Method for analyzing glycan on single extracellular vesicle in fluid sample

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for analyzing glycan on single extracellular vesicles in a fluid sample. The method comprises the following steps: capturing and manipulating EV by using magnetic nanoparticles, singly separating the captured EV by using polydisperse micro-droplets based on the specific affinity effect of lectin on a specific glycan structure, realizing local signal amplification in the droplets through an enzymatic reaction, and performing image acquisition on the droplets by using a fluorescence microscope. Therefore, recognition and analysis of glycan components on a single EV are realized. According to the preparation process for forming the micro-droplets through the oscillation emulsification method, the whole detection process is simpler, more convenient and quicker, no micro-fluidic chip or special device is needed, the detection process can be completed in a centrifugal tube, meanwhile, all the micro-droplets are parallel and quickly generated, and the quantity of the micro-droplets can be flexibly expanded. Compared with the traditional ELISA, the method provided by the invention has the advantages of high sensitivity, low detection limit and small sample consumption.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for analyzing polysaccharides on a single extracellular vesicle in a fluid sample. Background Art

[0002] Glycosylation is an important and widespread modification process in organisms, that is, the sugar residue structure is transferred to specific molecules (such as proteins and lipid molecules) under the action of glycosyltransferases to form glycan structures. Glycosylation is the most common post-translational modification process on proteins. It affects the physical and chemical properties and functions of proteins by participating in the polymerization, folding, maturation and transport of protein-peptide chains. Glycans can participate in many physiological and pathological processes such as cell communication, immune regulation, and tumor metastasis, and abnormal glycosylation modification is closely related to the occurrence and development of various diseases (such as cancer, leukemia, Alzheimer's disease, etc.). For example, compared with healthy cells, the proteoglycan structure on the surface of tumor cells is richer, and some specific glycan structures (such as Tn antigen, T antigen, sTn antigen) are often expressed. These antigens are expressed in large quantities 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 assist in disease diagnosis, discovery of new biomarkers, development of specific therapeutic targets, and many other aspects, showing good potential for clinical application.

[0003] Extracellular vesicles (EVs) are lipid bilayer membrane structure particles generated by cells and released into the extracellular space. They 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 rich glycan structures. The glycan structure on EVs is closely related to its parent cells, and has a similar glycan expression profile to the parent cells, which can reflect the information of the parent cells from which it originates. Therefore, EVs are important carriers of glycan active substances. In recent years, EV glycans have received increasing attention from researchers in many aspects, such as revealing the basic biological functions of EVs, diagnosing diseases (such as cancer), developing biomarkers, and real-time monitoring and prognosis of disease treatment effects.

[0004] Efficient identification and analysis of EV glycans is the primary prerequisite for developing their application value. However, due to the highly variable structure and diverse modification sites of glycans themselves, as well as the small size (submicron level), multiple types, and diverse components of EVs themselves, in-depth and accurate analysis of EV glycan information still faces huge challenges. Current detection methods for EV glycans mainly include mass spectrometry, liquid chromatography, lectin-specific recognition, etc. These methods often have complicated technical processes and high testing costs. More importantly, most of the current analytical studies on EV glycans are group analyses of EVs, and the glycan information analyzed is comprehensive and can only reflect the average characteristics of the EV population. Since cell secretion of EVs is affected by many factors such as cell type, physiological state, external stimulation, and generation method, EVs are highly heterogeneous. The aforementioned group analysis can only obtain comprehensive EV glycan information, which often results in the masking of certain glycan information of specific EV subpopulations that has high application value but low abundance.

