A method for the analysis of glycans on individual extracellular vesicles in a fluid sample

Abstract

The application belongs to the technical field of biology and specifically relates to a method for analyzing glycans on single extracellular vesicles in a fluid sample. The method uses magnetic nanoparticles to capture and manipulate EVs, based on the specific affinity of lectins for specific glycan structures, uses polydisperse microdroplets to separate the captured EVs, realizes local signal amplification in the droplets through enzymatic reaction, adopts fluorescence microscopy to collect images of the droplets, and thus realizes identification and analysis of the glycan components on single EVs. The preparation process of the microdroplets formed by the oscillating emulsification method of the application makes the entire detection process more simple and fast, does not require any microfluidic chip and special device, and can be completed in a centrifugal tube, and all the microdroplets are generated in parallel and quickly, and the amount of microdroplets can be flexibly expanded. Moreover, compared with the traditional ELISA, the method has high sensitivity, low detection limit and small sample consumption.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for analyzing glycans on single extracellular vesicles in a fluid sample. BACKGROUND

[0002] Glycosylation is an important and widespread modification process in organisms, that is, under the action of glycosyltransferase, a sugar residue structure is transferred to a specific molecule (such as a protein and a lipid molecule) to form a glycan structure. Glycosylation is the most common post-translational modification process on proteins, and affects the physicochemical properties and functions of proteins by participating in processes such as protein-peptide chain polymerization, folding, maturation and transport. Glycans can participate in many physiological and pathological processes such as cell communication, immune regulation, tumor metastasis, and abnormal glycosylation modification is closely related to the occurrence and development of many diseases (such as cancer, leukemia, Alzheimer's disease, etc.). For example, compared with healthy cells, tumor cells have more abundant protein glycan structures on the cell surface, and often express some specific glycan structures (such as Tn antigen, T antigen, sTn antigen), and these antigens are expressed in many types of cancer (breast cancer, lung cancer, prostate cancer, pancreatic cancer, gastrointestinal cancer, ovarian cancer and colorectal cancer). Therefore, the identification and analysis of glycan structures (especially abnormal glycans) can effectively help disease diagnosis, new biomarker discovery, specific therapeutic target development and other aspects, and show good clinical application potential.

[0003] Extracellular vesicles (EVs) are lipid bilayer membrane structure particles generated by cells and released into the extracellular space, and play an important role in many physiological processes, and are widely present in various body fluids (blood, urine, saliva, tears, milk, ascites, etc.). EVs carry a variety of active substances, including abundant glycan structures. The glycan structures on EVs are closely related to their parent cells, and have similar glycan expression profiles as the parent cells, and can reflect the information of the source parent cells. Therefore, EVs are important carriers of glycan active substances. In recent years, EV glycans have attracted more and more attention from researchers in the aspects of revealing the basic biology of EVs, diagnosing diseases (such as cancer), developing biomarkers, real-time monitoring and prognosis of disease treatment effects, etc.

[0004] High-efficiency identification and analysis of EV glycans is the primary prerequisite for developing their application value. However, due to the highly variable structure of glycans and the diversity of modification sites, combined with the small size (sub-micron level) of EV, the variety of EV, and the diversity of components, the in-depth and accurate analysis of EV glycan information still faces great challenges. Current detection methods for EV glycans mainly include mass spectrometry, liquid chromatography, and lectin-specific recognition. These methods often have complex technical processes and high testing costs. More importantly, current research on EV glycans is mostly a population analysis of EV, and the glycan information obtained is comprehensive, only reflecting the average characteristics of the EV population. Due to the influence of cell type, physiological state, external stimulus, and generation method, EV has high heterogeneity. The aforementioned population analysis can only obtain comprehensive EV glycan information, which often leads to the masking of some low-abundance glycans with high application value in specific EV subpopulations.

[0005] Currently, the main analysis methods for EV glycans are liquid chromatography, mass spectrometry, lectin microarray, enzyme-linked immunosorbent assay (ELISA), and the newly developed GMR magnetic analysis method based on giant magnetoresistance effect (CN114845811A Magnetic analysis of extracellular vesicle glycans), magnetic nanomaterial-based glycan analysis method (CN116819058A, A method for analyzing extracellular vesicle glycans in a fluid sample, and an apparatus and application thereof), and nanoparticle-mediated single extracellular vesicle glycan analysis method (CN202311029628, A method for analyzing glycans on a single extracellular vesicle).

[0006] Among the above methods, except for the nanoparticle-mediated single extracellular vesicle glycan analysis method, the other methods can only analyze the whole EV sample, obtain the average signal of the EV population, and cannot provide glycan information on the heterogeneity between individual vesicles. Moreover, these methods also face other shortcomings. For example, liquid chromatography and mass spectrometry require the destruction of EV structure to obtain glycan sugar chain structure, and these two methods have large sample consumption, complex analysis process, time-consuming, and require expensive instruments, professional operation, and data analysis skills. Enzyme-linked immunosorbent assay (ELISA) and lectin array chip are analysis methods based on the specific affinity of lectin and glycan structure. ELISA has low EV sample adsorption efficiency, low sample loading freedom, and high demand (≥50 microliters), and the entire analysis process is time-consuming. Commercial lectin array chips based on lectin-glycan affinity are generally expensive and require specific instruments. The nanoparticle-mediated single extracellular vesicle glycan analysis method (the only method for glycan analysis at the single EV level so far) can analyze glycans at the single EV level, but the signal cannot be amplified, and some low-abundance glycan structures cannot be effectively detected.

