered double-metal hydroxide structure coated with a double-metal organic framework. The functionalized dual aptamers can specifically recognize two protein markers on the surface of the same exosome. An aptEGFR and HFeH signal tag is assembled on the surface of the magnetic bead modified with the aptCD63 through the recognition effect of the aptEGFR and the exosome, and the'sandwich type 'aptamer biosensor aiming at tumor-derived exosome detection is constructed. In the application, the method is applied to three high-sensitivity and high-specificity signal output platforms of chemiluminescence, electrochemical signal and chemical colorimetry. The method has the advantage of avoiding interference of free target protein, and provides new technical support for clinical diagnosis application and scientific research of exosome-related tumor biomarkers.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biological detection technology and nanosensor technology, and particularly relates to a method for detecting tumor exosomes based on hierarchical iron-based heterojunction nanozymes. Background Art

[0002] Exosomes are extracellular vesicles with a diameter of approximately 30 - 150 nm, and their main components include lipids, proteins, and nucleic acids, which have important biological functions and significance. As signal molecules, exosomes can not only regulate cell growth and differentiation but also participate in biological processes such as inflammation transmission, tumor metastasis, and tissue repair, playing an important role in the occurrence and development of diseases. Recent studies have found that exosomes derived from tumor cells are important biomarkers for cancer diagnosis, classification, and prognosis, such as lung cancer, pancreatic cancer, liver cancer, etc. Although tumor-derived exosomes are regarded as a novel and ideal tumor biomarker, due to their nano-scale size, high heterogeneity, and other characteristics, they still face some challenges in the detection of biological samples. So far, traditional exosome detection methods represented by Western blot and ELISA have the disadvantages of cumbersome operation and expensive antibodies. In recent years, emerging detection technologies such as fluorescence, colorimetry, electrochemistry, and chemiluminescence have received extensive attention due to their advantages of simplicity, sensitivity, economy, and high efficiency. High-sensitivity detection platforms represented by chemiluminescence detection technology have been widely used in detecting tumor-derived exosomes in biological samples due to their superior controllability and high sensitivity.

[0003] In order to significantly improve the detection ability of chemiluminescent biosensors, nanozymes, as a kind of nanomaterial with the catalytic characteristics of mimicking natural enzymes, have attracted much attention due to their economy, stability, and other characteristics. Among them, metal-organic frameworks (MOFs) formed by different types of metal units and organic ligands have shown great application potential due to their large specific surface area, high pore density, and easy structural regulation. More and more MOF nanozymes have been widely developed and applied in the fields of analytical biosensing and cancer treatment. Compared with single-metal MOFs, MOF nanozymes can show more significant catalytic activity advantages when acting synergistically with other functionalized metals. For example, the bimetallic Cu / Zr-MOF-818 system has been proven to have ultra-high superoxide dismutase-like activity. In addition, the incorporation of Zr can significantly improve the peroxidase-like activity of UiO-66(Ce / Zr). Although composite nanozymes can greatly enhance the analytical performance, it is still worth exploring to construct nano-composites with higher catalytic activity based on MOFs.

[0004] Considering the heterogeneous characteristics of tumor-derived exosomes, functionalized dual-aptamer probes are used to improve the recognition ability of target exosomes. Compared with traditional antibodies, aptamers are single-stranded oligonucleotides (ssDNAs) containing 10-100 bases and are more economical and efficient. Based on this, the use of nucleic acid aptamers as molecular recognition elements shows strong competitiveness in the construction of biosensors, especially in the biomedical field with broad application prospects. Inspired by the double-antibody sandwich detection, the use of functionalized dual-aptamers to simultaneously recognize two protein markers on the same exosome can exclude the interference caused by non-target exosomes and soluble target proteins to improve the detection specificity. Summary of the Invention

[0005] Based on the above background, we designed a multi-mode biosensing system integrating "separation-detection" for the detection of tumor-derived exosomes.

