A method for quantitatively detecting extracellular vesicles based on self-interference spectrum reconstruction
By employing a self-interference spectral reconstruction method, combined with a digital microfluidic chip and a self-interference spectral imaging system, the problem of insufficient imaging accuracy of extracellular vesicles was solved, achieving high-precision quantitative detection of single vesicles and detection of surface markers, while simplifying the optical system structure.
Patent Information
- Application Number
- CN202510937345.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2026-02-27
- Estimated Expiration
- 2045-07-08
AI Technical Summary
Existing technologies suffer from insufficient imaging accuracy in quantitative imaging of extracellular vesicles, especially in the quantitative detection of single vesicles, where precise measurement and detection of surface markers cannot be achieved, and the use of complex optical components must be avoided.
The self-interference spectral reconstruction method is used to achieve quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles through digital microfluidic chip and self-interference spectral imaging system. The self-interference spectral imaging system is used for spectral reconstruction and Fourier transform analysis to generate high-contrast reconstructed images and quantitatively calculate vesicle volume.
It achieves high-precision single-vesicle imaging without the need for complex optical sensors, is compatible with surface biomarker detection, significantly improves imaging accuracy and detection efficiency, and avoids interference from fluorescent labels.
Smart Images
Figure CN120702932B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biomedical detection and optical engineering, and particularly relates to a method for quantitatively detecting extracellular vesicles based on self-interference spectrum reconstruction. BACKGROUND
[0002] Among numerous early diagnosis and treatment technologies, liquid biopsy is widely used in clinical practice due to its advantages such as no need for surgery or puncture sampling, small trauma, low risk, etc. The technology realizes the screening, diagnosis and classification of tumors by detecting the content of specific biomarkers in blood samples, etc. It not only has high timeliness and dynamic monitoring capability, but also shows high sensitivity and specificity. In particular, the detection of surface protein markers based on extracellular vesicles has wider clinical applicability and higher detection accuracy because it can provide more comprehensive tumor information.
[0003] However, the current single-vesicle quantitative imaging technology still faces the key technical bottleneck of insufficient imaging accuracy. In view of the precise detection requirement of extracellular vesicles, the existing technology needs to break through in the following aspects: it is necessary to develop a non-fluorescent marker-dependent optical imaging method, which can realize the accurate measurement of vesicle particle size and the parallel detection of surface markers, while ensuring the stability of optical signals and avoiding the use of complex optical elements.
[0004] In view of the above technical gap, it is necessary to develop an innovative detection method with non-marked optical quantitative imaging capability to break through the dual technical bottlenecks of single-vesicle quantitative detection accuracy and imaging accuracy in the existing technology. SUMMARY
[0005] The purpose of the present application is to provide a method for quantitatively detecting extracellular vesicles based on self-interference spectrum reconstruction, which can realize the quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles at the same time, significantly improve the single-vesicle quantitative detection accuracy and imaging accuracy, and provide a new technical solution for efficient detection of extracellular vesicles.
[0006] To achieve the above purpose, the present application provides a method for quantitatively detecting extracellular vesicles based on self-interference spectrum reconstruction, comprising the following steps:
[0007] Step S1, adding a clinical sample into a digital microfluidic chip, and separating sample droplets and droplets with functionalized modified magnetic beads through electrodes.
[0008] Step S2, after mixing the two droplets, adding vesicle binding solution and mixing, and then incubating for 5 minutes.
[0009] Step S3, fixing the magnetic beads by magnetic control, removing the solution, and then adding deionized water to clean the magnetic beads.
[0010] Step S4, after removing the magnetic bead cleaning solution, add the vesicle elution solution and incubate with the magnetic beads for 3 min.
[0011] Step S5, separate the droplet containing extracellular vesicles.
[0012] Step S6, transfer the droplet containing extracellular vesicles above the gold-coated glass slide, remove the droplet after incubation for 30 min.
[0013] Step S7, wash the hybridized gold-coated glass slide with deionized water twice.
[0014] Step S8, detect the number of vesicles and their size distribution using a self-interference spectral imaging system.
[0015] Preferably, in step S1, the digital microfluidic chip is composed of an upper plate, a droplet operation layer, and a lower plate.
[0016] Preferably, the upper plate uses a gold-coated glass slide with an array of immobilized immune probes as a self-interference immunohybridization substrate; the lower plate is integrated with a reservoir electrode, a control electrode, and a connection electrode.