[0007] In recent years, researchers have proposed various analysis methods to analyze the molecular characteristics of individual EVs, such as total internal reflection fluorescence TIRF imaging, proximity ligation PCR technology, nanoflow detection, microdroplet-based enzymatic reaction detection, in situ fixation fluorescence detection, and in situ rolling circle amplification. These techniques have their own advantages and disadvantages and are suitable for different analysis scenarios. It should be noted that current analysis of EVs using these techniques is mainly focused on protein and nucleic acid components within EVs, and there is little research on glycan analysis at the individual EV level.

[0008] Among the foregoing methods, microdroplet-based enzymatic reaction detection is an important single-target detection and analysis technology. This technology is a new technology that distributes target objects in a fluid to a large number of independent and parallel micro-reaction units (i.e., microdroplets), so that each reaction unit contains as many target molecules as possible, and then performs signal amplification, detection, and distribution statistics to achieve absolute counting of target molecules. This technology has been successfully applied to the analysis of nucleic acids and proteins in EVs, but there is no report on the analysis of EV glycans. In addition, most current research on the analysis of EVs using microdroplet technology uses microfluidic technology to generate monodisperse microdroplet units, which usually requires high control of microfluidics and special microfluidic chip design and expensive core control devices. At the same time, monodisperse microdroplets are generated by microfluidic emulsification in time sequence, which makes the number of droplets vary only with the running time, and is limited by the microfluidic chip and control equipment in expanding the generation of droplets. Moreover, the loading of EVs in this technology usually uses commercial micrometer-sized large-size microspheres, and the surface area of such microspheres is much larger than that of EVs. In order to ensure that the number of EVs on each microsphere does not exceed one, limited by the Poisson distribution, 90% of the microspheres are invalid microspheres without EVs. Further, the microdroplet partitioning of individual microspheres is completely random, and in order to ensure that the number of microspheres in each microdroplet does not exceed one, usually only less than 10% of the partitions are effective droplets containing one microsphere, and 90% of the droplets are empty droplets without encapsulated microspheres. That is, overall, less than 1% of the microdroplets contain EVs, and more than 99% of the microdroplets are redundant. These redundant empty droplets will cause a large waste of analysis resources in subsequent detection and analysis, reducing the detection throughput and analysis efficiency. In addition, this technology requires a certain level of professional technical skills from the operator, and still has limitations in universality, versatility, and ease of operation. SUMMARY

[0009] To solve the above problems in the prior art, the following improvements are made in the present application:

[0010] The first aspect of the present application aims to provide a method for analyzing individual extracellular vesicle glycans in a fluid sample.

[0011] The second aspect of the present application aims to provide a device for analyzing a single extracellular vesicle glycan in a fluid sample.

[0012] The third aspect of the present application aims to provide an application.

[0013] In order to achieve the above-mentioned purposes of the present application, the technical scheme adopted by the present application is:

[0014] The first aspect of the present application provides a method for analyzing a single extracellular vesicle glycan in a fluid sample, comprising the following steps:

[0015] S1: mixing the EV particle-containing fluid sample with functionalized magnetic nanoparticles to obtain MNP@poly-EV complex;

[0016] S2: blocking the MNP@poly-EV complex, and then mixing with biotin-coupled lectin to obtain enzyme-linked immunosorbent nanocomposite;

[0017] S3: mixing the enzyme-linked immunosorbent nanocomposite with a substrate solution and an oil phase liquid to prepare a polydisperse droplet;

[0018] S4: optical signal detection.

[0019] In some embodiments of the present application, in S1, the mixing time of the EV particle-containing fluid sample and the functionalized magnetic nanoparticles is at least 10 min; preferably 10 min to 40 min.

[0020] In some embodiments of the present application, in S1, the blocking treatment time is at least 15 min; preferably 10 min to 90 min.

[0021] In some embodiments of the present application, in S2, the mixing time of the biotinylated EV and the streptavidin-coupled enzyme molecule is at least 10 min; preferably 10 min to 30 min.

[0022] In some embodiments of the present application, in S1, the mass ratio of EV particles to functionalized magnetic nanoparticles in the EV particle-containing fluid sample is (0.005-0.1):1.

[0023] In some embodiments of the present application, in S2, after the blocking and mixing, the steps of solid-liquid separation and washing are further included.

[0024] In some embodiments of the application, in S2, the blocking reagent includes, but is not limited to, at least one of commercial quick blocking reagent, bovine serum albumin (BSA), skim milk, casein, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP).

[0025] In some embodiments of the application, in S2, the lectin includes Concanavalin A (ConA), Lens culinaris agglutinin (LCA), Pea lectin (PSA), Helix lucorum agglutinin (AAL), Ulex europaeus agglutinin I (UEA-I), Wheat germ agglutinin (WGA), Tomato lectin (LEL), Potato lectin (STL), Datura stramonium lectin (DSL), Succinylated WGA, Grifola frondosa agglutinin II (GSL-II), Erythrocyte agglutinin (PHA-E), Leukocyte agglutinin (PHA-L), Ricinus communis agglutinin I (RCA-I), Erythrina cristagalli lectin (ECL), Jacalin, Peanut agglutinin (PNA), Diffuse bands agglutinin (DBA), Soybean agglutinin (SBA), Vicia villosa lectin (VVL), Grifola frondosa agglutinin I (GSL-I), Sambucus nigra lectin (SNL), all biotin-modified lectins or glycosyl recognition antibodies.