[0006] The inventive method includes the following steps:

[0007] (1) An iron-based nanozyme composed of FeZr-MOF and CuFe-LDH wrapped on the outer layer

[0008] (FeZr-MOF@CuFe-LDH)

[0009] (2) Mix magnetic beads with aptamer apt CD63 to obtain magnetic bead-modified aptamer apt CD63 (apt CD63 @MBs);

[0010] (3) Mix hierarchical iron-based heterojunction nanozyme with aptamer apt EGFR to obtain hierarchical iron-based heterojunction nanozyme-modified aptamer apt EGFR (apt EGFR @HFeH);

[0011] (4) Simultaneously recognize two protein biomarkers on exosomes through magnetic bead-modified aptamer apt CD63 and hierarchical iron-based heterojunction nanozyme-modified aptamer apt EGFR to form a dual-aptamer sandwich composite structure, and respectively catalyze the chemiluminescence signal of the luminol / H2O2 system, the electrochemical signal generated by the decomposition of H2O2,

[0012] the colorimetric signal of the TMB / H2O2 color development system changing from colorless to blue, and achieve multi-mode high-efficiency signal output of tumor-derived exosomes on the chemiluminescence, electrochemical, and colorimetric platforms.

[0013] Preferably, the linking aptamer is the sequence shown in SeqNo.1-2.

[0014] Preferably, the reaction buffer solution is a PBST solution, and its components are: 10 mM Na2HPO4, 2 mM NaH2PO4, 135 mM NaCl, 4.7 mM KCl, 5 mM Tween 20, pH 7.4.

[0015] Preferably, the detection methods are carried out on a chemiluminescence platform, an electrochemical platform and a colorimetric platform respectively.

[0016] Preferably, the reaction time of steps (2) and (3) is 30 minutes.

[0017] Preferably, the reaction temperature of steps (2) and (3) is 37 °C.

[0018] Preferably, the exosome sample to be detected is prepared from any one of tumor cells or serum.

[0019] Preferably, the method for preparing the exosome sample to be detected includes any one of the ultracentrifugation method or the kit extraction method, preferably the centrifugation method.

[0020] Compared with the current exosome detection methods, the advantages of the present invention are as follows:

[0021] Compared with the traditional method for detecting exosomes using a step-by-step signal amplification platform, the present method realizes an exosome detection mode of "separation - detection" in one. The operation of the one-step method is simple and fast; it shortens the detection window period for disease screening and assists clinical diagnosis in improving the disease diagnosis rate.

[0022] Compared with the traditional single-platform and single-marker detection method, the detection signal of the present method is specific and comprehensive; it realizes multi-mode biosensing detection of serum tumor-derived exosomes, provides tumor information in multiple dimensions of time and space, and the information of multiple platforms is complementary and integrated to improve the signal detection accuracy. This biosensing system not only has the catalytic advantages of composite nanozymes but also has the advantages of realizing highly specific analysis of tumor-derived exosomes and avoiding interference from free target proteins. This provides a new idea for detecting exosome contents and provides new technical support for the clinical diagnosis application and scientific research of exosome-related tumor biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the solution for a method for detecting tumor exosomes based on a hierarchical iron-based heterojunction nanozyme according to the present invention.

[0024] Figure 2 It is a TEM characterization diagram of the hierarchical iron-based heterojunction nanozyme in the present invention.

[0025] Figure 3 It is the feasibility test result of detecting tumor exosomes based on a chemiluminescence platform, an electrochemical platform and a colorimetric platform according to the present invention.

[0026] Figure 4 This is the optimized result of the detection of tumor exosomes based on the hierarchical iron-based heterojunction nanozyme in the present invention.

[0027] Figure 5 This is the sensitivity result of the detection of tumor exosomes based on the chemiluminescence platform, electrochemical platform and colorimetric platform in the present invention and the specificity result based on the chemiluminescence platform

[0028] Figure 6 This is the detection result of the present invention based on the hierarchical iron-based heterojunction nanozyme for clinical specimens (10 healthy people and 10 lung cancer patients).

