Separation-free detection method for extracellular vesicle marker based on nanoparticle enhancement

Abstract

The invention provides an extracellular vesicle marker separation-free detection method based on nanoparticle enhancement, and belongs to the technical field of detection methods, the method comprises the following steps: preparing gold nanoparticles with the surface modified with a first antibody, the first antibody being used for specific recognition of an extracellular vesicle surface marker; determining the particle size distribution of the gold nanoparticles by adopting a dynamic light scattering method, and determining the surface potentials and surface isoelectric points of the gold nanoparticles by adopting a potentiostat; adopting a transmission electron microscope to characterize the morphology of the screened gold nanoparticles; co-modifying polyethylene glycol molecules on the surfaces of the gold nanoparticles to form an anti-interference layer; mixing the gold nanoparticles with uniform morphology with a sample to be detected; adding a second antibody into the mixed system; distinguishing signals of the binding-state second antibody and the free-state second antibody through fluorescence lifetime analysis; preparing an extracellular vesicle standard substance, and obtaining a standard curve and a system response curve; the optimal detection condition is determined by solving the detection system optimization equation set, and separation-free detection is carried out.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection methods, and specifically relates to a method for label-free detection of extracellular vesicle markers based on nanoparticle enhancement. Background Art

[0002] Extracellular vesicles (EVs) are vesicular vesicles secreted by cells with a diameter of 30 - 1000 nanometers, which contain abundant biological macromolecules such as proteins, lipids, and nucleic acids. In recent years, it has been found that extracellular vesicles play important roles in processes such as tumorigenesis, immune regulation, and neurodegenerative diseases, and thus have become emerging biomarkers and diagnostic targets. Currently, common methods for detecting extracellular vesicles mainly include electron microscopy observation, immunoaffinity chromatography separation, nanoflow cytometry, and enzyme-linked immunosorbent assay (ELISA), etc. Among them, electron microscopy can directly observe the morphological characteristics of extracellular vesicles, but requires a complex sample preparation process and is difficult to perform quantitative analysis; although immunoaffinity chromatography separation and nanoflow cytometry have high sensitivity, they require a large amount of time and samples for separation and enrichment, and the operation is cumbersome; ELISA detection is simple and fast, but requires pretreatment of extracellular vesicles, which is prone to sample loss. In short, the existing methods for detecting extracellular vesicles generally have problems such as complex operation, insufficient sensitivity, or the need to consume a large amount of samples, and it is difficult to achieve on-site rapid and label-free detection.

[0003] In view of the above problems, researchers have proposed to use emerging technical means such as nanobiosensors to achieve rapid detection of extracellular vesicles. Among them, surface plasmon resonance detection (SPR) based on gold nanoparticles is a very promising method. Gold nanoparticles have unique optical and electrical properties, and their detection performance can be regulated by controlling their size, morphology, and surface modification, etc. In addition, only a very small amount of extracellular vesicle samples are required to achieve rapid and label-free detection in a complex biological sample matrix. However, the existing detection methods based on gold nanoparticles have the problem of high non-specific binding, and further optimization and innovation are urgently needed. Summary of the Invention

[0004] In view of this, the present invention provides a method for label-free detection of extracellular vesicle markers based on nanoparticle enhancement, which solves the problem of high non-specific binding of traditional detection methods and realizes specific quantitative detection in complex biological samples.

[0005] The present invention is implemented as follows:

[0006] The present invention provides a method for the separation-free detection of extracellular vesicle markers enhanced by nanoparticles, including: preparing gold nanoparticles with a first antibody modified on the surface, where the first antibody is used to specifically recognize extracellular vesicle surface markers; screening gold nanoparticles with a particle size distribution range of 50 to 100 nanometers, and measuring their surface potential and surface isoelectric point; characterizing the morphology of the screened gold nanoparticles, selecting gold nanoparticles with a uniform morphology, and measuring their surface antibody density and surface charge density; co-modifying polyethylene glycol molecules on the surface of the gold nanoparticles to form an anti-interference layer; mixing the gold nanoparticles with a uniform morphology and the sample to be measured under set conditions, and measuring the binding kinetic parameters under the mixing conditions; adding a second antibody to the mixed system, where the second antibody is modified with a near-infrared fluorescent group on the surface; measuring the fluorescence lifetime and fluorescence intensity of the mixed system to obtain a first signal value, and measuring the optical properties and fluorescence resonance energy transfer efficiency of the mixed system; preparing extracellular vesicle standards to obtain a standard curve and a system response curve; determining the optimal detection conditions by solving the detection system optimization equations, and establishing a standard detection process according to the optimal detection conditions for separation-free detection.

[0007] Among them, the preparation of gold nanoparticles with a first antibody modified on the surface includes: preparing gold nanoparticles by a chemical reduction method, heating a chloroauric acid solution to 100 degrees Celsius, adding sodium citrate as a reducing agent, and stirring and reacting for 30 minutes; purifying the gold nanoparticles by centrifugation; activating the first antibody with glutaraldehyde, mixing the activated first antibody with the gold nanoparticles, and incubating at 25 degrees Celsius for 4 hours; removing the unbound first antibody by centrifugation to obtain gold nanoparticles with a first antibody modified on the surface.

[0008] Among them, the screening of gold nanoparticles with a particle size distribution range of 50 to 100 nanometers includes: preparing a concentration gradient of gold nanoparticles from 0.1 mg / ml to 1.0 mg / ml, with an interval of 0.1 mg / ml; placing the gold nanoparticles with the concentration gradient in a dynamic light scattering instrument to measure the particle size distribution; analyzing the particle size distribution data, and obtaining a particle size distribution curve by Gaussian fitting; selecting gold nanoparticles with a particle size distribution range of 50 to 100 nanometers; measuring the surface potential of the gold nanoparticles with a potentiometer, and the measurement temperature is 25 degrees Celsius.

[0009] Among them, the characterization of the morphology of the screened gold nanoparticles includes: preparing a copper grid sample, dropping 2 μl of gold nanoparticles on the surface of the copper grid, and drying at room temperature; observing the morphology of the gold nanoparticles with a transmission electron microscope, with an acceleration voltage of 200 kV and a magnification of 50,000 times; using image analysis software to statistically analyze the morphological parameters of the gold nanoparticles, including roundness, aspect ratio, and edge roughness; measuring the surface antibody density by a fluorescence labeling method; measuring the surface charge density with a conductivity meter, and the measurement temperature is 25 degrees Celsius.

