Quantitative detection method for extracellular vesicles based on self-interference spectrum reconstruction

Abstract

The invention provides a quantitative detection method for extracellular vesicles based on self-interference spectrum reconstruction, and relates to the crossing field of biomedical detection and optical engineering. The method comprises the following steps: adding a clinical sample into a digital micro-fluidic chip, and separating sample liquid drops and liquid drops with functional modified magnetic beads through an electrode; mixing the two liquid drops, adding a vesicle binding solution, mixing, and incubating; fixing the magnetic beads through magnetic control, removing the solution, and adding deionized water to clean the magnetic beads; after removing the magnetic bead cleaning solution, adding a vesicle eluent and the magnetic beads for incubation; liquid drops containing extracellular vesicles are separated out; transferring the liquid drops containing the extracellular vesicles to the upper part of a gold-plated slide, incubating and then removing the liquid drops; washing the hybridized gold-plated glass slide with deionized water; and detecting the number and particle size distribution of the vesicles by using a self-interference spectral imaging system. According to the invention, quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles can be realized at the same time, and quantitative imaging precision and detection efficiency of single vesicles are improved.

Description

Technical Field

[0001] The present invention relates to the intersecting field of biomedical detection and optical engineering, and in particular to a method for quantitatively detecting extracellular vesicles based on self-interference spectroscopy reconstruction. Background Art

[0002] Among the many early diagnosis and treatment technologies, liquid biopsy has gained widespread clinical application due to its advantages, including the lack of surgical or puncture sampling, minimal invasiveness, and low risk. This technology screens, diagnoses, and classifies tumors by detecting specific biomarkers in samples such as blood. It offers not only high timeliness and dynamic monitoring capabilities, but also high sensitivity and specificity. In particular, detection of surface protein markers based on extracellular vesicles (EVs) offers broader clinical applicability and higher accuracy, as it provides more comprehensive tumor information.

[0003] However, current single-vesicle quantitative imaging techniques still face a key technical bottleneck: insufficient imaging accuracy. To meet the demand for precise detection of extracellular vesicles, existing technologies urgently need breakthroughs in the following areas: developing optical imaging methods that are independent of fluorescence labeling, enabling precise measurement of vesicle size and concurrent detection of surface markers, while ensuring optical signal stability and avoiding the use of complex optical components.

[0004] To address the above-mentioned technological gaps, there is an urgent need to develop an innovative detection method with label-free optical quantitative imaging capabilities to break through the dual technical bottlenecks of existing technologies in single vesicle quantitative detection accuracy and imaging precision. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, which can simultaneously realize the quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles, significantly improve the accuracy of single vesicle quantitative detection and imaging accuracy, and provide a new technical solution for the efficient detection of extracellular vesicles.

[0006] To achieve the above objectives, the present invention provides a method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, comprising the following steps:

[0007] Step S1: Add clinical samples to the digital microfluidic chip, and separate sample droplets and droplets with functionalized modified magnetic beads through electrodes.

[0008] Step S2: After the two droplets are mixed, vesicle binding solution is added, mixed, and incubated for 5 minutes.

[0009] Step S3: Fix the magnetic beads by magnetic control, remove the solution and add deionized water to wash the magnetic beads.

[0010] Step S4: After removing the magnetic bead washing solution, add the vesicle elution solution and incubate the magnetic beads for 3 minutes.

[0011] Step S5: Separate the droplets containing extracellular vesicles.

[0012] Step S6: Transfer the droplet containing extracellular vesicles onto a gold-coated glass slide, incubate for 30 minutes, and then remove the droplet.

[0013] Step S7: Wash the hybridized gold-plated glass slide twice with deionized water.

[0014] Step S8: Detect the number of vesicles and their particle size distribution using a self-interference spectroscopy imaging system.

[0015] Preferably, in step S1, the digital microfluidic chip consists of three parts: an upper electrode plate, a droplet operation layer and a lower electrode plate.

[0016] Preferably, the upper electrode plate uses a gold-plated glass slide fixed with an immune probe array as a self-interference immune hybridization substrate; the lower electrode plate is integrated with a liquid storage electrode, a control electrode and a connection electrode.

[0017] Preferably, in step S1, sodium alginate is used as the anionic polysaccharide modification material of the magnetic beads.

[0018] Preferably, in step S1, the specific steps of functionalizing and modifying the magnetic beads are as follows:

[0019] Step S101: Activate the surface of the magnetic beads.