[0029] CuFe-LDH: Copper-iron layered double hydroxide

[0030] FeZr-MOF: Iron-zirconium metal-organic framework

[0031] HFeH: Hierarchical iron-based heterojunction nanozyme

[0032] Apt: Aptamer

[0033] CD63: Lysosome-associated membrane protein 3

[0034] EGFR: Epidermal growth factor receptor Detailed implementation manners

[0035] In this study, we designed a highly specific and efficient multimode biosensing system capable of realizing the "separation-detection" function for ultrasensitive detection of tumor-derived tEVs. The core of this system is a highly efficient iron-based nanozyme (HFeH), which consists of an inner layer of FeZr-MOF and an outer layer of CuFe-LDH. During the synthesis process, FeZr-MOF and CuFe-LDH are first prepared separately, and then the two are mixed and stirred for 12 hours to form a composite nanomaterial HFeH, that is, a layered double metal hydroxide (CuFe-LDH) coated with a bimetallic organic framework (FeZr-MOF), as Figure 1 shown in A.

[0036] Inspired by the double antibody sandwich method, functionalized double aptamers can simultaneously recognize two protein markers on the surface of the same tEVs, thus eliminating the interference of non-target tEVs and soluble target proteins and improving the specificity of detection. As Figure 1 shown in B

[0037] CD63@MBs). aptCD63 specifically recognizes and captures tEVs, and then assembles the apt EGFR recognition of tEVs to assemble the apt EGFR @HFeH signal tags onto the surface of magnetic beads. This construction forms a "sandwich-type" aptamer biosensor for the detection of tumor-derived tEVs, which can produce strong signal outputs in various forms, including chemiluminescence (through the luminol / H2O2 system), electrochemistry (H2O2 system), and colorimetry

[0038] (TMB / H2O2 system). This biosensing system not only utilizes the catalytic performance of the composite nanozyme to achieve highly specific analysis of tumor-derived tEVs, but also effectively reduces the interference of free target proteins.

[0039] Experimental Materials

[0040] The HPLC-purified oligonucleotide chains, hydrogen peroxide (H2O2), and PBS buffer solution used were purchased from Sangon Biotech (Shanghai) Co., Ltd. Zirconium chloride (ZrCl4), sodium hydroxide (NaOH), luminol, and N,N-dimethylformamide (DMF) were purchased from Aladdin (Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O) and copper nitrate trihydrate Cu(NO3)2·3H2O were purchased from Macklin (Shanghai, China), Tween 20 and 2-aminoterephthalic acid (H2BDC-NH2) were purchased from Sigma-Aldrich (USA), and ferric nitrate nonahydrate (Fe(NO3)3·9H2O) was purchased from Guangzhou Kemon Biotechnology Co., Ltd. (Shanghai, China). Breast cancer cell line (MCF-7), non-small cell lung cancer cell line (A549), human melanoma cell line (A375), and normal liver cell line (L02) were purchased from ATCC (USA). All chemical reagents were of analytical grade and used directly without further purification. All aqueous solutions were prepared with deionized water.

[0041] Experimental Instruments

[0042] The MPI-A type electrochemiluminescence analyzer (Xi'an Remai Instrument Co., Ltd., China) was used for signal detection on the chemiluminescence platform, the CHI 660E electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China) was used for signal detection on the electrochemical platform, the Agilent Synergy H1 multi-functional microplate reader (Agilent, USA) was used for signal detection on the colorimetric platform, and the FEI Tecnai G2 12 transmission electron microscope (FEI Company, USA).

[0043] It should be understood that the following examples are only for illustrative explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0044] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.

[0045] The nucleotide sequences involved in the present invention are shown in Table 1.

[0046] Table 1

[0047]

[0048] Example 1: Synthesis of Hierarchical Iron-Based Heterojunction Nanozyme and Construction of Detection Element

[0049] 1.1 Synthesis of FeZr-MOF

[0050] It is prepared by a one-step hydrothermal method. Specifically, solutions of ZrCl4, FeCl3·6H2O, and H2BDC-NH2 with equal molar concentrations are prepared, and the solvent is N,N-dimethylformamide (DMF). Subsequently, 5 mL of ZrCl4 solution, 5 mL of FeCl3·6H2O solution, 10 mL of H2BDC-NH2 solution, and 5 mL of AcOH are mixed evenly. After magnetic stirring for 20 minutes, the reaction is carried out in an oil bath at 120 °C for 12 hours. After the reaction is completed, the solution is cooled to room temperature. The product is centrifuged and washed three times with methanol and ethanol each, and then dried overnight in a vacuum drying oven at 60 °C. The prepared yellow-brown FeZr-MOF powder is collected for subsequent use.