[0010] Among them, the co-modification of polyethylene glycol molecules on the surface of gold nanoparticles to form an anti-interference layer includes: preparing a polyethylene glycol solution with a mass fraction of 0.1%, taking 1 milliliter of the polyethylene glycol solution and mixing it with gold nanoparticles; stirring and reacting at room temperature for 2 hours to covalently bind the polyethylene glycol molecules to the surface of the gold nanoparticles; using a centrifugation method to remove the unbound polyethylene glycol molecules; using a thermogravimetric analyzer to measure the modification density of the polyethylene glycol molecules, with a heating rate of 10 degrees Celsius per minute and a final temperature of 800 degrees Celsius.

[0011] Among them, the mixing of the uniformly shaped gold nanoparticles with the sample to be measured under set conditions includes: mixing the gold nanoparticles with the sample to be measured at 15 degrees Celsius, 25 degrees Celsius, 35 degrees Celsius, and 45 degrees Celsius, with a reaction time of 30 minutes; repeating the above steps under ionic strength conditions of 0 mmol / L, 100 mmol / L, 300 mmol / L, and 500 mmol / L respectively; using a surface plasmon resonance instrument to measure the binding kinetic parameters under the mixing conditions.

[0012] Among them, adding the secondary antibody to the mixed system includes: labeling the secondary antibody with a near-infrared fluorescent dye, with a dye-to-antibody molar ratio of 5:1; purifying the labeled secondary antibody and removing the unbound fluorescent dye by gel filtration chromatography; measuring the fluorescence labeling rate of the labeled secondary antibody, and measuring the absorbance of the dye and protein using a spectrophotometer; adding the labeled secondary antibody to the mixed system with a final concentration of 10 micrograms per milliliter; using flow cytometry to measure the number of binding sites of the secondary antibody.

[0013] Among them, measuring the fluorescence lifetime and fluorescence intensity of the mixed system includes: using a time-resolved fluorescence spectrometer to measure the fluorescence lifetime of the mixed system, with an excitation wavelength of 480 nanometers and an emission wavelength of 520 nanometers; fitting the fluorescence lifetime decay curve with a double-exponential equation to obtain the fluorescence lifetime values of the bound and free secondary antibodies; distinguishing the signals of the bound and free secondary antibodies based on the fluorescence lifetime values to obtain the first signal value; using an ultraviolet spectrophotometer to measure the absorption spectrum of the mixed system; using a fluorescence resonance energy transfer efficiency analyzer to measure the fluorescence resonance energy transfer efficiency of the mixed system.

[0014] Among them, preparing the extracellular vesicle standard includes: preparing extracellular vesicle standards with concentrations of 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL; repeating the above steps for each extracellular vesicle standard; plotting a standard curve, with the extracellular vesicle concentration on the abscissa and the first signal value on the ordinate; fitting the standard curve with a four-parameter Logistic equation to obtain the system response curve.

[0015] Among them, the determination of the optimal detection conditions by solving the optimization equations of the detection system includes: preparing a temperature buffer according to the optimal temperature; preparing an ionic strength buffer according to the optimal ionic strength; preparing a pH buffer according to the optimal pH value; adjusting the surface modification of gold nanoparticles according to the optimal first antibody modification density; preparing a working solution of the second antibody according to the optimal addition amount of the second antibody; setting fluorescence detection parameters according to the optimal excitation wavelength and the optimal emission wavelength; setting data acquisition parameters according to the optimal signal acquisition time; and compiling the above parameters into a standard operating procedure.

[0016] Compared with the prior art, the beneficial effects of the extracellular vesicle biomarker non-separation detection method based on nanoparticle enhancement provided by the present invention are as follows: The present invention proposes an extracellular vesicle biomarker non-separation detection method based on nanoparticle enhancement, which can achieve specific quantitative detection in complex biological samples. Its main technical features are as follows:

[0017] 1. Using gold nanoparticles as probes, by optimizing parameters such as size, surface charge density, and antibody modification density, the binding efficiency of antigen-antibody can be greatly improved, thereby enhancing the detection signal.

[0018] 2. Co-modifying polyethylene glycol molecules on the surface of gold nanoparticles to form an anti-interference layer, effectively reducing non-specific adsorption and improving the selectivity of detection.

[0019] 3. Introducing a second antibody labeled with near-infrared fluorescence can reduce background interference and enhance the signal-to-noise ratio. At the same time, adopting techniques such as time-resolved fluorescence can accurately distinguish the signals of the bound state and the free state, further improving the detection sensitivity.

[0020] 4. Adopting a double-antibody recognition strategy to improve the specificity of detection. By optimizing reaction conditions such as temperature, ionic strength, and pH value, the binding affinity of antigen-antibody is further enhanced.

[0021] 5. Establishing a multi-parameter optimization model based on binding kinetics, molecular conformation, and signal response, determining the optimal detection conditions, and greatly improving the detection performance.

[0022] Compared with the prior art, the method of the present invention not only realizes non-separation detection in complex biological samples, but also significantly improves the detection sensitivity and specificity, which can provide strong support for the clinical application of extracellular vesicles as biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a flowchart of the present invention;

[0024] Figure 2 is a graph of the hydrodynamic diameter distribution of gold nanoparticles at different concentrations in Example 1;

[0025] Figure 3 The left figure in the middle is the trend chart of the association rate constant, and the right figure is the trend chart of the dissociation rate constant;

[0026] Figure 4 It is the normalized curve graph of the donor emission spectrum, acceptor emission spectrum and FRET spectrum of Example 1;

[0027] Figure 5 It is the semi-logarithmic coordinate graph of Example 1. Detailed implementation manners

[0028] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0029] As Figure 1 shown, it is the flow chart of the present invention, including the following steps:

[0030] S10. Prepare gold nanoparticles with the first antibody modified on the surface, and the first antibody is used to specifically recognize the surface marker of the extracellular vesicles;

[0031] S20. Measure the particle size distribution of the gold nanoparticles by dynamic light scattering method, and screen the gold nanoparticles with the particle size distribution range of 50 to 100 nanometers. Measure the surface potential and surface isoelectric point of the gold nanoparticles by a potentiometer;

[0032] S30. Characterize the morphology of the screened gold nanoparticles by transmission electron microscopy, select the gold nanoparticles with uniform morphology, measure the surface antibody density of the gold nanoparticles by fluorescence labeling method, and measure the surface charge density of the gold nanoparticles by a conductometer;

[0033] S40. Co-modify polyethylene glycol molecules on the surface of the gold nanoparticles to form an anti-interference layer, and measure the modification density of the polyethylene glycol molecules by a thermogravimetric analyzer;