[0026] In some embodiments of the application, in S3, the enzyme is a streptavidin-conjugated enzyme molecule, and theoretically, the enzyme species is not limited, such as β-galactosidase, horseradish peroxidase, etc. Correspondingly, the enzyme substrate selected corresponds to the enzyme used. In the examples of the present application, β-galactosidase is selected, and the substrate is fluorescein di-β-D-galactopyranoside (FDG). If horseradish peroxidase is selected, the substrate can be selected from commercial chemiluminescent substrates or 3,3',5,5'-tetramethylbenzidine.

[0027] In some embodiments of the application, in S3, the method for preparing the polydisperse droplets includes one or more combinations of oscillation, stirring, whipping, impingement, and ultrasonic.

[0028] In some embodiments of the application, in S3, the oil phase liquid includes a surfactant and an oily liquid.

[0029] In some embodiments of the application, in S3, the surfactant includes one or more combinations of Triton X-100, Tween 20, Span 80, EM90, DC5200, 5225C, SF1328, BM-12.

[0030] In some embodiments of the present application, in S3, the oily liquid comprises at least one of fluorinated oil, mineral oil, silicone oil, ester, and liquid alkane.

[0031] In some embodiments of the present application, in S3, the volume ratio of the oily phase liquid to the aqueous phase ingredient is (1-50):1. Preferably, it is (10-20):1.

[0032] In some embodiments of the present application, in S3, the contact time of the oil phase and the aqueous phase is ≥5 seconds, preferably 10-30 seconds; and the reaction time after the enzyme contacts with the substrate is ≥5 minutes, and in the examples of the present application, 30 minutes is used.

[0033] In some embodiments of the present application, in S4, the optical signal detection comprises the steps of image acquisition, processing, and analysis.

[0034] In some embodiments of the present application, in S4, the slide is a commercial cover glass and cover glass with a hydrophilic treated surface, and in theory, its thickness and material are not limited, and the thickness includes but is not limited to 1.0-1.2 millimeters or 0.13-0.22 millimeters; the slide material includes but is not limited to ultra-white glass, quartz, borosilicate glass, etc.

[0035] In some embodiments of the present application, in S4, a fluorescence microscope is used to capture images of the polydisperse droplets in the bright field and fluorescence channels, and the types of the microscope used include but are not limited to wide-field fluorescence microscope, total internal reflection microscope, confocal microscope, etc. The wide-field fluorescence microscope is preferred in the present application.

[0036] In some embodiments of the present application, in S4, the image signal of the target droplet loaded with EV is collected by the fluorescence microscope, and the number of target particles collected in each microscope field of view (FOV) can be freely adjusted. In the examples of the present application, the number of droplets in each FOV is greater than 1000, and the number of droplets analyzed in the analysis process is not limited and can be freely adjusted.

[0037] In some embodiments of the present application, in S4, the processing of the droplet pictures collected by the fluorescence microscope and the derivation of the picture data values are not limited in theory, and the types of the picture processing software include but are not limited to image J / fiji and ZEN Blue 3.5, etc. In the examples of the present application, ZEN Blue 3.5 analysis software is used to perform bright field recognition of the polydisperse droplets and fluorescence value analysis on the collected multi-channel pictures, and the conversion and derivation of the data values obtained in the analysis are performed.

[0038] In some embodiments of the present application, in S1, the functionalized magnetic nanoparticles used are of core-shell structure, and the surface functionalization modification can use, but is not limited to, polydopamine, silicon dioxide, polyethyleneimine, polyacrylic acid, PEG, etc.; and the synthesis and preparation method can use, but is not limited to, hydrothermal method, solvothermal method, coprecipitation method, thermal decomposition method, sol-gel method, microemulsion method, gas phase precipitation method, etc.

[0039] In a second aspect of the present application, a device for analyzing a single extracellular vesicle glycan in a fluid sample is provided.

[0040] A device for analyzing a single extracellular vesicle glycan, comprising:

[0041] Functionalized magnetic nanoparticles for capturing extracellular vesicles in an EV-containing particle fluid sample, obtaining MNP@poly-EV complexes;

[0042] A blocking solution for blocking processing the MNP@poly-EV complexes, obtaining blocked MNP@poly-EV complexes;

[0043] A biotin-modified lectin for affinity labeling the blocked MNP@poly-EV complexes, obtaining biotinylated EVs;

[0044] A streptavidin-coupled enzyme molecule for specific binding with the biotinylated EVs, obtaining enzyme-labeled magnetic complexes;

[0045] An enzyme molecule corresponding substrate for reacting with the enzyme-labeled magnetic complexes;

[0046] An oil phase liquid for preparing polydisperse droplets.

[0047] In some embodiments of the present application, the device for analyzing a single extracellular vesicle glycan in a fluid sample comprises:

[0048] An inlet unit configured to receive an EV-containing particle fluid sample;

[0049] A mixing unit configured with:

[0050] Functionalized magnetic nanoparticles for capturing extracellular vesicles in the EV-containing particle fluid sample, obtaining MNP@poly-EV complexes;

[0051] A blocking solution for blocking processing the MNP@poly-EV complexes, obtaining blocked MNP@poly-EV complexes;

[0052] biotin-modified lectin for affinity labeling the closed MNP@poly-EV complex to obtain biotinylated EVs;

[0053] streptavidin-conjugated enzyme molecule for specific binding to the biotinylated EVs to obtain enzyme-labeled magnetic complex;

[0054] substrate for the enzyme molecule for reaction with the enzyme-labeled magnetic complex;

[0055] oil phase liquid for preparing polydisperse droplets;

[0056] magnetic separation unit configured to sequentially magnetically separate the MNP@poly-EV complex, the biotinylated EVs, and the enzyme-labeled magnetic complex;

[0057] detection unit configured to measure the fluorescence signal of the polydisperse droplets.