[0051] 1.2 Synthesis of CuFe-LDH

[0052] Weigh 0.5 mol L -1 Cu(NO3)2·3H2O and 0.25 mol L -1 Fe(NO3)3·9H2O and dissolve them in 40 mL of deionized water. Then prepare 0.5 mol L -1 NaOH solution for adjusting the pH. Mix well with a magnetic stirrer at 80 °C and adjust the pH to 5.5, then mix slowly for 15 minutes. After that, heat the solution in an oil bath at 80 °C for 12 h. After the reaction is completed, cool it to room temperature, wash it 3 times with deionized water and ethanol to remove free metal ions, centrifuge at a speed of 12000 rpm / min for 5 min, and dry it in a vacuum at 60 °C for standby.

[0053] Synthesis of 1.3HFeH nanozyme

[0054] Add 1 mg of vacuum-dried FeZr-MOF and CuFe-LDH powders into 1 mL of water. After ultrasonic treatment for 15 minutes until fully dissolved, mix them in a ratio of 1:100 and incubate at room temperature for 48 hours to allow full binding through electrostatic adsorption for later use.

[0055] 1.4apt CD63 Preparation of @MBs

[0056] Take 3 μL of magnetic beads with a particle size of 300 nm, wash them three times by magnetic separation with PBST solution, and resuspend them in 100 μL of PBS; add 1.5 μL of 100 μM single-stranded CD63 aptamer, incubate at 37 °C for 2 hours, wash three times by magnetic separation with PBS solution, and resuspend in 50 μL of PBS solution for later use.

[0057] 1.5apt EGFR Preparation of @HFeH

[0058] Take 1 mg of HFeH and resuspend it in 100 μL of PBST solution. Add 1.5 μL of 100 μM single-stranded EGFR aptamer, incubate at 37 °C for 2 hours, centrifuge and wash three times with PBS solution, and resuspend in 50 μL of PBS solution for later use.

[0059] 1.6 Material characterization

[0060] Wash the liquid HFeH sample with ethanol to remove surface contaminants, dry it at 60 °C, take 1 mg, clamp the sample on a copper grid, place it in the electrolyte, and thin the sample to dozens of nanometers through the action of current. Place it on the copper grid to provide support and reduce electron scattering. Thoroughly remove moisture, load the prepared sample onto the TEM sample stage for imaging analysis. As Figure 2 , the TEM characterization image of the HFeH nanozyme with FeZr-MOF sized 70 - 100 nm surrounding and adsorbing on CuFe-LDH sized 500 nm - 1 μm.

[0061] Example 2: Multi-platform construction of a detection method based on hierarchical iron-based heterojunction nanozymes

[0062] 2.1 Detection method of exosomes on the chemiluminescence platform using hierarchical iron-based heterojunction nanozymes

[0063] Use the hierarchical iron-based heterojunction nanozymes and detection components synthesized in Example 1 for detection verification on the chemochromic platform, as Figure 3 shown in A. The control group is without the target, and the experimental positive group is with the target. Take 50 μL each of the prepared CD63 aptamer-modified magnetic beads and EGFR aptamer-modified materials, mix them, add 10 μL of exosomes (1×1010 particles / mL -1 ) Incubate at 37 °C for 1 hour, wash three times by magnetic separation, and resuspend with 100 μL of PBS. Take a 96-well white plate, add 80 μL of 15 μM luminol working solution and 20 μL of the mixture to be tested, place it at the inlet of the multifunctional microplate reader, add 100 μL of 30 μM hydrogen peroxide before injection, and detect immediately. As shown in the results, the experimental positive group with the target has a better signal, while the signal is lower without the target, successfully verifying the feasibility of the chemiluminescence platform for detection.