[0020] Step S102: using 3-aminopropyltriethoxysilane to perform aminosilanization modification on the surface-activated magnetic beads, and then removing the solution.

[0021] Step S103: adding EDC-NHS carboxyl activation solution to the MES buffer solution in which sodium alginate is dissolved, and mixing the mixture evenly.

[0022] Step S104: adding the aminosilanized modified magnetic beads to the mixed solution obtained in step S103, incubating the mixture, and removing the solution after the modification of the carboxyl-activated anionic polysaccharide is completed.

[0023] Step S105: adding the magnetic beads modified with anionic polysaccharides into an EDC-NHS carboxyl activation solution dissolved in glacial acetic acid solution to block excess amino groups.

[0024] Step S106: washing the blocked magnetic beads with deionized water to complete the functional modification of the magnetic beads.

[0025] Preferably, in step S2, the vesicle binding solution consists of a 2 mol / L betaine solution with a pH of 4.5, 50 mmol / L ethylenediaminetetraacetic acid and 0.5% acetic acid solution.

[0026] Preferably, in step S4, the vesicle eluent is a 200 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 8.0.

[0027] Preferably, the deionized water washing time is 5 minutes.

[0028] Preferably, in step S8, the self-interference spectral imaging system adopts an inverted fluorescence microscope structure, which is composed of a gold-coated glass slide, an aperture objective lens, a dichroic prism, a wide-field lens, a reflector, a collimated broadband light source, an imaging lens and a planar array spectral camera.

[0029] Preferably, in step S8, the specific steps of using the self-interference spectroscopy imaging system to detect the number of vesicles and their particle size distribution are as follows:

[0030] Step S801: The broadband white light emitted by the collimated broadband light source is reflected by a reflector and collimated by a wide-field lens, then transmitted through a beam splitter prism and focused by an aperture objective lens onto the extracellular vesicles on the hybridized gold-coated glass slide, thereby stimulating the interference effect between the scattered light of the extracellular vesicles and the reflected light of the substrate, thereby forming interference light.

[0031] Step S802: After the interference light returns through the aperture objective lens and the beam splitter prism, it is focused by the imaging lens to the area array spectral camera, and the area array spectral camera collects interference images of different wavelengths.

[0032] Step S803: The computer performs spectrum reconstruction algorithm processing on the interference images of different wavelengths to generate a reconstructed image.

[0033] Step S804: The computer performs Fourier transform analysis on the interference signal to quantitatively calculate the volume of the extracellular vesicles.

[0034] Therefore, the present invention adopts the above-mentioned method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, and the beneficial technical effects are as follows:

[0035] In response to the limitations of existing technologies that rely on fluorescent labeling and cannot achieve quantitative detection of single vesicles, the present invention proposes a new technology for label-free single extracellular vesicle quantitative detection based on self-interference spectral reconstruction, which achieves high-contrast imaging of single extracellular vesicles and can accurately quantify the vesicle volume. It has three major technical advantages: first, it does not require complex nanostructured optical sensors or modulation equipment, and the system structure is simplified; second, it is compatible with the co-localization detection of surface biomarkers of extracellular vesicles and has strong functional scalability; third, it adopts a white light illumination scheme to effectively avoid laser speckle interference and significantly improve the contrast of vesicle scattering images, ultimately achieving label-free quantitative imaging of weakly scattering media under sub-diffraction limit conditions, which is original and innovative at the methodological level. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an overall flow chart of a method for quantitatively detecting extracellular vesicles based on self-interference spectroscopy reconstruction according to the present invention;

[0037] Figure 2 Schematic diagram of the structure of the digital microfluidic chip;

[0038] Figure 3 Schematic diagram of the structure of the self-interference spectroscopy imaging system;

[0039] Figure 4 This is the simulation result of the self-interference spectrum of extracellular vesicles on a gold-coated glass slide;

[0040] Figure 5 This is the simulation result diagram of the linear relationship between the extracellular vesicle particle size and the interference term intensity;

[0041] Figure 6 This is a comparison chart of the separation effects of the method of the present invention and conventional ultracentrifugation on extracellular vesicles, wherein: Figure 6 (a) is an electron micrograph of the extracellular vesicles isolated and obtained by the method of the present invention; Figure 6 (b) is a graph showing the size distribution of extracellular vesicles isolated by the method of the present invention; Figure 6 (c) is an electron micrograph of extracellular vesicles separated by conventional ultracentrifugation; Figure 6 (d) in the figure is the particle size distribution result obtained by conventional ultracentrifugation separation.