[0017] Preferably, in step S1, sodium alginate is used as a cationic polysaccharide modification material for magnetic beads.
[0018] Preferably, in step S1, the specific steps for functional modification of magnetic beads are as follows:
[0019] Step S101: Surface activation of magnetic beads.
[0020] Step S102: Amino-silane modification of the surface-activated magnetic beads using 3-aminopropyl triethoxysilane, and then remove the solution.
[0021] Step S103: Add EDC-NHS carboxyl activation solution to the MES buffer solution containing dissolved sodium alginate, and mix well.
[0022] Step S104: Add the amino-silane modified magnetic beads to the mixture obtained in step S103, incubate, and then remove the solution after carboxyl activation of the cationic polysaccharide modification.
[0023] Step S105: Add the cationic polysaccharide modified magnetic beads to the EDC-NHS carboxyl activation solution containing dissolved glacial acetic acid solution to block excess amino groups.
[0024] Step S106: Wash 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 is composed of 2 mol / L betaine solution with pH=4.5, and 50 mmol / L ethylenediaminetetraacetic acid and 0.5% acetic acid solution.
[0026] Preferably, in step S4, the vesicle elution solution is 200 mmol / L tris-hydroxymethyl aminomethane hydrochloride buffer solution with pH=8.0.
[0027] Preferably, the time for cleaning with deionized water is 5 min.
[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 sheet, an aperture objective lens, a light splitting prism, a wide-field lens, a mirror, a collimated broadband light source, an imaging lens, and a plane array spectral camera.
[0029] Preferably, in step S8, the specific steps of detecting the number and particle size distribution of the vesicles by using the self-interference spectral imaging system are as follows:
[0030] Step S801: the broadband white light emitted by the collimated broadband light source is reflected by the mirror and collimated by the wide-field lens, then transmitted by the light splitting prism, and focused by the aperture objective lens to irradiate the extracellular vesicles on the hybridized gold-coated glass sheet, so that the interference effect of the extracellular vesicle scattered light and the substrate reflected light is excited to form interference light.
[0031] Step S802: the interference light returns after the aperture objective lens and the light splitting prism, and is focused by the imaging lens to the plane array spectral camera, and the plane array spectral camera collects interference images of different wavelengths.
[0032] Step S803: the computer performs spectral 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 extracellular vesicle quantitative detection method based on self-interference spectral reconstruction has the following beneficial technical effects:
[0035] In view of the limitations of the prior art relying on fluorescent labeling and being unable to realize single-vesicle quantitative detection, the present application proposes a new technology of non-labeled single extracellular vesicle quantitative detection based on self-interference spectral reconstruction, realizes high-contrast imaging of single extracellular vesicle, and can accurately quantify the volume of the vesicle, which has three technical advantages: first, the system structure is simplified without complex nano-structure optical sensor or modulation equipment; second, it is compatible with co-localization detection of surface biomarkers of extracellular vesicles, and has strong functional expansion; third, the white light illumination scheme is adopted, which effectively avoids laser speckle interference and significantly improves the contrast of vesicle scattering images, and finally realizes non-labeled quantitative imaging of weak scattering medium under sub-diffraction limit conditions, which has original innovation in the methodological level. BRIEF DESCRIPTION OF DRAWINGS
[0036] Figure 1 It is a whole flow chart of the extracellular vesicle quantitative detection method based on self-interference spectral reconstruction of the present application;
[0037] Figure 2 It is a structural schematic diagram of a digital microfluidic chip;
[0038] Figure 3 It is a structural schematic diagram of a self-interference spectral imaging system;
[0039] Figure 4 It is a self-interference spectral simulation result diagram of extracellular vesicles on a gold-coated glass slide;
[0040] Figure 5 It is a linear relationship simulation result diagram of the particle size of extracellular vesicles and the intensity of interference terms;
[0041] Figure 6 It is a comparison diagram of the separation effects of the method of the present application and the conventional ultracentrifugation method on extracellular vesicles, wherein, Figure 6 (a) in the figure is an electron micrograph of extracellular vesicles obtained by the method of the present application; Figure 6 (b) in the figure is a particle size distribution result diagram of extracellular vesicles obtained by the method of the present application; Figure 6 (c) in the figure is an electron micrograph of extracellular vesicles obtained by the conventional ultracentrifugation method; Figure 6 (d) in the figure is a particle size distribution result diagram obtained by the conventional ultracentrifugation method.