[0064] 2.2 Detection method of exosomes on the electrochemical platform using hierarchical iron-based heterojunction nanozymes

[0065] Magnetic glassy carbon electrode (MGCE, diameter 3 mm), polish it with alumina (0.3 and 0.05 μm) until the surface is clean for 5 minutes, wash it with deionized water, and dry the polished electrode with nitrogen. Take 25 μL each of the prepared CD63 aptamer-modified magnetic beads and EGFR aptamer-modified material, mix them, take 10 μL and drop it on the magnetic electrode, let it stand for 20 minutes, and add 5 μL of exosomes (1×10 10 particles / mL -1 ) Incubate at 37 °C for 1 hour, wash with PBS to remove unbound aptamers and materials. Electrochemical detection is carried out by differential pulse voltammetry (DPV) in PBS buffer containing 10 mM H2O2. The potential range is from -0.4 V to -1.0 V, the pulse amplitude is 0.07 V, the pulse width is 0.05 V, and the sampling width is 0.0167 V. As Figure 3 shown in B, the control group is without the target, and the experimental positive group is with the target. When the target exists, the apt CD63 -modified magnetic beads adsorbed on the magnetic electrode specifically recognize and capture specific exosomes. The exosomes act as intermediates, and the surface proteins recognize and connect the apt EGFR -modified materials, and due to the adsorption of the materials on the electrode, a DPV peak signal is generated; in the results, the intensity of the experimental positive group is higher than that of the group without the target, successfully verifying the feasibility of the electrochemical platform.

[0066] 2.3 Detection method of exosomes on the chemical colorimetric platform using hierarchical iron-based heterojunction nanozymes

[0067] Use the hierarchical iron-based heterojunction nanozymes and detection elements synthesized in Example 1 to verify the detection on the chemical colorimetric platform, as Figure 3 shown in C. The control group is without the target, and the experimental positive group is with the target. Take 50 μL each of the prepared CD63 aptamer-modified magnetic beads and EGFR aptamer-modified material, mix them, and add 10 μL of exosomes (1×10 10particles per mL -1 ) Incubate at 37 °C for 1 hour, wash three times by magnetic separation, and resuspend in 100 μL PBS. Add 10 μL of 0.5 M H2O2, 80 μL of TMB, and 10 μL of the mixture to be tested into a 96-well transparent plate, mix and react for 3 minutes, set the excitation wavelength to 650 nm, and detect on the machine. As shown in the results, the signal of the experimental positive group with the target is better, and the signal is lower without the target, successfully verifying the feasibility of the chemical colorimetric platform.

[0068] Example 3: Optimization of the conditions for the detection method of tumor exosomes based on hierarchical iron-based heterojunction nanozymes Optimize the conditions for exosome detection on the chemiluminescence detection platform established in Example 2 to achieve the best analytical performance of the tEVs detection platform. The robustness of the optimized parameters ensures the smooth progress of future experiments. Optimize the key experimental conditions using chemiluminescence as the detection mode. These conditions include the ratio of HFeH nanozyme to Apt in the Apt EGFR @HFeH signal probe, the ratio of magnetic beads (MBs) to Apt in the Apt EGFR @MBs capture probe, and the reaction time for the recognition and binding of tEVs to Apt CD63 @MBs. When the ratio of HFeH to Apt CD63 is 3.3 / 1, the ratio of MBs to Apt EGFR @HFeH aptamer and Apt CD63 @MBs is 1 / 5, and the reaction time is 90 minutes, the best reaction efficiency can be achieved, and the signal-to-noise ratios are increased to 10.81, 11.55, and 12.17 ( EGFR A–4C), respectively. Subsequent experiments were carried out based on these optimized conditions.

[0069] Example 4: Performance verification of the detection method of tumor exosomes based on hierarchical iron-based heterojunction nanozymes

[0070] 4.1 Verification of the sensitivity of exosome detection based on hierarchical iron-based heterojunction nanozymes on the chemiluminescence platform

[0071] Figure 5 Verify the sensitivity performance of tumor exosome detection on the chemiluminescence detection platform established in Example 2, as shown in

[0072] A. Detect the relationship between the chemiluminescence signal and the exosome concentration, and set 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 8 , 1×109 , 1×10 10 particle / mL -1 nine exosome concentration gradients, and the results show that in the range of 1×10 2 particle / mL -1 ~1×10 10 particle / mL -1 , the chemiluminescence intensity has a good linear relationship with the logarithm of exosome concentration. The fitted linear equation is y = 167609.469lgC - 57841.409 (R = 0.9889), where y is the chemiluminescence intensity, C is the exosome concentration, and the detection limit is 40.738 particles / mL, successfully verifying the good sensitivity performance in the chemiluminescence platform.