[0042] Reference numerals

[0043] 1. Gold-coated glass slide; 2. Aperture objective lens; 3. Beam splitter prism; 4. Wide-field lens; 5. Reflector; 6. Collimated broadband light source; 7. Imaging lens; 8. Area array spectral camera; 9. Computer; 10. Upper electrode plate; 11. Lower electrode plate; 12. Conductive electrode; 13. Reservoir electrode; 14. Control electrode; 15. Connecting electrode. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0045] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0046] Example 1

[0047] like Figure 1 As shown, the present invention provides a method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, comprising the following steps:

[0048] Step S1: Add a clinical sample to a digital microfluidic chip, and separate a sample droplet and a droplet with functionalized modified magnetic beads through electrodes.

[0049] The present invention uses sodium alginate as the anionic polysaccharide modification material of the magnetic beads, and the specific steps of functionalizing the modified magnetic beads are as follows:

[0050] Step S101: Activate the surface of the magnetic beads.

[0051] Step S102: using 3-aminopropyltriethoxysilane to perform aminosilanization modification on the surface-activated magnetic beads, and then removing the solution.

[0052] Step S103: adding EDC-NHS carboxyl activation solution to the MES buffer solution in which sodium alginate is dissolved, and mixing the mixture evenly.

[0053] Step S104: adding the aminosilanized modified magnetic beads to the mixed solution obtained in step S103, incubating the mixture, and removing the solution after the modification of the carboxyl-activated anionic polysaccharide is completed.

[0054] Step S105: adding the magnetic beads modified with anionic polysaccharides into an EDC-NHS carboxyl activation solution dissolved in glacial acetic acid solution to block excess amino groups.

[0055] Step S106: washing the blocked magnetic beads with deionized water to complete the functional modification of the magnetic beads.

[0056] Among them, the digital microfluidic chip uses electrowetting technology to achieve precise control of sample and reagent droplets. Its core structure is a "sandwich" design, which consists of: an upper electrode plate 10, a droplet operation layer and a lower electrode plate 11 from top to bottom. Figure 2 As shown:

[0057] The top layer is the upper electrode 10, which uses a gold-plated glass slide fixed with an immune probe array as a self-interference immune hybridization substrate. The substrate uses carboxyl-modified fixed antibodies to capture extracellular vesicles carrying specific protein markers on the surface; the non-immune probe fixing area of ​​the upper electrode 10 is hydrophobically modified with Teflon material and forms a stable electrical connection with the long conductive electrodes 12 on both sides of the lower electrode 11 through conductive tape.

[0058] The middle layer is the droplet operation layer, the immune probe is suspended above the lower electrode plate 11, and the droplet to be tested realizes "probe-liquid" contact through the lower electrode.

[0059] The bottom electrode plate of the bottom layer integrates three electrodes: a liquid storage electrode 13, a control electrode 14 and a connecting electrode 15: the liquid storage electrode 13 is used for adding samples and reagents, the control electrode 14 is used to control the movement of droplets, and the connecting electrode 15 connects the liquid storage electrode 13 and the control electrode 14 to the control system to achieve system integration.

[0060] Step S2: After the two droplets are mixed, vesicle binding solution is added, and the mixture is mixed by running in circles on the chip and then incubated for 5 minutes.

[0061] The vesicle binding solution consists of a 2 mol / L betaine solution with a pH of 4.5, 50 mmol / L ethylenediaminetetraacetic acid and 0.5% acetic acid solution.

[0062] Step S3: Fix the magnetic beads by magnetic control, remove the solution and add deionized water to wash the magnetic beads for 5 minutes.

[0063] Step S4: retain the magnetic beads, remove the magnetic bead washing solution, add the vesicle elution solution and incubate the magnetic beads for 3 minutes.

[0064] The vesicle eluent is a 200 mmol / L tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 8.0.

[0065] Step S5: Separate the droplets containing extracellular vesicles, add water to the retained magnetic beads and move them to a waste liquid pool.

[0066] Step S6: Transfer the droplet containing extracellular vesicles onto the gold-coated glass slide 1, incubate for 30 minutes, and then remove the droplet.

[0067] Step S7: Wash the hybridized gold-plated glass slide 1 twice with deionized water, each time for 5 minutes.

[0068] Step S8: After the chip is dried, the number of vesicles and their size distribution are detected using a self-interference spectroscopy imaging system.