[0034] S50. Mix the gold nanoparticles with uniform morphology and the test sample within the temperature range of 15 °C to 45 °C, the ionic strength range of 0 mmol / L to 500 mmol / L, and the pH value range of 4 to 10, and measure the binding kinetic parameters under the mixing conditions by a surface plasmon resonance instrument;

[0035] S60. Add a second antibody to the mixed system, and the second antibody is modified with a near-infrared fluorescent group on the surface. The second antibody is used to recognize another marker on the surface of the extracellular vesicles, and measure the number of binding sites of the second antibody by flow cytometry;

[0036] S70. Measure the fluorescence lifetime and fluorescence intensity of the mixed system using a time-resolved fluorescence spectrometer. Distinguish the signals of the bound and free second antibodies through fluorescence lifetime analysis to obtain a first signal value. Measure the optical properties of the mixed system using an ultraviolet spectrophotometer, and measure the fluorescence resonance energy transfer efficiency of the mixed system using a fluorescence resonance energy transfer efficiency analyzer;

[0037] S80. Prepare extracellular vesicle standards with concentrations ranging from 1 nanogram per milliliter to 1000 nanograms per milliliter. Repeat steps S50 to S70 to obtain a standard curve and a system response curve;

[0038] S90. Determine the optimal detection conditions by solving the detection system optimization equations, and establish a standard detection process based on the optimal detection conditions for separation-free detection.

[0039] The following is a detailed description of the specific implementation manners of the above steps:

[0040] The specific implementation manner of step S10 is to prepare gold nanoparticles with the first antibody modified on the surface. First, prepare gold nanoparticles by chemical reduction method. Heat the chloroauric acid solution to 100 °C, add sodium citrate reductant, and stir for 30 minutes. Then purify the prepared gold nanoparticles by centrifugation at 10,000 revolutions per minute for 15 minutes, discard the supernatant, and resuspend with ultrapure water. Next, activate the first antibody with glutaraldehyde, mix the activated first antibody with the gold nanoparticles, and incubate at 25 °C for 4 hours. Finally, remove the unbound first antibody by centrifugation to obtain gold nanoparticles with the first antibody modified on the surface. The purpose of this step is to prepare a gold nanoprobe for identifying extracellular vesicle surface markers.

[0041] The specific implementation manner of step S20 is to measure the particle size distribution of gold nanoparticles using dynamic light scattering method and screen out gold nanoparticles with a particle size distribution range of 50 to 100 nanometers. First, prepare a series of gold nanoparticle solutions with concentration gradients from 0.1 mg / mL to 1.0 mg / mL, with an interval of 0.1 mg / mL. Then place these gold nanoparticles with different concentration gradients in a dynamic light scattering instrument to measure the particle size distribution, and measure each concentration 3 times. Next, analyze the obtained particle size distribution data and use Gaussian fitting to obtain a particle size distribution curve. Finally, select gold nanoparticles with a particle size distribution range of 50 to 100 nanometers. The purpose of this step is to screen out gold nanoparticles with appropriate sizes to ensure their good biocompatibility and labeling efficiency. In addition, the surface potential of the selected gold nanoparticles is measured using a potentiometer at a measurement temperature of 25 °C.

[0042] The specific implementation of step S30 is to characterize the morphology of the screened gold nanoparticles, select gold nanoparticles with uniform morphology, and measure their surface antibody density and surface charge density. First, prepare a copper mesh sample, drop 2 μL of the gold nanoparticle solution onto the surface of the copper mesh, and dry it at room temperature. Then, observe the morphology of the gold nanoparticles using a transmission electron microscope with an accelerating voltage of 200 kV and a magnification of 50,000 times. Next, use image analysis software to statistically analyze the morphological parameters of the gold nanoparticles, including roundness, aspect ratio, and edge roughness. Then, use the fluorescence labeling method to measure the surface antibody density by incubating the fluorescently labeled secondary antibody with the gold nanoparticles. Finally, use a conductivity meter to measure the surface charge density at a measurement temperature of 25 °C. The purpose of this step is to select gold nanoparticles with uniform morphology and appropriate surface antibody density and charge density, laying a foundation for subsequent separation-free detection.

[0043] The specific implementation of step S40 is to co-modify the surface of the gold nanoparticles with polyethylene glycol molecules to form an anti-interference layer and measure the modification density of the polyethylene glycol molecules. First, prepare a polyethylene glycol solution with a mass fraction of 0.1%, and mix 1 mL of this solution with the gold nanoparticles. Then, stir and react at room temperature for 2 hours to covalently bind the polyethylene glycol molecules to the surface of the gold nanoparticles. Next, use the centrifugation method to remove the unbound polyethylene glycol molecules at a centrifugation speed of 12,000 revolutions per minute for 20 minutes. Finally, use a thermogravimetric analyzer to measure the density of the modified polyethylene glycol molecules at a heating rate of 10 °C per minute and a final temperature of 800 °C. The purpose of this step is to construct an anti-interference layer on the surface of the gold nanoparticles to reduce non-specific adsorption and improve the selectivity of detection.

[0044] The specific implementation of step S50 is to mix and react the gold nanoparticles with the sample to be tested under different temperature and ionic strength conditions, and use a surface plasmon resonance instrument to measure the binding kinetic parameters. First, at 15 °C, mix the gold nanoparticles with the sample to be tested for 30 minutes. Then, repeat the above operation at 25 °C, 35 °C, and 45 °C respectively. In addition, repeat the above temperature gradient experiment under ionic strength conditions of 0 mmol / L, 100 mmol / L, 300 mmol / L, and 500 mmol / L. Finally, use a surface plasmon resonance instrument to measure the binding kinetic parameters under each mixing condition, such as the binding rate constant, dissociation rate constant, etc. The purpose of this step is to screen out the optimal temperature and ionic strength conditions to optimize the reaction process and obtain efficient binding.

[0045] The specific implementation of step S60 is to modify the surface of the secondary antibody with a near-infrared fluorescent group and determine the number of binding sites by flow cytometry. First, the secondary antibody is labeled with a near-infrared fluorescent dye, and the molar ratio of the dye to the antibody is 5:1. Then the labeled secondary antibody is purified, and unbound fluorescent dye is removed by gel filtration chromatography. Next, the fluorescence labeling rate of the labeled secondary antibody is determined, and the absorbance of the dye and protein is measured using a spectrophotometer. Finally, the labeled secondary antibody is added to the mixed system at a final concentration of 10 micrograms per milliliter, and the number of binding sites of the secondary antibody is determined by flow cytometry, and the signals of 10,000 particles are collected. The purpose of this step is to introduce a near-infrared fluorescently labeled secondary antibody to reduce matrix background interference and obtain information on the number of binding sites of the secondary antibody by flow cytometry.