[0072] 4.2 Sensitivity verification of exosome detection based on hierarchical iron-based heterojunction nanozyme on an electrochemical platform

[0073] The sensitivity performance of the electrochemical detection platform built in Example 2 was verified for exosome detection, as shown in Figure 5 Figure B. The relationship between the electrochemical signal and exosome concentration was detected. Seven exosome concentration gradients of 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 particles / mL were set respectively. The results show that in the range of 1×10 2 particle / mL -1 ~1×10 10 particle / mL -1 , the electrochemical signal intensity has a good linear relationship with the logarithm of exosome concentration. The fitted linear equation is y = 2.413lgC + 10.628 (R = 0.9762), where y is the chemiluminescence intensity, C is the exosome concentration, and the detection limit is 1.135×10 3 particles / mL, successfully verifying the good sensitivity performance in the electrochemical platform.

[0074] 4.3 Sensitivity verification of exosome detection based on hierarchical iron-based heterojunction nanozyme on a chemical colorimetric platform

[0075] The sensitivity performance of the chemical colorimetric detection platform built in Example 2 was verified for exosome detection, as shown in Figure 5 Figure C. The relationship between the chemiluminescence signal and exosome concentration was detected. 1×10 5, 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 particles / mL for seven exosome concentration gradients. The results show that in the range of 1×10 5 particles / mL to 1×10 11 particles / mL, the chemochromic signal intensity has a good linear relationship with the logarithm of exosome concentration. The fitted linear equation is y = 0.183lgC - 0.283 (R = 0.9844), where y is the chemiluminescence intensity and C is the exosome concentration. The detection limit is 6.457×10 4 particles / mL, successfully verifying the good sensitivity performance of the chemochromic platform.

[0076] 4.4 Specificity verification of exosome detection based on hierarchical iron-based heterojunction nanozyme

[0077] The specificity performance of the chemiluminescence platform established in Example 2 was verified, as Figure 5 shown in D. Exosomes derived from human hepatocytes (L02), breast cancer cells (MCF-7), melanoma cells (A375), and non-small cell lung cancer (A549) were used as control groups. The responses of L02, MCF-7, and A375 to the constructed sandwich chemiluminescence sensor were negligible, while the detection signal responses of CD63-positive and EGFR-positive exosomes were good. The above results indicate that the developed aptamer sensor has good selectivity and anti-interference ability for CD63-positive and EGFR-positive exosomes.

[0078] Example 5: Detection of clinical specimens using the method for detecting tumor exosomes based on hierarchical iron-based heterojunction nanozyme

[0079] The method for detecting tumor exosomes based on hierarchical iron-based heterojunction nanozyme constructed in Example 2 was used to detect clinical specimens. Ten serum samples from breast cancer patients and ten serum samples from healthy individuals were collected. Exosomes were extracted, and 5 μL of exosomes were added as detection markers to the chemiluminescence detection platform to detect signals and statistically analyze the data. Paired t-test analysis showed that there was a significant difference in the CL signals between exosomes (EVs) derived from lung cancer and the healthy control group (p < 0.001), as Figure 6 shown. Therefore, the robust clinical discrimination ability of the developed exosome detection platform in the above clinical samples was verified.