[0058] In some embodiments of the present application, the inlet unit, the mixing unit, and the magnetic separation unit are the same unit.

[0059] In some embodiments of the present application, the mixing unit is fluidically coupled to the inlet unit; and the magnetic separation unit is fluidically coupled to the mixing unit.

[0060] In some embodiments of the present application, the EV-containing particle-containing fluid sample includes an EV-containing particle-containing body fluid, tissue, or cell culture supernatant; preferably, the body fluid includes, but is not limited to, one or more of blood, saliva, urine, cerebrospinal fluid, peritoneal effusion, pleural effusion, sweat, semen, lymph; and the body fluid source includes, but is not limited to, human, monkey, mouse, rat, rabbit, pig, monkey, dog, and the like.

[0061] In some embodiments of the present application, the EV-containing particle-containing fluid sample includes a cell secretion or is obtained by at least one ultrasonic fragmentation including cell fragments, living cells.

[0062] In some embodiments of the present application, the EV-containing particle-containing fluid sample includes a solution containing EV particles obtained by differential centrifugal freezing, density gradient centrifugation, polymer-based precipitation, ultrafiltration, size exclusion chromatography, immunoseparation, and the like; preferably, the EV-containing particle-containing fluid sample includes a solution containing EV particles obtained by differential centrifugal freezing, the centrifugal force of the differential centrifugal freezing is 2000g-200000g, and the time is 30min-200min; preferably, the centrifugal force of the differential centrifugal freezing is 15000g-200000g, and the time is 30min-150min.

[0063] In some embodiments of the present application, the device for analyzing the extracellular vesicle glycan of the fluid sample comprises a detection kit, a magnetic separation system, a detection system, etc.

[0064] In a third aspect of the present application, the use of the method of the first aspect of the present application and / or the device of the second aspect of the present application in analyzing a single extracellular vesicle glycan is provided.

[0065] The present application has the following beneficial effects:

[0066] The present application first proposes an analysis strategy for simplifying and rapidly detecting a single EV glycan based on polydisperse microdroplets.

[0067] The present application overcomes the limitation of traditional EV glycan analysis methods that can only perform overall analysis of EV glycans, and can identify and analyze the glycan components from the level of a single EV, providing glycan information of the heterogeneity between individual vesicles in the EV sample, while having higher sensitivity and lower detection limit. The entire testing process does not require special experimental instruments, devices, reagents, does not involve any microfluidic chip and microfluidic equipment, is simple, convenient and fast, has low detection cost and good economy.

[0068] The method of the present application uses nanoscale magnetic particles as a medium for manipulating EVs, making the capture and glycan labeling process of EVs convenient to operate. More importantly, the present application uses nanoscale magnetic particles to replace the micrometer-sized microbeads used in conventional microdroplet detection methods, and uses the excellent monodispersity of nanoscale particles in solution to help achieve the effect of directly dispersing a single EV particle into a single droplet, eliminating the limitations of magnetic beads in microdroplet detection methods, and improving the utilization efficiency of microdroplets.

[0069] The entire detection process of the present application can be completed on a centrifuge tube and a commercial glass slide, and the preparation of multiple droplets can be achieved by simple shaking, generating all droplets in parallel without the need for microfluidic chips and devices. The signal acquisition of the method of the present application uses a conventional fluorescence microscope, without the need for high-end and expensive microscopic imaging devices such as a super-resolution microscope and a total internal reflection microscope. The method of the present application has good repeatability, low sample requirement, and the sample operation volume can be freely adjusted. The number of target particles collected in each microscope image field is greater than 1000, and the number of image collections is also unlimited. The detection limit of the method of the present application is significantly lower than that of the overall analysis method such as the conventional ELISA method, and the detection performance of EV glycans is more sensitive. Moreover, the EV glycan information obtained by the method of the present application is more specific and refined, and can fully reflect the heterogeneity of the glycan characteristics between different EV particles. BRIEF DESCRIPTION OF DRAWINGS

[0070] The present application will be further described below in conjunction with the drawings and examples, in which:

[0071] Figure 1 For single EV glycan analysis based on polydisperse droplets.

[0072] Figure 2 For characterization of functionalized magnetic nanoparticles (MNP@PDA): (A) is the particle concentration and size distribution; (B) is the SEM image of MNP@PDA particles.

[0073] Figure 3 (A) is the TEM morphology of EV particles; (B) is the NTA concentration and size distribution of EV particles.

[0074] Figure 4 For polydisperse droplets prepared in Example 3 (A) and their droplet diameter distribution (B).

[0075] Figure 5 For the capture of EVs by functionalized magnetic nanoparticles and the dispersion results of EVs in droplets.