[0005] Currently, the main analysis methods for EV glycans include liquid chromatography, mass spectrometry, lectin microarray, enzyme-linked immunosorbent assay (ELISA), and the recently developed GMR magnetic analysis method based on the 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 fluid samples, its device and application), 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-cell extracellular vesicle glycan analysis method, other methods can only analyze EV samples as a whole and obtain the average signal of the EV population, and cannot provide glycan information of heterogeneity between individual vesicles. In addition, these methods also face other shortcomings. For example, liquid chromatography and mass spectrometry need to destroy the structure of EVs to obtain the sugar chain structure of glycans. In addition, these two methods consume a large amount of samples, and the analysis process is complicated and time-consuming, requiring expensive instruments, professional operation and data analysis skills. Enzyme-linked immunosorbent assay (ELISA) and lectin array chips are analysis methods based on the specific affinity of lectins and glycan structures. Among them, the ELISA method has low adsorption efficiency for EV samples, low freedom of sample loading and large 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. Although the nanoparticle-mediated single extracellular vesicle glycan analysis method (the only method to date for glycan analysis at the level of a single EV) performs glycan analysis at the level of a single EV, the signal cannot be amplified and some low-abundance glycan structures cannot be effectively detected.

[0007] In recent years, researchers have proposed a variety of analytical methods to analyze the molecular characteristics of single EVs, such as total internal reflection fluorescence (TIRF) imaging, proximity encoded PCR technology, nanoflow cytometry, droplet-based enzymatic reaction detection, in situ fixed fluorescence detection, in situ rolling circle amplification, etc. These technologies have their own advantages and disadvantages and are suitable for different analytical scenarios. It should be pointed out that the current analysis of EVs using these technologies is mainly aimed at the protein and nucleic acid components in EVs, and there are very few studies on the analysis of glycans at the single EV level.

[0008] Among the above methods, microdroplet-based enzymatic reaction detection is an important single-target detection and analysis technology. This technology is an emerging technology that distributes the target in the fluid into a large number of independent and parallel microreaction units (i.e., microdroplets), so that each reaction unit contains as much as possible a target molecule, and on this basis, 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 are no reports on EV glycan analysis. In addition, most of the current studies on EV analysis using microdroplet technology use microfluidics to generate monodisperse microdroplet units. This technology usually has extremely high requirements for microfluidic control and requires special microfluidic chip design and expensive core control devices. At the same time, monodisperse microdroplets are generated by time-sequential emulsification of microfluidics, which makes the number of droplets change only with the running time, and is limited by microfluidic chips and control equipment in terms of expanding the amount of droplet generation. Moreover, the EV load in this technology usually uses large-sized commercial microspheres at the micrometer level. 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, 90% of the microspheres are invalid microspheres that do not carry EVs due to the Poisson distribution. Furthermore, the microdroplet partitions are completely random in encapsulating a single microsphere. 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 valid droplets containing one microsphere, and 90% of the droplets are empty droplets without encapsulated microspheres. In other words, 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 analytical resources in subsequent detection and analysis, reducing detection throughput and analysis efficiency. In addition, this technology has certain requirements on the professional and technical level of the operator, and still has limitations in terms of versatility, universality, and ease of operation. Summary of the invention

[0009] In order to solve the above problems existing in the prior art, the present invention has made the following improvements: The first aspect of the present invention aims to provide a method for analyzing glycans in single extracellular vesicles in a fluid sample.

[0010] The second aspect of the present invention aims to provide a device for analyzing glycans in single extracellular vesicles in a fluid sample.

[0011] The third aspect of the present invention aims to provide an application.

[0012] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is: In a first aspect of the present invention, a method for analyzing glycans in a single extracellular vesicle in a fluid sample is provided, comprising the following steps: S1: Mixing the fluid sample containing EV particles with functionalized magnetic nanoparticles to obtain MNP@poly-EV complex; S2: blocking treatment of MNP@poly-EV complex, followed by mixing with biotin-coupled lectin to obtain ELISA nanocomplex; S3: mixing the ELISA nanocomplex with a substrate solution and an oil phase liquid to prepare polydisperse droplets; S4: Optical signal detection.

[0013] In some embodiments of the present invention, in S1, the mixing time of the fluid sample containing EV particles and the functionalized magnetic nanoparticles is at least 10 min, preferably 10 min to 40 min.

[0014] In some embodiments of the present invention, in S1, the sealing treatment time is at least 15 minutes, preferably 10 minutes to 90 minutes.