[0042] Reference signs
[0043] 1, gold-coated glass slide; 2, aperture objective lens; 3, light splitting prism; 4, wide field lens; 5, mirror; 6, collimating 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, liquid storage electrode; 14, control electrode; 15, connecting electrode. DETAILED DESCRIPTION
[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0045] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
[0046] Example 1
[0047] like Figure 1 As shown, this invention provides a method for quantitative detection of extracellular vesicles based on self-interference spectral reconstruction, comprising the following steps:
[0048] Step S1: Add the clinical sample to the digital microfluidic chip and separate a sample droplet and a droplet with a functionalized magnetic bead through electrodes.
[0049] This invention uses sodium alginate as the anionic polysaccharide modification material for magnetic beads. The specific steps for functionalizing and modifying the magnetic beads are as follows:
[0050] Step S101: Surface activation of the magnetic beads.
[0051] Step S102: The surface-activated magnetic beads are modified with aminosilanization using 3-aminopropyltriethoxysilane, and the solution is removed after completion.
[0052] Step S103: Add EDC-NHS carboxyl activation solution to the MES buffer solution containing dissolved sodium alginate, and mix well.
[0053] Step S104: Add the aminosilanized magnetic beads to the mixture obtained in step S103, incubate, and remove the solution after the carboxyl-activated anionic polysaccharide modification is completed.
[0054] Step S105: Add the anionic polysaccharide-modified magnetic beads to the EDC-NHS carboxyl activation solution containing dissolved glacial acetic acid to block excess amino groups.
[0055] Step S106: Wash the sealed magnetic beads with deionized water to complete the functional modification of the magnetic beads.
[0056] The digital microfluidic chip employs electrowetting technology to achieve precise control of sample and reagent microdroplets. Its core structure is a "sandwich" design, consisting of, from top to bottom: an upper electrode 10, a droplet manipulation layer, and a lower electrode 11. Figure 2 As shown:
[0057] The top layer is the upper plate 10, which uses a gold-plated glass fixed with an immune probe array as a self-interference immune hybridization substrate. The substrate is fixed with an antibody modified by a carboxyl group to capture extracellular vesicles carrying specific protein markers on the surface. The non-immune probe fixing area of the upper plate 10 is hydrophobically modified by Teflon material and forms stable electrical connection with the long strip-shaped conductive electrodes 12 on both sides of the lower plate 11 through conductive adhesive tape.
[0058] The middle layer is a droplet operation layer, and the immune probe is suspended above the lower plate 11. The droplet to be tested realizes “probe-liquid” contact through the lower electrode.
[0059] The lower plate of the bottom layer is integrated with three electrodes: a liquid storage electrode 13, a control electrode 14, and a connecting electrode 15. The liquid storage electrode 13 is used for sample and reagent addition, the control electrode 14 is used for droplet movement control, and the connecting electrode 15 connects the liquid storage electrode 13 and the control electrode 14 to the control system, realizing system integration.
[0060] Step S2, after mixing the two droplets, add vesicle binding solution, and after mixing and running around on the chip, incubate for 5 min.
[0061] The vesicle binding solution is composed of 2 mol / L betaine solution with pH = 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 then add deionized water to clean the magnetic beads for 5 min.
[0063] Step S4, retain the magnetic beads, remove the magnetic bead cleaning solution, and then add vesicle eluent to incubate with the magnetic beads for 3 min.
[0064] The vesicle eluent is 200 mmol / L tris-hydroxymethyl aminomethane hydrochloride buffer with pH = 8.0.
[0065] Step S5, separate the droplet containing extracellular vesicles, and then move the retained magnetic beads to the waste pool after adding water.
[0066] Step S6, move the droplet containing extracellular vesicles above the gold-plated glass 1, incubate for 30 min, and then remove the droplet.
[0067] Step S7, clean the hybridized gold-plated glass 1 with deionized water for 2 times, each time for 5 min.
[0068] Step S8, after the chip is dried, use a self-interference spectral imaging system to detect the number of vesicles and their particle size distribution.
[0069] As Figure 3As shown, the self-interference spectral imaging system adopts an inverted fluorescence microscope structure, which is composed of a gold-coated glass sheet 1, an aperture objective lens 2, a beam splitting prism 3, a wide-field lens 4, a mirror 5, a collimated broadband light source 6, an imaging lens 7, and a surface array spectral camera 8.