[0076] Figure 6 For the detection and analysis of single EV glycan in Example 5: (A) fluorescence microscope images of polydisperse droplets without EVs and loaded with single EVs (Bright bright field image, AF488 fluorescence image and Merge superimposed image); (B) single EV glycan fluorescence intensity pile-up distribution and (C) statistical distribution; the lectin used in this figure is SNL.

[0077] Figure 7 For representative polydisperse droplet fluorescence images of different concentrations of EVs, the concentrations of EVs are 0, 1 x 10 8 , 1 x 10 9 , 1 x 10 10 particles / mL, respectively.

[0078] Figure 8 For the correlation between the single EV glycan detection method based on polydisperse droplets and the ELISA method; seven lectins, ConA, DBA, GSL-I, PHA-L, sWGA, DSL, ECL, etc., were used in this figure.

[0079] Figure 9 For the EV glycan information collected by the single EV glycan detection method based on polydisperse droplets (the method of the present application) and the ELISA method; each point of the data obtained by the method of the present application in the figure represents the fluorescence intensity of one droplet, and a total of 22 different lectins were used. DETAILED DESCRIPTION

[0080] The concept and technical effects of the present application will be described clearly and completely in combination with the embodiments below, so as to fully understand the purposes, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0081] The overall analysis process of the present application is shown in Figure 1 The technical concept is as follows:

[0082] Step one: EV immobilization. Functionalized magnetic nanoparticles (MNP@PDA) are mixed with EV solution to capture EVs in the solution, obtaining magnetic nanoparticles loaded with EVs (denoted as MNP@PDA-EV).

[0083] Step two: preparation of enzyme-linked immunosorbent nanocomposites. First, the MNP@PDA-EV is subjected to surface blocking treatment. The biotin-coupled lectin is used to specifically bind to the surface glycan of the EV in the nanocomposite, and then the streptavidin-coupled enzyme molecule is combined with the lectin, thereby obtaining the enzyme-linked immunosorbent nanocomposite. During the entire preparation process, all processes involving the separation of solid phase (magnetic nanoparticles) from liquid phase are carried out by magnetic separation with the aid of a magnet.

[0084] Step three: preparation of polydisperse droplets. After uniformly dispersing the enzyme-linked immunosorbent nanocomposite prepared in the foregoing in the substrate solution, it is rapidly mixed with the oil phase and fully oscillated until the solution turns milky white, thereby obtaining polydisperse droplets encapsulating the nanocomposite and the substrate solution.

[0085] Step four: droplet imaging and image acquisition. A certain volume of polydisperse droplets prepared in step three is sucked into a sample cell, so that the droplets are dispersed in a single layer in the sample cell. Under light shielding conditions, the enzyme molecules in the enzyme-linked immunosorbent nanocomposite catalyze the substrate to produce a fluorescent signal. The droplets in the sample cell are imaged by a fluorescence microscope. Image processing and data analysis. After quality screening and processing of the collected images, the related data of the droplets in the images are extracted and collected, and are subjected to statistics and analysis.

[0086] Example 1: Preparation of functionalized magnetic nanoparticles (MNP@PDA)

[0087] Firstly, the magnetic core was synthesized by solvothermal method. 1.6 grams of ferric chloride, 3 grams of sodium acetate, 0.6 grams of sodium citrate, 1 milliliter of water were dissolved in 50 milliliters of ethylene glycol, mixed thoroughly, and then reacted at 180°C for 12 hours to obtain magnetic nanoparticles, denoted as MNP. Then the surface of MNP was coated with polydopamine to realize functionalization. That is, 3 milligrams of MNP particles were dispersed in 100 milliliters of water under the assistance of ultrasonic dispersion, and then 100 milligrams of polydopamine and 1 milliliter of 28% ammonia water were added in turn. After mixing thoroughly, the mixture was left to react at room temperature for 4 hours, and finally the functionalized magnetic nanoparticles with core-shell structure were obtained, denoted as MNP@PDA.

[0088] The particle concentration and particle size distribution of MNP@PDA were characterized by nanoparticle tracking analysis (NTA), as shown in FIGS. 1A and 1B. Figure 2 As shown in FIG. 1A, the average particle size of MNP@PDA particles was about 150 nanometers. The morphology of MNP@PDA was characterized by scanning electron microscopy (SEM), as shown in FIG. 1B. Figure 2 As can be seen from FIG. 1B, MNP@PDA has a relatively smooth surface, and the size has good matching with the NTA test.

[0089] Example 2 Separation and purification of EV

[0090] The continuous differential centrifugation method was used to separate and purify EV. For the A549 cell culture medium solution containing EV particles, first centrifuged at 2500g for 1 hour, then centrifuged the supernatant at 20000g for 1 hour, and then further centrifuged the supernatant at 167000g for 2 hours. Finally, the extracellular vesicle precipitate at the bottom of the centrifuge tube was collected and denoted as EV, which was used for subsequent glycan analysis. Wherein, g represents the acceleration of gravity.

[0091] The morphology of the aforementioned collected EV sample was characterized by transmission electron microscopy (TEM), as shown in FIG. 2A. Figure 3 As can be seen from FIG. 2A, it has a typical vesicle structure. The particle concentration and particle size distribution of the EV sample were characterized by nanoparticle tracking analysis, as shown in FIGS. 2B and 2C. Figure 3 As shown in FIG. 2B, the average size of EV particles was about 130 nanometers.