[0015] In some embodiments of the present invention, 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.

[0016] In some embodiments of the present invention, the mass ratio of EV particles to functionalized magnetic nanoparticles in the fluid sample containing EV particles in S1 is (0.005~0.1):1.

[0017] In some embodiments of the present invention, in S2, the sealing and mixing further include the steps of solid-liquid separation and washing.

[0018] In some embodiments of the present invention, in S2, the blocking solution includes but is not limited to at least one of commercial rapid blocking solution, bovine serum albumin (BSA), skimmed milk powder, casein, gelatin, polyethylene glycol (PEG), and polyvinyl pyrrolidone (PVP).

[0019] In some embodiments of the present invention, in S2, the lectin includes concanavalin A (ConA), lentil agglutinin (LCA), pea agglutinin (PSA), aurantiacus aurantium agglutinin (AAL), urea oleifera agglutinin I (UEA-I), wheat germ agglutinin (WGA), tomato agglutinin (LEL), potato agglutinin (STL), stramonium stramonium agglutinin (DSL), succinylated wheat germ agglutinin (Succinylated The present invention can be applied to the following lectins: WGA), Griffonia seed agglutinin II (GSL-II), hemagglutinin (PHA-E), leukocyte agglutinin (PHA-L), Ricinus communis agglutinin I (RCA-I), Erythrina chinensis agglutinin (ECL), jacalin, peanut agglutinin (PNA), dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), Vicia villosum agglutinin (VVL), Griffonia seed agglutinin I (GSL-I), and elderberry agglutinin (SNL). In addition to the above lectins, all biotin-modified lectins or saccharide recognition antibodies can be applied to the method of the present invention.

[0020] In some embodiments of the present invention, in S3, the enzyme is a streptavidin-coupled enzyme molecule. Theoretically, there is no limit to the type of enzyme, such as β-galactosidase, horseradish peroxidase, etc. Correspondingly, the selected enzyme substrate corresponds to the enzyme used. In the example of the present invention, β-galactosidase is selected, and the substrate is fluorescein di-β-D-pyranogalactoside (FDG). If horseradish peroxidase is selected, the substrate can be a commercial chemiluminescent substrate or 3,3',5,5'-tetramethylbenzidine.

[0021] In some embodiments of the present invention, in S3, the method for preparing the polydisperse droplets comprises one or more combinations of oscillation, stirring, blowing, impact, and ultrasound.

[0022] In some embodiments of the present invention, in S3, the oil phase liquid includes a surfactant and an oily liquid.

[0023] In some embodiments of the present invention, in S3, the surfactant includes a combination of one or more of Triton X-100, Tween20, Span 80, EM90, DC5200, 5225C, SF1328, and BM-12.

[0024] In some embodiments of the present invention, in S3, the oily liquid includes at least one of fluorinated oil, mineral oil, silicone oil, esters, and liquid alkanes.

[0025] In some embodiments of the present invention, in S3, the volume ratio of the oil phase liquid to the water phase component is (1-50):1. Preferably, it is (10-20):1.

[0026] In some embodiments of the present invention, in S3, the contact time between the oil phase and the water phase is ≥5 seconds, preferably 10 to 30 seconds; the reaction time after the enzyme contacts the substrate is ≥5 minutes, and 30 minutes is used in the example of the present invention.

[0027] In some embodiments of the present invention, in S4, the optical signal detection includes the steps of image acquisition, processing and analysis.

[0028] In some embodiments of the present invention, in S4, the glass slide is a commercial slide and cover glass with a hydrophilic surface treatment. Theoretically, there is no limit on its thickness and material, and its thickness includes but is not limited to 1.0~1.2 mm or 0.13~0.22 mm; the material of the glass slide includes but is not limited to ultra-white glass, quartz, borosilicate glass, etc.

[0029] In some embodiments of the present invention, in S4, a fluorescence microscope is used to capture bright field and fluorescence channel images of the polydisperse droplets, and the types of microscopes used include but are not limited to wide-field fluorescence microscopes, total internal reflection microscopes, confocal microscopes, etc. The present invention preferably uses a wide-field fluorescence microscope.