[0069] like Figure 3As shown, the self-interference spectroscopy imaging system adopts an inverted fluorescence microscope structure, which consists of a gold-coated glass slide 1, an aperture objective lens 2, a beam splitter prism 3, a wide-field lens 4, a reflector 5, a collimated broadband light source 6, an imaging lens 7 and a planar array spectral camera 8.

[0070] In addition, the self-interference spectral imaging system uses a hyperspectral camera as the area array spectral camera 8 and is equipped with a 100× air mirror and an aperture objective lens 2 with a numerical aperture of 0.9.

[0071] The specific steps are as follows:

[0072] Step S801: Broadband white light emitted by a collimated broadband light source 6 is reflected by a reflector 5, collimated into parallel light by a wide-field lens 4, and directed toward a beam splitter prism 3. After being transmitted by the beam splitter prism 3, the parallel light continues along the optical path, is focused by an aperture objective lens 2, and illuminates the extracellular vesicles on the hybridized gold-coated slide 1. This stimulates interference between the scattered light from the extracellular vesicles and the reflected light from the substrate, generating interference light and forming an interference signal that carries the detection information.

[0073] Step S802: The interference light returns along the original optical path, passes through aperture objective lens 2 and beam splitter prism 3, and is focused by imaging lens 7 onto area array spectral camera 8. Area array spectral camera 8 achieves optical detection of extracellular vesicles by collecting interference images at different wavelengths.

[0074] The specific contents are as follows:

[0075] The interference signal detected by the area array spectral camera 8 at a position without extracellular vesicles only contains the substrate reflected light, and the corresponding background light intensity I bg The expression of (λ) is:

[0076]

[0077] Among them, E i (λ) represents the intensity of the broadband white light electric field incident on the gold-coated glass slide after hybridization; r represents the reflection efficiency; λ represents the wavelength of the illumination light; E r (λ) represents the intensity of the optical electric field reflected from the substrate.

[0078] At the fixed position of the extracellular vesicles, the interference signal detected by the area array spectral camera 8 includes the substrate reflected light and the extracellular vesicle scattered light, and the corresponding total light intensity I det The expression of (λ) is:

[0079] I det (λ)=|E r (λ)| 2 +|E s (λ)| 2 +2|E r (λ)||E s(λ)|cos(Δφ);

[0080] Among them, E s (λ) represents the intensity of the electric field of extracellular vesicle scattered light; Δφ represents E r (λ) and E s The phase difference between the two (λ) is proportional to the illumination wave number and is expressed as Λ represents the coefficient related to the axial position of the extracellular vesicles.

[0081] Step S803: The computer 9 performs spectrum reconstruction algorithm processing on the interference images of different wavelengths to generate a reconstructed image. The specific contents are as follows:

[0082] After the self-interference phenomenon occurs, the image contrast σ det The expression of (λ) is:

[0083]

[0084] where s represents the scattering cross section of the extracellular vesicles.

[0085] Through analysis, we can see that the self-interference phenomenon adds interference terms to the image contrast. Due to the nanoscale size of extracellular vesicles and their weak scattering cross section lead to The value is very low, making Much greater than Therefore, under specific wavelength conditions, self-interference can significantly enhance image contrast. However, images obtained by conventional broadband illumination microscopy are the result of a linear superposition of images at each wavelength. Because the interference term is a trigonometric function, the images of extracellular vesicles at different wavelengths appear different in brightness compared to the background, making it difficult to directly observe extracellular vesicles in the superimposed image.

[0086] To solve this problem, the present invention adopts a spectral reconstruction algorithm to subtract background light from interference images of different wavelengths, and then takes their absolute values ​​and superimposes them to generate a high-contrast reconstructed image.

[0087] Assuming (x, y) represents the two-dimensional coordinates of the image, the reconstructed image I recons The expression for (x,y) is:

[0088] I recons (x,y)=∑ λ |I det (x,y,λ)-I bg (x,y,λ)|;

[0089] Among them, I det(x,y,λ) represents the total light intensity detected at the coordinate (x,y) with a wavelength of λ; I bg (x,y,λ) represents the background light intensity detected at coordinate (x,y) with wavelength λ.

[0090] Step S804: The computer 9 performs Fourier transform analysis on the interference signal to quantitatively calculate the volume of the extracellular vesicles. The specific contents are as follows:

[0091] By the image contrast σ det The expression of (λ) can be sorted out as follows:

[0092]

[0093] Because the size of extracellular vesicles is smaller than the wavelength of visible light, according to relevant literature, they can be approximately regarded as Rayleigh scattering media. Therefore, the polarizability α of extracellular vesicles in air can be expressed as:

[0094]

[0095] Where V represents the volume of the extracellular vesicles; n represents the refractive index of the extracellular vesicles, n≈1.37.