[0092] Example 3 Immobilization of EV and identification and labeling of the surface glycan of EV

[0093] 5 microliters of MNP@PDA particle mother liquor (1 milligram / milliliter) and 50 microliters of PBS solution were taken in a 0.6 milliliter centrifuge tube. After washing and magnetic separation of the nanoparticles, the washing solution was removed. 50 microliters of EV sample was incubated with MNP@PDA particles for 30 minutes to allow MNP@PDA particles to fully capture EV in the solution, and MNP@PDA-EV complex was obtained.

[0094] After magnetic separation of the complex, the liquid phase was removed, and 100 μΐ of a 1% (mass fraction) bovine serum albumin solution was added, and the MNP was subjected to surface blocking treatment at room temperature for 1 h. After the blocking solution was removed, 50 μΐ of a 5 μg / mi biotin-modified SNL solution was added, and the mixture was incubated at room temperature for 30 min to ensure effective recognition of the sialic acid glycan on the EV by the SNL. After magnetic separation, 50 μΐ of a 4 μg / mi streptavidin-conjugated β-galactosidase solution was added to the particles, and the mixture was incubated for 30 min, so that the enzyme molecules were combined with the SNL based on the biotin-streptavidin affinity, and then the EV glycan was labeled with the enzyme. After the excess solution was removed by magnetic separation, the particles were washed with 100 μΐ of PBS in a centrifuge tube, and the washing was repeated three times. The washed particles were redispersed in 30 μΐ of a PBS solution and transferred to a 1.5-ml centrifuge tube for standby.

[0095] Operation time: (1) The EV capture time of the MNP@PDA particles in step one was ≥10 min, and 30 min was used in this example; (2) The surface blocking treatment time of the MNP@PDA-EV particles in step two was ≥15 min, and 60 min was used in this example; (3) The combination time of the biotin-modified lectin and the EV glycan structure in step two was ≥10 min, and 30 min was used in this example; (4) The combination time of the streptavidin-conjugated enzyme molecules and the lectin in step two was ≥10 min, and 30 min was used in this example.

[0096] This example further tested 22 kinds of lectins, including concanavalin A (ConA), lens culinaris agglutinin (LCA), pea agglutinin (PSA), Helicoglosum oryzae agglutinin (AAL), Ulex europaeus agglutinin I (UEA-I), wheat germ agglutinin (WGA), tomato agglutinin (LEL), potato agglutinin (STL), datura stramonium agglutinin (DSL), succinylated wheat germ agglutinin (Succinylated WGA), Grifola frondosa agglutinin II (GSL-II), erythrocyte agglutinin (PHA-E), leukocyte agglutinin (PHA-L), ricinus communis agglutinin I (RCA-I), erythrina coralliodendron agglutinin (ECL), jacalin, peanut agglutinin (PNA), double flower mung bean agglutinin (DBA), soybean agglutinin (SBA), velvet vetch agglutinin (VVL), Grifola frondosa agglutinin I (GSL-I), and Sambucus nigra agglutinin (SNL).

[0097] Example 4 Preparation of polydisperse droplets

[0098] The water phase was removed from the particle suspension in the 1.5 ml centrifuge tube after magnetic separation, 20 μl of substrate solution (fluorescein di-β-D-galactopyranoside with a concentration of 50 μmol / ml) was added, and the magnetic nanoparticle complex was mixed with the substrate solution. 200 μl of oil phase (a mixture of mineral oil, 0.1% (mass fraction) Triton X-100 and 3% (mass fraction) EM90) was added into the centrifuge tube immediately, the centrifuge tube was shaken for 20 seconds to emulsify the oil and water phases in the centrifuge tube to form water-in-oil polydisperse droplets. The reaction time of the enzyme and the substrate was recorded from the mixing of the magnetic nanoparticle complex and the substrate solution.

[0099] The operation time in this step: the contact time of the oil phase and the water phase ≥5 seconds, and 10-30 seconds are preferred in this example; the reaction time of the enzyme and the substrate after contact ≥5 minutes, and 30 minutes are used in this example.

[0100] Example 5: Imaging of the droplets, picture collection and data analysis

[0101] 5 μl of the prepared polydisperse droplets were taken into a sample cell with a length of 25 mm, a width of 10 mm and a depth of 20 μm, and a cover glass was gently placed on the liquid surface to seal the sample cell, and the droplets were dispersed into a single layer in the sample cell by capillary action. The bottom carrier of the sample cell was a standard glass slide with a thickness of 1.0±0.2 mm, and the top was an ultrawhite glass cover glass with a thickness of 0.17±0.01 mm. The sample cell was placed in the dark, and the image collection of the droplets in the sample cell was performed using a wide-field fluorescence microscope when the contact reaction time of the enzyme and the substrate reached 30 minutes. A 10x objective was used to identify and position the droplets in the bright field, and the fluorescence channel image of the droplets was collected at the same time. 300-500 pictures with a size of 35 mm×20 mm were collected for each sample.

[0102] The commercially available standard glass slide with a thickness of 1.0±0.2 mm and the commercially available ultrawhite glass cover glass with a thickness of 0.17±0.01 mm were used in this example. The depth of the sample cell was 20 μm.

[0103] The collected pictures were quickly screened for quality, and after the high-quality images were confirmed, the data extraction stage was entered, the collected images were processed using Zen Blue 3.5, the droplets in the images were identified, and the data related to the droplets (such as the number of droplets, size parameters, fluorescence intensity values, etc.) were automatically analyzed and extracted, and a csv file was generated for subsequent data analysis and processing.