[0070] In addition, the self-interference spectral imaging system adopts a hyperspectral camera as the surface array spectral camera 8, and is equipped with a 100x air lens and an aperture objective lens 2 with a numerical aperture of 0.9.
[0071] The specific steps are as follows:
[0072] Step S801: The broadband white light emitted by the collimated broadband light source 6 is reflected by the mirror 5, collimated into parallel light by the wide-field lens 4, and then irradiated to the beam splitting prism 3. The parallel light is transmitted by the beam splitting prism 3, continues to propagate along the light path, is focused by the aperture objective lens 2, and irradiates the extracellular vesicles on the hybridized gold-coated glass sheet 1, so as to excite the interference effect of the extracellular vesicle scattered light and the substrate reflected light, generate interference light, and form interference signals carrying detection information.
[0073] Step S802: The interference light returns along the original light path, passes through the aperture objective lens 2 and the beam splitting prism 3 in turn, and is focused by the imaging lens 7 to the surface array spectral camera 8. The surface array spectral camera 8 realizes optical detection of the extracellular vesicles by collecting interference images of different wavelengths.
[0074] The specific content is as follows:
[0075] The interference signal detected by the surface array spectral camera 8 at the position without extracellular vesicles only contains the substrate reflected light, and the corresponding background light intensity I bg The expression of I
[0076]
[0077] Wherein, E i (λ) represents the electric field intensity of the broadband white light incident on the hybridized gold-coated glass sheet; r represents the reflection efficiency; λ represents the illumination light wavelength; E r (λ) represents the electric field intensity of the substrate reflected light.
[0078] And at the position of the fixed extracellular vesicles, the interference signal detected by the surface array spectral camera 8 contains the substrate reflected light and the extracellular vesicle scattered light, and the corresponding total light intensity I det The expression of I
[0079] I det (λ) = |E r (λ)| 2 + |E s (λ)| 2 + 2 |E r (λ) |E s(λ) | cos(Δφ) ;
[0080] wherein E s (λ) represents the scattered electric field intensity of extracellular vesicles; Δφ represents the phase difference between E r (λ) and E s (λ), which is proportional to the number of illumination waves, is expressed as Λ represents a coefficient related to the axial position of extracellular vesicles.
[0081] Step S803: The computer 9 performs spectral reconstruction algorithm processing on the interference images of different wavelengths to generate a reconstructed image. The specific content is as follows:
[0082] After the self-interference phenomenon occurs, the image contrast σ det (λ) is expressed as:
[0083]
[0084] wherein s represents the scattering cross section of extracellular vesicles.
[0085] Through analysis, it can be seen that the self-interference phenomenon increases the interference term Due to the nanoscale size characteristics of extracellular vesicles and their weak scattering cross section results in a very low value, so that is much larger than Therefore, under certain wavelength conditions, the self-interference phenomenon can significantly enhance the image contrast. However, the image obtained by the ordinary broadband illumination microscope is the result of linear superposition of images of different wavelengths. Due to the trigonometric function form of the interference term, the images of extracellular vesicles under different wavelengths have different brightness compared to the background, which further leads to the difficulty in directly observing extracellular vesicles in the superimposed image.
[0086] To solve this problem, the spectral reconstruction algorithm is used to subtract the background light of the interference images of different wavelengths, and then the absolute value is superimposed to generate a reconstructed image with high contrast.
[0087] Let (x, y) represent the two-dimensional coordinates of the image, then the expression of the generated reconstructed image I recons (x, y) is:
[0088] I recons (x, y) = ∑ λ |I det (x, y, λ) - I bg (x, y, λ) |;
[0089] wherein I det(x, y, l) represents the total light intensity detected at coordinates (x, y) and wavelength l; I bg (x, y, l) represents the background light intensity detected at coordinates (x, y) and wavelength l.
[0090] Step S804: The computer 9 performs Fourier transform analysis on the interference signal and quantitatively calculates the volume of the extracellular vesicle. The specific content is as follows:
[0091] The expression of the image contrast σ det is obtained by rearrangement:
[0092]
[0093] Since the size of the extracellular vesicle is smaller than the wavelength of visible light, it can be approximated as a Rayleigh scattering medium according to the relevant literature, and therefore the polarizability a of the extracellular vesicle in air can be expressed as:
[0094]
[0095] where V represents the volume of the extracellular vesicle; n represents the refractive index of the extracellular vesicle, n≈1.37.