[0096] The scattering cross section s of extracellular vesicles is related to the wavelength λ of the illumination light and the polarizability α. The specific expression is:

[0097]

[0098] Here, ∝ indicates proportionality.

[0099] Therefore, the scattering cross section s of extracellular vesicles is set to:

[0100]

[0101] Where β represents the coefficient related to the optical system.

[0102] Due to s 2 The value is low and can be approximately ignored in the calculation process. The expression of spectral reflectance R(λ) is:

[0103]

[0104] Among them, λ c Indicates the central wavelength of the illumination light.

[0105] The spectral reflectance R(λ) is decomposed into the DC component r 2 and trigonometric functions expanded along the wavenumber dimension

[0106] The interference term amplitude is obtained by Fourier transform The numerical value of .

[0107] Since n, λ c 、r、E i (λ) are all known or calibrable parameters. The amplitude of the interference term is proportional to the extracellular vesicle volume V. The expression of the extracellular vesicle volume V is derived as follows:

[0108]

[0109] Where FFT(·) means Fourier transform of the spectral reflectance along the wavenumber dimension; abs(·) means taking the modulus of the Fourier transform result.

[0110] Standard silica nanoparticles with known particle size are used to calibrate the system parameters to obtain V and The proportional coefficient k between them gives the expression of the extracellular vesicle volume V as follows:

[0111]

[0112] Example 2

[0113] This example uses spectral interference to reconstruct the particle size of silica nanoparticles of different sizes. The specific experimental conditions are: 1 μm above the gold-coated glass slide, r = 0.5, using an aperture objective lens 2 with a numerical aperture NA = 0.9, and performing normalized reflectance spectrum simulation on silica nanoparticles of 50nm, 75nm, 90nm, 105nm, and 120nm, respectively. The results are as follows: Figure 4 shown.

[0114] Example 3

[0115] The simulation parameters of Example 2 were used to simulate the relative amplitude of the reflectance spectra of extracellular vesicles with different particle sizes. The simulation results are as follows: Figure 5 shown.

[0116] In the specific experiment, the reflectance spectra of each pixel of the extracellular vesicles were added together and then Fourier transformed. Finally, the ratio of the interference term amplitude to the DC component was used as the vertical axis, that is, the normalized signal.

[0117] Simulation results show that the relative amplitude of the interference term is linearly related to the extracellular vesicle volume. Furthermore, because the amplitude and phase obtained by Fourier transform are separated, the amplitude of the interference term is independent of the axial position of the particle for focused extracellular vesicles. Therefore, the calculated extracellular vesicle volume does not change with changes in the axial position, fundamentally ensuring the accuracy of the extracellular vesicle volume measurement method of the present invention.

[0118] Example 4

[0119] In this example, the method of the present invention for preparing anionic polysaccharide-modified magnetic beads and the conventional ultracentrifugation method were used to treat HeLa cell culture fluid, and the separation effect of extracellular vesicles was compared. Figure 6 shown.

[0120] in, Figure 6 (a) is an electron micrograph of the extracellular vesicles isolated and obtained by the method of the present invention; Figure 6 (b) is a graph showing the size distribution of extracellular vesicles isolated by the method of the present invention; Figure 6 (c) is an electron micrograph of extracellular vesicles separated by conventional ultracentrifugation; Figure 6 (d) in the figure is the particle size distribution result obtained by conventional ultracentrifugation separation.

[0121] The experimental results show that the method of the present invention can capture and release extracellular vesicles, and the separated extracellular vesicle structure is not obviously broken.

[0122] The extracellular vesicles obtained by the two methods showed little difference in morphology, but the number of extracellular vesicles isolated per milliliter of sample differed significantly. The efficiency of extracellular vesicle isolation using the present method was approximately 7-8 times that of conventional ultracentrifugation, and the particle size distribution was consistent with experimental results using ultracentrifugation.

[0123] The above difference in separation efficiency stems from the different separation principles of the two methods: the method of the present invention relies on the density of the vesicles themselves to separate them, and the smaller the extracellular vesicles, the greater the difficulty of separation. However, the method of the present invention uses electrostatic adsorption, and the nanoscale size of extracellular vesicles reduces steric hindrance, increasing the probability of capture. In summary, the method of the present invention has significant advantages over conventional ultracentrifugation.