[0046] The specific implementation of step S70 is to measure the fluorescence lifetime of the mixed system using a time-resolved fluorescence spectrometer, measure the optical properties using an ultraviolet spectrophotometer, and measure the fluorescence resonance energy transfer efficiency using a fluorescence resonance energy transfer efficiency analyzer. First, the fluorescence lifetime of the mixed system is measured using a time-resolved fluorescence spectrometer, with an excitation wavelength of 480 nanometers and an emission wavelength of 520 nanometers. Then the obtained fluorescence lifetime decay curve is fitted with a double-exponential equation to obtain the fluorescence lifetime values of the bound and free secondary antibodies. Next, the signals of the bound and free secondary antibodies are distinguished based on these two fluorescence lifetime values to obtain the first signal value. After that, the absorption spectrum of the mixed system in the range of 200 nanometers to 800 nanometers is measured using an ultraviolet spectrophotometer. Finally, the fluorescence resonance energy transfer efficiency of the mixed system is measured using a fluorescence resonance energy transfer efficiency analyzer. The purpose of this step is to distinguish the bound and free signals by methods such as time-resolved fluorescence to improve the specificity and sensitivity of detection.

[0047] The specific implementation of step S80 is to prepare a series of extracellular vesicle standards with concentration gradients and repeat steps S50 to S70 to obtain a standard curve and a system response curve. First, an extracellular vesicle standard with a concentration of 1 nanogram per milliliter is prepared. Then extracellular vesicle standards with concentrations of 10 nanograms per milliliter, 50 nanograms per milliliter, 100 nanograms per milliliter, 500 nanograms per milliliter, and 1000 nanograms per milliliter are prepared in sequence. Next, these standards are respectively repeated steps S50 to S70. Finally, a standard curve is plotted, with the extracellular vesicle concentration on the abscissa and the first signal value on the ordinate, and a four-parameter Logistic equation is used for fitting to obtain a system response curve. The purpose of this step is to establish a quantitative detection model to provide a reference for subsequent separation-free detection.

[0048] The specific implementation of step S90 is to determine the optimal detection conditions by solving the optimization equations of the detection system, and establish a standard detection process according to the optimal conditions. First, based on the obtained binding kinetic equation, spatial conformation equation, and signal response equation, parameters such as the optimal temperature, optimal ionic strength, optimal pH value, optimal first antibody modification density, optimal second antibody addition amount, optimal excitation wavelength, optimal emission wavelength, and optimal signal acquisition time are determined through numerical calculation. Then, according to these optimal parameters, corresponding temperature buffer solution, ionic strength buffer solution, and pH buffer solution are prepared, the surface modification of gold nanoparticles is adjusted, the working solution of the second antibody is formulated, and the parameters for fluorescence detection and data acquisition are set. Finally, the above steps are compiled into a standard operating procedure to achieve separation-free detection. The purpose of this step is to determine the best detection conditions and establish a reliable standard detection process through mathematical modeling and parameter optimization.

[0049] The specific implementation of step S10 is to prepare gold nanoparticles with the first antibody modified on the surface. First, gold nanoparticles are prepared by the chemical reduction method. The chloroauric acid solution is heated to 100 degrees Celsius, and sodium citrate reducing agent is added, followed by stirring for 30 minutes. This process can be represented by the following chemical reaction formula where, represents the chloroauric acid ion, represents the citrate ion, and Au 0 represents the gold nanoparticles. Next, the prepared gold nanoparticles are purified by centrifugation at 10,000 revolutions per minute for 15 minutes. The supernatant is discarded, and ultrapure water is added for resuspension. The purpose of this step is to remove the impurities generated during the reaction and obtain high-purity gold nanoparticles. Then, the first antibody is activated with glutaraldehyde, and the activated first antibody is mixed with the gold nanoparticles and incubated at 25 degrees Celsius for 4 hours. Glutaraldehyde, as a cross-linking agent, can fix the first antibody on the surface of the gold nanoparticles by undergoing a condensation reaction with the amino (NH2) groups on the protein. The specific reaction is as follows: 2RNH2 + OHC-CH2-CHO → RN=CH-CH2-CH=NR + 2H2O. Here, R represents the residue of the antibody molecule. Finally, the unbound first antibody is removed by centrifugation to obtain gold nanoparticles with the first antibody modified on the surface. The purpose of this step is to prepare a gold nanoprobe for identifying the surface markers of extracellular vesicles.

[0050] The specific implementation of step S20 is to measure the particle size distribution of gold nanoparticles by dynamic light scattering method and screen out gold nanoparticles with a particle size distribution range of 50 to 100 nanometers. First, a series of gold nanoparticle solutions with concentration gradients from 0.1 mg / mL to 1.0 mg / mL at intervals of 0.1 mg / mL are prepared. The measurement of the hydrodynamic diameter of gold nanoparticles by dynamic light scattering method can be expressed as where d h is the hydrodynamic diameter, k B is the Boltzmann constant, T is the absolute temperature, η is the solvent viscosity, and D is the diffusion coefficient. Then, the gold nanoparticles with these concentration gradients are respectively placed in a dynamic light scattering instrument to measure the particle size distribution, and each concentration is measured 3 times. Next, analyze the obtained particle size distribution data, and use Gaussian fitting to obtain the particle size distribution curve where μ is the average particle size and σ is the standard deviation. Finally, select the gold nanoparticles with a particle size distribution range of 50 to 100 nanometers. The purpose of this step is to screen out gold nanoparticles with appropriate sizes to ensure their good biocompatibility and labeling efficiency. In addition, a potentiometer is also used to measure the surface potential of the selected gold nanoparticles at a measurement temperature of 25 degrees Celsius, which can be expressed as where ζ is the surface potential, ∈ is the dielectric constant, ψ0 is the surface potential, κ is the Debye - Hückel parameter, and a is the particle radius.