[0096] The scattering cross section s of the extracellular vesicle is related to the wavelength l of the illumination light and the polarizability a, and the specific expression is:
[0097]
[0098] where a represents a proportional relationship.
[0099] Therefore, the scattering cross section s of the extracellular vesicle is set as:
[0100]
[0101] where β represents a coefficient related to the optical system.
[0102] Since s 2 is relatively low, it can be approximately ignored in the calculation process, and therefore the expression of the spectral reflectance R(l) is:
[0103]
[0104] where l c represents the center wavelength of the illumination light.
[0105] The spectral reflectance R(l) is decomposed into a direct current component r 2 and a trigonometric function expanded along the wave number dimension
[0106] The value of the interference term amplitude is obtained by Fourier transform.
[0107] Since n, l c , r, E i ( l ) are known or calibrated parameters, the amplitude of the interference term is proportional to the volume of the extracellular vesicle V, and the expression of the volume of the extracellular vesicle V is derived as follows:
[0108]
[0109] where FFT(·) represents the Fourier transform of the spectral reflectance along the wavenumber dimension; and abs(·) represents the modulus of the Fourier transform result.
[0110] The known particle size of the standard silica nanoparticles is used to calibrate the system parameters, and the proportional coefficient k between V and is obtained, and the expression of the volume of the extracellular vesicle V is as follows:
[0111]
[0112] Example Two
[0113] In this embodiment, the spectral interference particle size reconstruction of silica nanoparticles with different particle sizes is carried out. The specific experimental conditions are as follows: at 1 pm above the gold-coated glass, r = 0.5, using an aperture objective 2 with a numerical aperture NA = 0.9, the normalized reflectance spectrum simulation of silica nanoparticles with particle sizes of 50 nm, 75 nm, 90 nm, 105 nm and 120 nm is carried out, and the results are shown in Figure 4 .
[0114] Example Three
[0115] The relative amplitude of the reflectance spectrum of extracellular vesicles with different particle sizes is simulated using the simulation parameter conditions of Example Two, and the simulation results are shown in Figure 5 .
[0116] In the specific experiment, the reflectance spectrum of each pixel of the extracellular vesicle is added, and then Fourier transform is performed, and finally the ratio of the amplitude of the interference term to the direct current component is taken as the vertical axis, that is, the normalized signal.
[0117] The simulation results show that the relative amplitude of the interference term is linearly related to the volume of the extracellular vesicle. In addition, since the amplitude and phase obtained by the Fourier transform are separated from each other, for the focused extracellular vesicle, the amplitude of the interference term is independent of the axial position of the particle, so the calculation result of the volume of the extracellular vesicle will not change due to the change of the axial position, which fundamentally ensures the measurement accuracy of the method of the present application for the volume of the extracellular vesicle.
[0118] Example Four
[0119] In this embodiment, the HeLa cell culture solution was treated by the method of the present application and the conventional ultracentrifugation method respectively, and the separation effects of extracellular vesicles were compared as shown in the following figures. Figure 6
[0120] Among them, Figure 6 (a) in the figure is an electron micrograph of extracellular vesicles separated by the method of the present application; Figure 6 (b) in the figure is a particle size distribution result graph of extracellular vesicles separated by the method of the present application; Figure 6 (c) in the figure is an electron micrograph of extracellular vesicles separated by the conventional ultracentrifugation method; Figure 6 (d) in the figure is a particle size distribution result graph of extracellular vesicles separated by the conventional ultracentrifugation method.
[0121] The experimental results show that the method of the present application can capture and release extracellular vesicles, and the structure of the separated extracellular vesicles is not obviously broken.
[0122] The extracellular vesicles obtained by the two methods have little difference in morphology, but there is a big difference in the number of extracellular vesicles separated per milliliter of sample. The efficiency of separating extracellular vesicles by the method of the present application is about 7-8 times that of the conventional ultracentrifugation method, and the particle size distribution conforms to the experimental results of the ultracentrifugation method.
[0123] The difference in separation efficiency is due to the different separation principles of the two methods: the separation of vesicles by the method of the present application depends on the density of the vesicles, and the smaller the size of the extracellular vesicles, the more difficult the separation. The electrostatic adsorption method used in the method of the present application has smaller steric hindrance due to the nanoscale size of the extracellular vesicles, so the probability of being captured is greater. In summary, the method of the present application has significant advantages compared with the conventional ultracentrifugation method.