[0124] Therefore, the present invention adopts the above-mentioned extracellular vesicle quantitative detection method based on self-interference spectral reconstruction, which effectively avoids the interference of fluorescent labels and can simultaneously realize the precise quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles, significantly improving the single vesicle quantitative imaging accuracy and detection efficiency, and providing a new technical solution for the efficient detection of extracellular vesicles.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, characterized in that: The following steps are involved: Step S1: adding clinical samples to a digital microfluidic chip, and separating sample droplets and droplets with functionalized modified magnetic beads through electrodes; Step S2: After the two droplets are mixed, vesicle binding solution is added and mixed, followed by incubation for 5 minutes; Step S3, fixing the magnetic beads by magnetic control, removing the solution and adding deionized water to wash the magnetic beads; Step S4: After removing the magnetic bead washing solution, add the vesicle eluate and incubate the magnetic beads for 3 minutes; Step S5, separating droplets containing extracellular vesicles; Step S6: Transfer the droplet containing extracellular vesicles onto a gold-coated glass slide, incubate for 30 minutes, and then remove the droplet; Step S7, washing the hybridized gold-coated glass slide twice with deionized water; Step S8: Detect the number of vesicles and their particle size distribution using a self-interference spectroscopy imaging system.

2. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: In step S1, the digital microfluidic chip consists of three parts: an upper electrode plate, a droplet operation layer, and a lower electrode plate.

3. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 2, characterized in that: The upper electrode plate adopts a gold-plated glass slide fixed with an immune probe array as a self-interference immune hybridization substrate; the lower electrode plate is integrated with a liquid storage electrode, a control electrode and a connection electrode.

4. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: In step S1, sodium alginate is used as the anionic polysaccharide modification material of the magnetic beads.

5. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 4, characterized in that: In step S1, the specific steps of functionalizing and modifying the magnetic beads are as follows: Step S101: Activating the surface of magnetic beads; Step S102: using 3-aminopropyltriethoxysilane to perform aminosilanization modification on the surface-activated magnetic beads, and removing the solution after completion; Step S103: adding EDC-NHS carboxyl activation solution to the MES buffer solution in which sodium alginate is dissolved, and mixing uniformly; Step S104: adding the aminosilanized modified magnetic beads to the mixed solution obtained in step S103, incubating the mixture, and removing the solution after the modification of the carboxyl-activated anionic polysaccharide is completed; Step S105: adding the magnetic beads modified with anionic polysaccharides to an EDC-NHS carboxyl activation solution dissolved in glacial acetic acid solution to block excess amino groups; Step S106: washing the blocked magnetic beads with deionized water to complete the functional modification of the magnetic beads.

6. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: In step S2, the vesicle binding solution consists of a 2 mol / L betaine solution with a pH of 4.5, 50 mmol / L ethylenediaminetetraacetic acid and 0.5% acetic acid solution.

7. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: In step S4, the vesicle eluent is a 200 mmol / L tris hydrochloride buffer solution with a pH of 8.

0.

8. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: The cleaning time using deionized water was 5 min.

9. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 1, characterized in that: In step S8, the self-interference spectral imaging system adopts an inverted fluorescence microscope structure, which is composed of a gold-coated glass slide, an aperture objective lens, a beam splitter prism, a wide-field lens, a reflector, a collimated broadband light source, an imaging lens and an area array spectral camera.

10. The method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction according to claim 9, characterized in that: In step S8, the specific steps of using the self-interference spectroscopy imaging system to detect the number of vesicles and their particle size distribution are as follows: Step S801: Broadband white light emitted by a collimated broadband light source is reflected by a reflector and collimated by a wide-field lens, then transmitted through a beam splitter prism and focused by an aperture objective lens onto the extracellular vesicles on the hybridized gold-coated glass slide, thereby stimulating the interference effect between the scattered light of the extracellular vesicles and the reflected light of the substrate, thereby forming interference light; Step S802: After the interference light returns through the aperture objective lens and the beam splitter prism, it is focused by the imaging lens to the area array spectral camera, which collects interference images of different wavelengths; Step S803: The computer performs spectrum reconstruction algorithm processing on the interference images of different wavelengths to generate a reconstructed image; Step S804: The computer performs Fourier transform analysis on the interference signal to quantitatively calculate the volume of the extracellular vesicles.

Images