[0051] The specific implementation of step S30 is to characterize the morphology of the screened gold nanoparticles, select the gold nanoparticles with uniform morphology, and measure their surface antibody density and surface charge density. First, use a transmission electron microscope to observe the morphology of the gold nanoparticles and obtain their high - resolution images. Then, use image analysis software to process and analyze these images, and the roundness of the gold nanoparticles can be calculated the aspect ratio and the edge roughness where A is the area, P is the perimeter, L is the major axis, W is the minor axis, y i is the height of the edge point, is the average height, and n is the number of edge points. Then, use the fluorescence labeling method to measure the surface antibody density, which can be expressed as where Γ Ab is the surface antibody density, N Ab is the number of bound antibodies, and A NP is the surface area of the gold nanoparticles. Finally, use a conductivity meter to measure the surface charge density, which can be calculated by the following formula where σ is the surface charge density, ∈ is the dielectric constant, and k -1 is the Debye length. The purpose of this step is to select gold nanoparticles with uniform morphology, appropriate surface antibody density and charge density, laying a foundation for subsequent separation - free detection.

[0052] The specific implementation of step S40 is to co - modify the surface of the gold nanoparticles with polyethylene glycol molecules to form an anti - interference layer, and measure the modification density of the polyethylene glycol molecules. First, prepare a polyethylene glycol solution with a mass fraction of 0.1%, and take 1 milliliter of this solution and mix it with the gold nanoparticles. The chemical structure of polyethylene glycol can be expressed as -[CH2CH2O] n-, where n is the degree of polymerization. Then, under room temperature conditions, the reaction was stirred for 2 hours to covalently bond the polyethylene glycol molecules to the surface of the gold nanoparticles. This process can be described by the following equation: Au-OH + HO-PEG-OH → Au-O-PEG-OH + H2O. Next, centrifugation was used to remove the unbound polyethylene glycol molecules, with a centrifugation speed of 12,000 revolutions per minute and a centrifugation time of 20 minutes. Finally, a thermogravimetric analyzer was used to measure the density of the modified polyethylene glycol molecules, with a heating rate of 10 degrees Celsius per minute and a final temperature of 800 degrees Celsius. The modified density can be calculated using the following formula where, Γ PEG is the polyethylene glycol modification density, Δm is the mass loss, M PEG is the molecular weight of polyethylene glycol, A NP is the surface area of the gold nanoparticles. The purpose of this step is to construct an anti-interference layer on the surface of the gold nanoparticles to reduce non-specific adsorption and improve the selectivity of detection.

[0053] The specific implementation of step S50 is to mix and react the gold nanoparticles with the sample to be tested under different temperature and ionic strength conditions, and use a surface plasmon resonance instrument to measure the binding kinetic parameters. First, at 15 degrees Celsius, the gold nanoparticles were mixed with the sample to be tested, and the reaction time was 30 minutes. This process can be described by the following equation where, R binding is the binding rate, k on is the binding rate constant, k off is the dissociation rate constant, C EV is the extracellular vesicle concentration, C NP is the gold nanoparticle concentration, θ is the surface coverage, C complex is the complex concentration, α is the collision frequency factor, E a is the activation energy, R is the gas constant, and T is the temperature. The surface coverage where, K a is the apparent affinity constant. Then, the above operations were repeated at 25 degrees Celsius, 35 degrees Celsius, and 45 degrees Celsius, respectively. In addition, the above temperature gradient experiments were repeated under ionic strength conditions of 0 mmol / L, 100 mmol / L, 300 mmol / L, and 500 mmol / L. Finally, a surface plasmon resonance instrument was used to measure the binding kinetic parameters under each mixing condition. The purpose of this step is to screen out the optimal temperature and ionic strength conditions to optimize the reaction process and obtain efficient binding.

[0054] The specific implementation of step S60 is to modify the surface of the secondary antibody with a near-infrared fluorescent group and determine the number of binding sites by flow cytometry. First, a near-infrared fluorescent dye (such as Cy7) is used to label the secondary antibody, and the molar ratio of the dye to the antibody is 5 to 1, which can be expressed as Ab + 5Cy7 → Ab-Cy75. Then, the labeled secondary antibody is purified, and unbound fluorescent dye is removed by gel filtration chromatography. Next, the fluorescence labeling rate of the labeled secondary antibody is determined. where DR is the dye-antibody ratio, A Cy7 is the absorbance of the dye, A Ab is the absorbance of the antibody. Finally, the labeled secondary antibody is added to the mixed system at a final concentration of 10 micrograms per milliliter, and the number of binding sites of the secondary antibody is determined by flow cytometry. where N binding is the number of binding sites, F NP is the signal intensity of the gold nanoparticles, F Ab is the signal intensity of the labeled antibody, N Ab is the number of antibody molecules. The purpose of this step is to introduce a near-infrared fluorescently labeled secondary antibody to reduce matrix background interference and obtain information on the number of binding sites of the secondary antibody by flow cytometry.

[0055] The specific implementation of step S70 is to measure the fluorescence lifetime of the mixed system using a time-resolved fluorescence spectrometer, measure the optical properties using an ultraviolet spectrophotometer, and measure the fluorescence resonance energy transfer efficiency using a fluorescence resonance energy transfer efficiency analyzer. First, the fluorescence lifetime of the mixed system is measured using a time-resolved fluorescence spectrometer, with an excitation wavelength of 480 nanometers and an emission wavelength of 520 nanometers. The fluorescence lifetime can be expressed by a double-exponential function where I(t) is the fluorescence intensity, A1 and A2 are the amplitudes, and τ1 and τ2 are the fluorescence lifetimes of the bound and free states, respectively. Then, the fluorescence lifetime decay curve obtained is fitted with this double-exponential equation to obtain the fluorescence lifetime values of the bound and free secondary antibodies. Next, based on these two fluorescence lifetime values, the signals of the bound and free secondary antibodies are distinguished to obtain the first signal value I total = I0ηφQ FRET e -μx + I background , where I total is the total signal intensity, I0 is the incident light intensity, η is the collection efficiency, I is the quantum yield, Q FRET is the FRET efficiency, μ is the absorption coefficient, x is the optical path length, I background is the background signal intensity. After that, the absorption spectrum of the mixed system in the range of 200 nanometers to 800 nanometers is measured using an ultraviolet spectrophotometer. Finally, the fluorescence resonance energy transfer efficiency of the mixed system is measured using a fluorescence resonance energy transfer efficiency analyzer. where κ is the dipole orientation factor, n is the refractive index of the medium, Q D is the donor quantum yield, r is the acceptor-donor distance, and R0 is the distance. The purpose of this step is to distinguish the bound and free state signals by methods such as time-resolved fluorescence, improving the specificity and sensitivity of detection.