Claims

1. A method for detecting tumor exosomes based on hierarchical iron-based heterojunction nanozymes, characterized in that, Including: a. Hierarchical iron-based heterojunction nanozyme, which has a structure of bimetallic organic framework (MOF) coated with layered double metal hydroxide (LDH), formed by mixing FeZr-MOF and CuFe-LDH, serving as a signal output source; b. Dual-aptamer sandwich structure for exosome recognition, where the CD63 aptamer apt CD63 and the EGFR aptamer apt EGFR can specifically recognize two protein receptors, CD63 and EGFR, on the surface of exosomes. After annealing the nucleic acid sequences of the dual aptamers, a CD63 aptamer magnetic bead complex is formed through biotin and streptavidin modification, apt EGFR The binding of apt to exosomes is through apt EGFR @HFeH signal tags are assembled on apt CD63 the surface of magnetic beads; c. The dual-aptamer biosensor is constructed in a "sandwich type" structure and is applicable to chemiluminescence platforms based on the luminol / H2O2 system, electrochemical platforms based on the H2O2 system, and colorimetric platforms based on the TMB / H2O2 system for the detection of tumor-derived exosomes.

2. The tumor exosome detection method based on a hierarchical iron-based heterojunction nanozyme according to claim 1, wherein The CD63 aptamer apt CD63 and the EGFR aptamer apt EGFR have nucleotide sequences shown in Table 1.

3. The preparation method of the "sandwich-type" aptamer biosensor according to claim 1, characterized in that, Including the following steps: 1) Heat the water bath to 95 °C and incubate the probe in an environment of 95 °C for 5 minutes; 2) Turn off the constant temperature function of the water bath, and take it out after the temperature slowly drops to room temperature; 3) Store the obtained product at 4 °C.

4. The operation according to any one of claims 1-3, characterized in that, Prepare a reaction buffer solution (PBST buffer), the main components: 10 mM Na2HPO4, 2 mM NaH2PO4, 135 mM NaCl, 4.7 mM KCl, 5 mM Tween 20, pH 7.

4.

5. The preparation method of the bimetallic organic framework-coated layered double metal hydroxide structure according to claim 1, wherein Including the following steps: 1) Prepare ZrCl4, FeCl3·6H2O and H2BDC-NH2 solutions, with the solvent being N,N-dimethylformamide (DMF), stir evenly, carry out a heating reaction in an oil bath, the stirring time is 10 - 20 minutes, the reaction temperature under oil bath conditions is 80 °C - 120 °C, and the reaction duration is 10 - 12 hours; 2) After the reaction, cool the solution to room temperature, centrifuge and separate the product, wash it 3 times with methanol and ethanol respectively, and finally dry it under vacuum to obtain FeZr-MOF; 3) Weigh Cu(NO3)2·3H2O and Fe(NO3)3·9H2O and dissolve them in deionized water, and prepare a NaOH solution; 4) Magnetically stir the mixture of Cu(NO3)2·3H2O and Fe(NO3)3·9H2O at 80 °C - 120 °C, adjust the pH to 5.0 - 8.0, and continuously mix for 5 - 15 minutes. 5) Heat the solution in an oil bath, the heating reaction temperature is 80 °C - 120 °C, and the heating duration is 10 - 12 hours. After the reaction, cool it to room temperature, wash it 3 times with deionized water and ethanol, and dry it under vacuum to obtain CuFe-LDH; 6) Add the vacuum-dried FeZr-MOF and CuFe-LDH powders to deionized water, perform ultrasonic treatment, wait until fully dissolved, oscillate at room temperature, and reserve after fully binding through electrostatic adsorption.

6. The preparation method according to claim 5, wherein Mix ZrCl4, FeCl3·6H2O and H2BDC-NH2 solutions According to the preparation method described in claim 5, it is characterized in that the concentration of the NaOH solution used to adjust the pH is 0.5 - 2.5 mol / L.

7. The preparation method according to claim 5, characterized in that, The mixing ratio of FeZr-MOF and CuFe-LDH powders is 1:100, the ultrasonic treatment duration for dissolution is 10 - 20 minutes, and the oscillation duration at room temperature is 24 - 48 hours.

8. A super-high-sensitivity tumor-derived exosome multimode biosensing system with a "separation-detection" function, which uses the "sandwich-type" structured dual-aptamer biosensor described in claim 1 and / or the method described in claim 5 to prepare a method for detecting tumor exosomes based on a hierarchical iron-based heterojunction nanozyme.

9. The method according to claim 8, wherein: An electrochemical workstation, an electrochemiluminescence analyzer or a multifunctional microplate reader is used.

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