[0104] The results are as follows:

[0105] Figure 4 In the above table, A is the bright field image of the polydisperse droplets prepared by the method of the present application, and the distribution of the diameters of the droplets is counted. Figure 4As shown in Figure B, the diameter of the obtained microdroplets is continuously distributed within the range of 1 to 54 micrometers, which conforms to the characteristics of polydisperse droplets. Among them, droplets with a diameter of 2 to 25 micrometers account for 98.8% of the total number of droplets. In this embodiment, microdroplets with a diameter of 5 to 20 micrometers are preferred for subsequent analysis of glycan characteristic information.

[0106] The nanoparticles that captured EVs were characterized using scanning electron microscopy (SEM). Figure 5 As shown in diagram A, the nanoparticles (black arrow) successfully captured EVs (red arrow). In the method of this invention, the nanoparticles capturing EVs are uniformly dispersed within a polydisperse droplet, and then... Figure 5 The bright-field plot of the polydisperse droplets in the middle B shows that each polydisperse droplet contains nanoparticles (red arrows).

[0107] When using the method of this invention to detect single EV polysaccharides, a fluorescence microscope is used to image polydisperse droplets, simultaneously obtaining bright-field images, fluorescence images, and their superimposed images of the polydisperse droplets. Figure 6 (A) It can be seen that when the droplet does not contain EV, the droplet has no obvious fluorescence signal ( Figure 6 (A, blank group); while when the droplets contain EV, the polydisperse droplets show obvious green fluorescence signals in the fluorescence image ( Figure 6 Middle A, EV group).

[0108] Statistical analysis of the droplet data extracted from the image yielded the fluorescence intensity packing distribution maps of microdroplets without EVs and loaded with a single EV. Figure 6 (B) and statistical distribution chart ( Figure 6 (C), using SNL as a representative lectin, the corresponding glycan signal in a single EV was collected. Figure 6 As can be seen from Figure B, the fluorescence signal intensity distribution range of droplets without EVs is mainly concentrated in the range of 164~5687, while the fluorescence signal distribution range of droplets loaded with a single EV is 282~15862, which is much larger than the signal intensity of droplets without EVs. Therefore, this indicates that the significant change in droplet fluorescence intensity comes from the EVs captured by the nanoparticles inside the droplets. Figure 6 The distribution of droplets in different fluorescence ranges was statistically analyzed in section C. It can be seen that the fluorescence signal distribution ranges of droplets without EVs and droplets loaded with EVs almost do not overlap, indicating that the method of this invention can effectively detect and collect glycan information on single EV particles. This invention preferably uses the average fluorescence intensity value of droplets without EVs plus three times the standard deviation as the threshold for subsequent analysis. Figure 6As can be seen from FIG. 6, the signals between EVs are separated from each other and do not interfere with each other. The fluorescence value of each microdroplet reflects the expression of glycans on the single EV contained therein. Different fluorescence values in different droplets exactly show that the expression of the same type of glycans is different between different EV particles. This shows that the method of the present application can reveal the heterogeneity of glycan expression on different EV particles, and this ability is not possessed by the bulk analysis method because the bulk analysis method can only give a single bulk value of the expression of glycans on all EV particles.

[0109] As Figure 7 shown, the polydisperse droplet fluorescence map of the present application method for detecting EVs of different concentrations. When the concentration of EVs is 0, there is no obvious signal in the droplets in the fluorescence map. When the concentration of EVs is 1 x 10 8 , 1 x 10 9 , 1 x 10 10 particles / mL, it can be seen that as the concentration of EVs increases, the number of obvious fluorescent droplets in the collected pictures also increases.

[0110] As Figure 8 shown, the fluorescence values of single microdroplets obtained by the method of the present application were summed and averaged, and then compared with the EV glycan signals corresponding to different lectins obtained by the ELISA method. The R 2 value obtained by curve fitting is 0.95, which shows that the present application method has good correlation with the gold standard ELISA method.

[0111] As Figure 9 shown, the present application method is suitable for the detection of different types of glycans. Using 22 different target glycan structure lectins, the glycan profile of the A549 EV sample collected by the method of the present application. It shows the difference of different glycan structures in the same EV sample; at the same time, it also shows the heterogeneity of glycan expression between different single EVs. At the same time, compared with the average signal of the EV population obtained by the ELISA method, the signal obtained by the method of the present application comes from a single EV.

[0112] Figure 8 and Figure 9The ELISA method used in the middle of the operation process as follows: take 50 microliters of EV samples in 96-well enzyme labeled plate. Incubate for 30 minutes, then remove the EV solution, add 100 microliters of 1% BSA solution to block the enzyme labeled plate. After blocking for 1 hour, remove the blocking solution, add 50 microliters of 5 micrograms / milliliter concentration of biotin modified lectin solution. Incubate for 30 minutes to target the binding of the lectin molecules to the glycan structure on the EV. Then remove the lectin supernatant, add 50 microliters of 4 micrograms / milliliter concentration of streptavidin coupled β-galactosidase solution, incubate for 30 minutes, and use the specific affinity of streptavidin and biotin to bind the enzyme molecules to the lectin molecules. Remove the enzyme solution, then wash the enzyme labeled plate with 100 microliters of PBS solution to remove the free enzyme molecules that are not bound to the lectin molecules. Finally, add 50 microliters of substrate solution (50 micromolar / milliliter concentration of fluorescein di-β-D-galactopyranoside solution), and after 30 minutes of reaction, collect the fluorescence signal with an enzyme labeled instrument.