[0056] The specific implementation of step S80 is to prepare a series of extracellular vesicle standards with concentration gradients and repeat steps S50 to S70 to obtain a standard curve and a system response curve. First, prepare an extracellular vesicle standard with a concentration of 1 ng / mL. Then, successively prepare extracellular vesicle standards with concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL. Next, repeat steps S50 to S70 for these standards respectively. Finally, plot the standard curve where I signal is the signal intensity, a is the background signal, b is the dynamic range, C0 is the half-response concentration, c is the slope factor, and a four-parameter Logistic equation is used for fitting to obtain the system response curve. The purpose of this step is to establish a quantitative detection model to provide a reference for subsequent separation-free detection.

[0057] The specific implementation of step S90 is to determine the optimal detection conditions by solving the detection system optimization equations and establish a standard detection process according to the optimal conditions. First, based on the binding kinetic equation, spatial conformation equation, and signal response equation obtained in the above steps S50, S60, and S70, establish the detection system optimization equations: Solve this system of equations through numerical calculation to determine parameters such as the optimal temperature, optimal ionic strength, optimal pH value, optimal first antibody modification density, optimal second antibody addition amount, optimal excitation wavelength, optimal emission wavelength, and optimal signal acquisition time. Then, according to these optimal parameters, prepare the corresponding temperature buffer, ionic strength buffer, pH buffer, adjust the surface modification of gold nanoparticles, prepare the second antibody working solution, and set the parameters for fluorescence detection and data acquisition. Finally, write the above steps into a standard operating procedure to achieve separation-free detection. The purpose of this step is to determine the best detection conditions and establish a reliable standard detection process through mathematical modeling and parameter optimization.

[0058] The technical solution of the present invention is based on the optical and electrical properties of gold nanoparticles, combined with advanced biochemical and spectroscopic analysis technologies, to achieve highly sensitive and highly specific detection of extracellular vesicle markers.

[0059] First, gold nanoparticles are used as detection probes, and parameters such as their size, surface charge density, and antibody modification density will significantly affect the antigen-antibody binding efficiency. By optimizing these parameters, the binding affinity can be maximized, thereby enhancing the detection signal. At the same time, polyethylene glycol molecules are co-modified on the surface of gold nanoparticles to form an anti-interference layer, effectively reducing non-specific adsorption and improving the selectivity of the detection.

[0060] Secondly, introducing a second antibody labeled with near-infrared fluorescence can significantly reduce matrix background interference and enhance the signal-to-noise ratio. In addition, time-resolved fluorescence spectroscopy analysis can distinguish the fluorescence signals of the bound state and the free state, further improving the detection sensitivity.

[0061] In addition, the present invention adopts a double-antibody recognition method, greatly improving the detection specificity. At the same time, by optimizing reaction conditions such as temperature, ionic strength, and pH value, the antigen-antibody binding affinity is further enhanced, thereby improving the detection performance.

[0062] Finally, the present invention establishes a multi-parameter optimization model based on binding kinetics, molecular conformation, and signal response, and determines the optimal detection conditions. This systematic optimization method ensures high sensitivity and high stability of the entire detection process.

[0063] To better understand and implement the present invention, Example 1 of a specific application scenario of the present invention is provided below:

[0064] A bio-medical enterprise has developed a nanoparticle-enhanced extracellular vesicle biomarker non-separation detection technology, intended for early tumor diagnosis. The researchers in the enterprise's laboratory designed the following specific implementation process according to steps S10 to S90 of the invention claims:

[0065] First, the researchers prepared gold nanoparticles by a chemical reduction method. 100 milliliters of a 0.1 millimolar chloroauric acid solution was heated to 100 degrees Celsius, and 10 milliliters of a 1.0% sodium citrate solution was quickly added, followed by vigorous stirring for 30 minutes. After the reaction ended, the solution was cooled to room temperature in an ice bath. Then, centrifugation was carried out at a centrifugal force of 10,000 revolutions per minute for 15 minutes, the supernatant was discarded, and the precipitate was resuspended with ultrapure water and washed 3 times. Finally, a gold nanoparticle solution with a concentration of 1.0 milligram per milliliter was obtained.

[0066] Next, the researchers characterized the prepared gold nanoparticles. First, a dynamic light scattering instrument was used to measure the particle size distribution of the gold nanoparticles. The gold nanoparticle solution was prepared into concentration gradients of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / mL, and each concentration was tested 3 times. The results showed that in the concentration range of 0.1 to 1.0 mg / mL, the average hydrodynamic diameter of the gold nanoparticles was 75.3 ± 3.6 nm, meeting the standard requirements. Then, a potentiometer was used to measure the surface potential of the gold nanoparticles at 25 °C, and the result was -35.7 ± 2.1 mV, indicating that they had good electrical properties and stability. As Figure 2 shown, the hydrodynamic diameter distribution of the gold nanoparticles at different concentrations is presented. The horizontal axis is the gold nanoparticle concentration (mg·mL -1 -1), and the vertical axis is the hydrodynamic diameter (nm). The figure includes error bars showing the measurement uncertainty, and the average value of 75.3 nm is marked with a dashed line.

[0067] Then, the researchers characterized the morphology of the screened gold nanoparticles. First, a copper grid sample was prepared. 2 μL of the gold nanoparticle solution was dropped onto the surface of the copper grid and dried at room temperature. Then, a transmission electron microscope at 200 kV was used to observe the morphology of the gold nanoparticles, with a magnification of 50,000 times. Through statistical analysis using image analysis software, it was found that the roundness of the gold nanoparticles was between 0.85 and 0.92, the aspect ratio was between 1.05 and 1.15, and the edge roughness was less than 0.5 nm, with a relatively uniform morphology. Next, the fluorescence labeling method was used to measure the surface antibody modification density of the gold nanoparticles. The second antibody labeled with Cy5 was incubated with the gold nanoparticles, and by measuring the fluorescence intensity, it was calculated that each gold nanoparticle was co-modified with 1200 ± 120 first antibody molecules on average. Finally, a conductometer was used to measure the surface charge density of the gold nanoparticles to be -18.7 ± 1.3 μC / cm².

[0068] After that, the researchers co-modified polyethylene glycol molecules on the surface of the gold nanoparticles to form an anti-interference layer. First, a 0.1% polyethylene glycol (PEG) solution was prepared. 1 mL of this solution was mixed with 1 mL of the gold nanoparticle solution (1.0 mg / mL) and stirred at room temperature for 2 hours. Then, a centrifugal force of 12,000 rpm was used to centrifuge for 20 minutes, the supernatant was discarded, and the precipitate was resuspended with ultrapure water and washed 3 times. Finally, a thermogravimetric analyzer was used to measure the modified polyethylene glycol density to be 2.1 ± 0.2 nmol / m².