[0124] Therefore, by using the above-mentioned one of the extracellular vesicle quantitative detection methods based on self-interference spectrum reconstruction, the interference of fluorescent labeling is effectively avoided, and precise quantitative counting, particle size measurement and surface protein marker detection of extracellular vesicles can be simultaneously realized, which significantly improves the single-vesicle quantitative imaging precision and detection efficiency, and provides a new technical scheme for 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 application but not to limit it, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or replaced by equivalents, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.
Claims
1. A method for quantitative detection of extracellular vesicles based on self-interference spectroscopy reconstruction, characterized in that, The method comprises the following steps: Step S1, adding a clinical sample into a digital microfluidic chip, and separating sample droplets and droplets with functionalized modified magnetic beads through electrodes; The digital microfluidic chip is composed of an upper electrode plate, a droplet operation layer and a lower electrode plate; The upper electrode plate uses a gold-coated glass plate with an array of immune probes fixed thereon as a self-interference immune hybridization substrate; and the lower electrode plate is integrated with a liquid storage electrode, a control electrode and a connection electrode; Step S2, after mixing the two droplets, adding a vesicle binding solution and mixing, and then incubating for 5 minutes; Step S3, fixing the magnetic beads through magnetic control, removing the solution, and then adding deionized water to clean the magnetic beads; Step S4, after removing the magnetic bead cleaning solution, adding a vesicle elution solution and incubating with the magnetic beads for 3 minutes; Step S5, separating the droplets containing extracellular vesicles; Step S6, transferring the droplets containing extracellular vesicles to above the gold-coated glass plate, incubating for 30 minutes, and then removing the droplets; Step S7, cleaning the hybridized gold-coated glass plate twice with deionized water; Step S8, detecting the number and particle size distribution of the vesicles by using a self-interference spectral imaging system; The specific steps of detecting the number and particle size distribution of the vesicles by using the self-interference spectral imaging system are as follows: Step S801: the wideband white light emitted by the collimated wideband light source is reflected by the reflector and collimated by the wide-field lens, then transmitted by the light splitting prism, and focused by the aperture objective lens to irradiate the extracellular vesicles on the hybridized gold-coated glass plate, so that the interference effect of the scattered light of the extracellular vesicles and the reflected light of the substrate is excited to form interference light; Step S802: the interference light returns after the aperture objective lens and the light splitting prism, and is focused to the area array spectral camera by the imaging lens, and the area array spectral camera collects interference images of different wavelengths; Step S803: the computer processes the interference images of different wavelengths by using a spectral reconstruction algorithm 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.
2. The method according to claim 1, wherein, In step S1, sodium alginate is used as a cationic polysaccharide modification material for the magnetic beads.
3. The method according to claim 2, wherein, In step S1, the specific steps of functionalizing and modifying the magnetic beads are as follows: Step S101: surface activation of the magnetic beads; Step S102: amino silanization modification of the surface-activated magnetic beads by using 3-aminopropyl triethoxysilane, and then removing the solution; 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 amino silanization modified magnetic beads to the mixed solution obtained in step S103, incubating, and then removing the solution after completing the carboxyl activation of the cationic polysaccharide modification; Step S105: adding the cationic polysaccharide modified magnetic beads to the EDC-NHS carboxyl activation solution in which glacial acetic acid solution is dissolved, to block the excess amino groups; Step S106: washing the blocked magnetic beads with deionized water to complete the functionalization and modification of the magnetic beads.
4. The method according to claim 1, wherein, In step S2, the vesicle binding solution is composed of 2 mol / L betaine solution with pH=4.5, 50 mmol / L ethylenediaminetetraacetic acid and 0.5% acetic acid solution.
5. The method of claim 1, wherein the method is characterized by, In step S4, the vesicle eluent is 200 mmol / L Tris-HCl buffer solution with pH=8.
0.
6. The method of claim 1, wherein the method is characterized by, The time for cleaning with deionized water was 5 min.
7. The method of claim 1, wherein the method is characterized by, In step S8, the interference spectral imaging system adopts an inverted fluorescence microscope structure, which is composed of a gold-coated glass, an aperture objective lens, a light splitting prism, a wide-field lens, a mirror, a collimated broadband light source, an imaging lens, and a face array spectral camera.