Claims

1. A method for analyzing a single extracellular vesicle glycan in a fluid sample, comprising the steps of: S1: mixing a fluid sample containing EV particles with functionalized magnetic nanoparticles to obtain MNP@PDA-EV complexes; the preparation method of the functionalized magnetic nanoparticles comprising the steps of: taking 1.6 grams of ferric chloride, 3 grams of sodium acetate, 0.6 grams of sodium citrate, 1 milliliter of water, dissolving in 50 milliliters of ethylene glycol, mixing thoroughly, and then reacting at 180℃ for 12 hours to obtain magnetic nanoparticles; under the assistance of ultrasonic dispersion, 3 milligrams of magnetic nanoparticles were dispersed in 100 milliliters of water, and then 100 milligrams of dopamine and 1 milliliter of 28% ammonia water were added in sequence, mixed thoroughly, and then reacted at room temperature for 4 hours to obtain functionalized magnetic nanoparticles; in S1, the mass ratio of EV particles to functionalized magnetic nanoparticles in the fluid sample containing EV particles is (0.005-0.1):1; S2: blocking the MNP@PDA-EV complex, and then mixing with biotin-coupled lectin to obtain biotinylated EV, and streptavidin-coupled enzyme molecules specifically bind to the biotinylated EV to obtain enzyme-linked immunosorbent nanocomposites; in S2, the blocking and mixing further comprise the steps of solid-liquid separation and washing; S3: mixing the enzyme-linked immunosorbent nanocomposites with a substrate solution and an oil phase liquid to prepare polydisperse droplets; in S3, the preparation method of the polydisperse droplets comprises one or more combinations of oscillation, stirring, whipping, impact, and ultrasonic; S4: optical signal detection; the method is not for the purpose of disease diagnosis and treatment. 2.The method of claim 1, wherein: the oil phase liquid comprises a surfactant and an oily liquid. 3.The method of claim 2, wherein: the surfactant comprises a combination of one or more of Triton X-100, Tween 20, Span 80, EM90, DC5200, 5225C, SF1328, and BM-12; the oily liquid comprises at least one of fluorinated oil, mineral oil, silicone oil, ester, and liquid alkane. 4.The method of claim 1, wherein: the volume ratio of the oil phase liquid to the aqueous phase component is (1-50):

1. 5.The method of claim 1, wherein: in S4, the optical signal detection comprises the steps of image acquisition, processing, and analysis. 6.An apparatus for analyzing a single extracellular vesicle glycan in a fluid sample, comprising: functionalized magnetic nanoparticles for capturing extracellular vesicles in a fluid sample containing EV particles to obtain MNP@PDA-EV complexes; the preparation method of the functionalized magnetic nanoparticles comprising the steps of: ​ ​ 1.6 g of ferric chloride, 3 g of sodium acetate, 0.6 g of sodium citrate, 1 ml of water were dissolved in 50 ml of ethylene glycol, mixed thoroughly, and then reacted at 180°C for 12 hours to obtain magnetic nanoparticles; 3 mg of the magnetic nanoparticles were dispersed in 100 ml of water under the assistance of ultrasonic dispersion, and then 100 mg of dopamine and 1 ml of 28% ammonia water were sequentially added, mixed thoroughly, and then left to react at room temperature for 4 hours to obtain functionalized magnetic nanoparticles; a blocking solution for blocking the MNP@PDA-EV complex, to obtain a blocked MNP@PDA-EV complex; a biotin-modified lectin for affinity labeling of the blocked MNP@PDA-EV complex, to obtain biotinylated EVs; a streptavidin-coupled enzyme molecule for specific binding with the biotinylated EVs, to obtain an enzyme-labeled magnetic complex; a substrate corresponding to the enzyme molecule for reaction with the enzyme-labeled magnetic complex; an oil phase liquid for preparing polydisperse droplets.

7. The device for analyzing a single extracellular vesicle glycan in a fluid sample according to claim 6, wherein: the device for analyzing a single extracellular vesicle glycan comprises: an inlet unit configured to receive an EV particle-containing fluid sample; a mixing unit configured with: 1) functionalized magnetic nanoparticles for capturing extracellular vesicles in the EV particle-containing fluid sample, to obtain an MNP@PDA-EV complex; 2) a blocking solution for blocking the MNP@PDA-EV complex, to obtain a blocked MNP@PDA-EV complex; 3) a biotin-modified lectin for affinity labeling of the blocked MNP@PDA-EV complex, to obtain biotinylated EVs; 4) a streptavidin-coupled enzyme molecule for specific binding with the biotinylated EVs, to obtain an enzyme-labeled magnetic complex; 5) a substrate corresponding to the enzyme molecule for reaction with the enzyme-labeled magnetic complex; 6) an oil phase liquid for preparing polydisperse droplets; a magnetic separation unit configured to sequentially magnetically separate the MNP@PDA-EV complex, the biotinylated EVs, and the enzyme-labeled magnetic complex; a detection unit configured to measure the fluorescence signal of the polydisperse droplets.

8. Use of the method for analyzing a single extracellular vesicle glycan in a fluid sample according to any one of claims 1 to 5, or the device for analyzing a single extracellular vesicle glycan in a fluid sample according to any one of claims 6 to 7, in analyzing a single extracellular vesicle glycan; the use is for non-disease diagnosis and treatment purposes.

Images