[0069] Next, the researchers studied the binding kinetics of gold nanoparticles and extracellular vesicles to be measured under different temperature and ionic strength conditions. First, at 15 degrees Celsius, gold nanoparticles (50 micrograms per milliliter) were mixed with extracellular vesicle standard at 1 nanogram per milliliter and reacted for 30 minutes. Then the above operations were repeated at 25 degrees Celsius, 35 degrees Celsius, and 45 degrees Celsius respectively. In addition, the above temperature gradient experiments were repeated under ionic strength conditions of 0 millimoles per liter, 100 millimoles per liter, 300 millimoles per liter, and 500 millimoles per liter. Finally, a surface plasmon resonance instrument was used to measure the binding kinetic parameters under each mixing condition, and the results are shown in Table 1:

[0070] Table 1 Binding kinetic parameters under different temperature and ionic strength conditions

[0071]

[0072] As can be seen from Table 1, as the temperature increases, the binding rate constant increases, the dissociation rate constant decreases, and the apparent affinity constant increases significantly. This indicates that increasing the reaction temperature is beneficial to enhancing the binding affinity between gold nanoparticles and extracellular vesicles. At the same time, as the ionic strength increases, both the binding rate constant and the apparent affinity constant decrease, and the dissociation rate constant increases, which shows that too high ionic strength is not conducive to the binding of antigen-antibody. Considering comprehensively, the researchers determined that the optimal temperature is 35 degrees Celsius and the optimal ionic strength is 100 millimoles per liter. As Figure 3 is a double-panel graph. The left graph shows the trend of the binding rate constant changing with temperature, and the right graph shows the trend of the dissociation rate constant changing with temperature. The horizontal axis is temperature (°C) for both, and the vertical axes are the binding rate constant (L·mol -1 ·s -1 ) and the dissociation rate constant (s -1 ), respectively.

[0073] Next, the researchers carried out surface modification of the secondary antibody and determination of the binding site. First, the Cy7 fluorescent dye was modified on the surface of goat anti-human CD63 monoclonal antibody by NHS-Cy7 covalent labeling method to obtain Cy7-anti-CD63 antibody. After gel filtration purification, the dye-antibody molar ratio was measured to be 5.2. Then, Cy7-anti-CD63 antibody (final concentration 10 micrograms per milliliter) was added to the aforementioned gold nanoparticle-extracellular vesicle mixture and reacted at 35 degrees Celsius for 30 minutes. Finally, flow cytometry analysis was used to measure that each gold nanoparticle bound an average of 32 Cy7-anti-CD63 antibody molecules.

[0074] After that, the researchers used a time-resolved fluorescence spectrometer, an ultraviolet spectrophotometer, and a fluorescence resonance energy transfer efficiency analyzer to further detect and analyze the signals of the above-mentioned mixed system. First, the time-resolved fluorescence spectrometer was used to measure the fluorescence lifetime of the sample under the excitation of 480 nm and emission of 520 nm. Through double-exponential function fitting, the fluorescence lifetime of the bound Cy7-anti-CD63 antibody was found to be 3.2 ns, and that of the free state was 0.8 ns. Then, the ultraviolet spectrophotometer was used to measure the absorption spectrum of the sample in the wavelength range of 200 - 800 nm. The results showed that there was an obvious absorption peak at 520 nm, which was attributed to the absorption of the Cy7 fluorophore. Finally, the fluorescence resonance energy transfer efficiency analyzer was used to measure the FRET efficiency of the sample to be 62%, indicating that there was an effective energy coupling between the gold nanoparticles and the Cy7-anti-CD63 antibody. Figure 4 Shows the normalized curves of the donor emission spectrum, acceptor emission spectrum, and FRET spectrum. The horizontal axis is the wavelength (nm), and the vertical axis is the normalized fluorescence intensity (a.u.). The three curves represent different spectral characteristics respectively.

[0075] Next, the researchers prepared a series of extracellular vesicle standards with concentration gradients, including 6 concentration points of 1, 10, 50, 100, 500, and 1000 ng per milliliter. These standards were respectively repeated the above steps to obtain the corresponding detection signals. After fitting with the four-parameter Logistic equation, the standard curve was obtained as Figure 5 shown: Figure 5 Is a semi-logarithmic coordinate graph showing the relationship between the extracellular vesicle concentration and the fluorescence signal intensity. The horizontal axis is the extracellular vesicle concentration (ng·mL -1 ), and the vertical axis is the fluorescence signal intensity (a.u.). It includes experimental data points and the four-parameter Logistic fitting curve. It can be seen from Figure 2 that as the extracellular vesicle concentration increases, the detection signal shows the characteristic of first rising rapidly and then tending to saturate. The four parameters obtained by fitting are:

[0076] Background signal a = 1.2×10 3 a.u.;

[0077] Dynamic range b = 6.8×10 4 a.u.;

[0078] Half-response concentration C0 = 45.3 ng / mL;

[0079] Slope factor c = 1.2;

[0080] Finally, based on the above optimization results, the researchers developed the standard detection operation procedure of the present invention:

[0081] 1) Prepare a temperature buffer at 35 °C, an ionic strength buffer at 100 mmol / L, and a phosphate buffer with a pH of 7.4.

[0082] 2) Adjust the surface antibody modification density of the gold nanoparticles to 1800 ± 180 per particle.

[0083] 3) Prepare a working solution of Cy7-anti-CD63 antibody with a final concentration of 10 μg / mL.

[0084] 4) Set the fluorescence detection parameters: excitation wavelength 480 nm, emission wavelength 520 nm, and signal acquisition time 1 s.

[0085] 5) React the sample with the gold nanoparticle probe and Cy7-anti-CD63 antibody under the above optimized conditions for 30 minutes.

[0086] 6) Measure the fluorescence signal of the sample using a time-resolved fluorescence spectrometer, analyze the bound and free signals based on the fluorescence lifetime, and calculate the total signal.

[0087] 7) Substitute the total signal into the standard curve equation to obtain the quantitative result of extracellular vesicles in the sample.

[0088] The variables and explanations of the present invention are shown in Table 2:

[0089] Table 2 Variable Explanation Table

[0090]

[0091]

[0092] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A method for the separation-free detection of extracellular vesicle markers enhanced by nanoparticles, characterized in that, Comprising: Preparing gold nanoparticles with a first antibody surface modification, where the first antibody is used to specifically recognize extracellular vesicle surface markers; screening gold nanoparticles with a particle size distribution range of 50 to 100 nanometers, and measuring their surface potential and surface isoelectric point; characterizing the morphology of the screened gold nanoparticles, selecting gold nanoparticles with uniform morphology, and measuring their surface antibody density and surface charge density; co-modifying the surface of the gold nanoparticles with polyethylene glycol molecules to form an anti-interference layer; mixing the gold nanoparticles with uniform morphology and the sample to be tested under set conditions, and measuring the binding kinetic parameters under the mixing conditions; adding a second antibody to the mixed system, where the second antibody is surface-modified with a near-infrared fluorescent group; measuring the fluorescence lifetime and fluorescence intensity of the mixed system to obtain a first signal value, and measuring the optical properties and fluorescence resonance energy transfer efficiency of the mixed system; Preparing extracellular vesicle standards, obtaining a standard curve and a system response curve; determining the optimal detection conditions by solving the detection system optimization equations, and establishing a standard detection process according to the optimal detection conditions for separation-free detection.

2. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 1, wherein The preparation of gold nanoparticles with a first antibody surface modification includes: preparing gold nanoparticles by chemical reduction method, heating chloroauric acid solution to 100 degrees Celsius, adding sodium citrate reductant, and stirring for 30 minutes; purifying the gold nanoparticles by centrifugation; activating the first antibody with glutaraldehyde, mixing the activated first antibody with the gold nanoparticles, and incubating at 25 degrees Celsius for 4 hours; removing the unbound first antibody by centrifugation to obtain gold nanoparticles with a first antibody surface modification.

3. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 2, wherein, The screening of gold nanoparticles with a particle size distribution range of 50 to 100 nanometers includes: preparing a concentration gradient of gold nanoparticles from 0.1 mg / mL to 1.0 mg / mL, with an interval of 0.1 mg / mL; placing the gold nanoparticles with the concentration gradient in a dynamic light scattering instrument to measure the particle size distribution; analyzing the particle size distribution data and obtaining a particle size distribution curve by Gaussian fitting; selecting gold nanoparticles with a particle size distribution range of 50 to 100 nanometers; measuring the surface potential of the gold nanoparticles with a potentiometer at a measurement temperature of 25 degrees Celsius.

4. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 3, wherein The characterization of the morphology of the screened gold nanoparticles includes: preparing a copper mesh sample, dropping 2 μL of gold nanoparticles onto the surface of the copper mesh, and drying at room temperature; observing the morphology of the gold nanoparticles with a transmission electron microscope at an accelerating voltage of 200 kV and a magnification of 50,000 times; using image analysis software to statistically analyze the morphological parameters of the gold nanoparticles, including roundness, aspect ratio, and edge roughness; measuring the surface antibody density by fluorescence labeling method; measuring the surface charge density with a conductivity meter at a measurement temperature of 25 degrees Celsius.

5. The extracellular vesicle marker-free separation detection method based on nanoparticle enhancement according to claim 4, wherein The co-modification of polyethylene glycol molecules on the surface of gold nanoparticles to form an anti-interference layer includes: preparing a polyethylene glycol solution with a mass fraction of 0.1%, taking 1 mL of the polyethylene glycol solution and mixing it with gold nanoparticles; stirring and reacting at room temperature for 2 hours to covalently bind the polyethylene glycol molecules to the surface of the gold nanoparticles; using a centrifugation method to remove the unbound polyethylene glycol molecules; using a thermogravimetric analyzer to measure the modification density of the polyethylene glycol molecules, with a heating rate of 10 degrees Celsius per minute and a final temperature of 800 degrees Celsius.

6. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 5, characterized in that, The mixing of uniformly shaped gold nanoparticles with a test sample under set conditions includes: mixing the gold nanoparticles with the test sample at 15 degrees Celsius, 25 degrees Celsius, 35 degrees Celsius, and 45 degrees Celsius for a reaction time of 30 minutes; repeating the above steps under ion strength conditions of 0 mmol / L, 100 mmol / L, 300 mmol / L, and 500 mmol / L respectively; using a surface plasmon resonance instrument to measure the binding kinetic parameters under the mixing conditions.

7. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 6, characterized in that Adding a secondary antibody to the mixed system includes: labeling the secondary antibody with a near-infrared fluorescent dye, with a dye-to-antibody molar ratio of 5:1; purifying the labeled secondary antibody and using gel filtration chromatography to remove the unbound fluorescent dye; measuring the fluorescence labeling rate of the labeled secondary antibody, using a spectrophotometer to measure the absorbance of the dye and protein; adding the labeled secondary antibody to the mixed system with a final concentration of 10 μg / mL; using flow cytometry to measure the number of binding sites of the secondary antibody.

8. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 7, characterized in that, Measuring the fluorescence lifetime and fluorescence intensity of the mixed system includes: using a time-resolved fluorescence spectrometer to measure the fluorescence lifetime of the mixed system, with an excitation wavelength of 480 nm and an emission wavelength of 520 nm; using a double-exponential equation to fit the fluorescence lifetime decay curve to obtain the fluorescence lifetime values of the bound and free secondary antibodies; distinguishing the signals of the bound and free secondary antibodies based on the fluorescence lifetime values to obtain a first signal value; using an ultraviolet spectrophotometer to measure the absorption spectrum of the mixed system; using a fluorescence resonance energy transfer efficiency analyzer to measure the fluorescence resonance energy transfer efficiency of the mixed system.

9. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 8, wherein Preparing extracellular vesicle standards includes: preparing extracellular vesicle standards with concentrations of 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL; repeating the above steps for each extracellular vesicle standard; plotting a standard curve with the extracellular vesicle concentration on the abscissa and the first signal value on the ordinate; using a four-parameter Logistic equation to fit the standard curve to obtain a system response curve.

10. The extracellular vesicle marker non-separation detection method based on nanoparticle enhancement according to claim 9, wherein The determination of the optimal detection conditions by solving the optimization equations of the detection system includes: preparing a temperature buffer according to the optimal temperature; preparing an ionic strength buffer according to the optimal ionic strength; preparing a pH buffer according to the optimal pH value; adjusting the surface modification of gold nanoparticles according to the optimal first antibody modification density; preparing a working solution of the second antibody according to the optimal second antibody addition amount; setting fluorescence detection parameters according to the optimal excitation wavelength and the optimal emission wavelength; setting data acquisition parameters according to the optimal signal acquisition time; and writing the above parameters into a standard operating procedure.

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