[0030] In some embodiments of the present invention, in S4, a fluorescence microscope is used to collect image signals of target droplets carrying EVs, and the number of target particles collected in each microscope image field of view (FOV) can be freely adjusted. In the example of the present invention, the number of droplets in each FOV is selected to be greater than 1000. During the analysis process, the number of droplets analyzed is not limited and can be freely adjusted.

[0031] In some embodiments of the present invention, in S4, the image of the droplets collected by the fluorescence microscope is processed and the image data values ​​are exported. Theoretically, there is no restriction on the type of image processing software, including but not limited to image J / fiji and ZEN Blue 3.5. In the example of the present invention, ZEN Blue 3.5 analysis software is used to perform bright field recognition and fluorescence value analysis of multi-dispersed droplets on the collected multi-channel images, and convert and export the various data values ​​obtained from the analysis.

[0032] In some embodiments of the present invention, in S1, functionalized magnetic nanoparticles with a core-shell structure are used, and their surface functionalization modification can adopt but is not limited to polydopamine, silica, polyethyleneimine, polyacrylic acid, PEG, etc.; their synthesis and preparation methods can adopt but are not limited to hydrothermal method, solvothermal method, coprecipitation method, thermal decomposition method, sol-gel method, microemulsion method, vapor phase precipitation method, etc.

[0033] In a second aspect of the present invention, a device for analyzing glycans in single extracellular vesicles in a fluid sample is provided.

[0034] A device for analyzing glycans in a single extracellular vesicle, comprising: Functionalized magnetic nanoparticles are used to capture extracellular vesicles in a fluid sample containing EV particles to obtain MNP@poly-EV complexes; A blocking solution, which is used to block the MNP@poly-EV complex to obtain a blocked MNP@poly-EV complex; A biotin-modified lectin, which is used to affinity label the closed MNP@poly-EV complex to obtain biotinylated EV; An enzyme molecule coupled with streptavidin, which is used to specifically bind to the biotinylated EV to obtain an enzyme-labeled magnetic complex; The enzyme molecule corresponds to a substrate, which is used to react with the enzyme-labeled magnetic complex; Oil phase liquid, which is used to prepare polydisperse droplets.

[0035] In some embodiments of the present invention, the device for analyzing glycans in a single extracellular vesicle in a fluid sample comprises: an inlet unit configured to receive a fluid sample containing EV particles; A mixing unit, the mixing unit being configured with: Functionalized magnetic nanoparticles are used to capture extracellular vesicles in the fluid sample containing EV particles to obtain MNP@poly-EV complexes; A blocking solution, which is used to block the MNP@poly-EV complex to obtain a blocked MNP@poly-EV complex; A biotin-modified lectin, which is used to affinity label the blocked MNP@poly-EV complex to obtain biotinylated EV; An enzyme molecule coupled with streptavidin, which is used to specifically bind to the biotinylated EV to obtain an enzyme-labeled magnetic complex; The enzyme molecule corresponds to a substrate, which is used to react with the enzyme-labeled magnetic complex; An oil phase liquid, which is used to prepare polydisperse droplets; A magnetic separation unit, wherein the magnetic separation unit is configured to sequentially magnetically separate and obtain the MNP@poly-EV complex, the biotinylated EV, and the enzyme-labeled magnetic complex; A detection unit is configured to measure the fluorescence signal of the polydisperse droplets.

[0036] In some embodiments of the present invention, the inlet unit, the mixing unit, and the magnetic separation unit are the same unit.

[0037] In some embodiments of the present invention, the fluid of the mixing unit is connected to the inlet unit; and the fluid of the magnetic separation unit is connected to the mixing unit.

[0038] In some embodiments of the present invention, the fluid sample containing EV particles includes body fluid, tissue or cell culture supernatant containing EV particles; 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, and lymph; the source of body fluid includes but is not limited to humans, monkeys, mice, rats, rabbits, pigs, monkeys, dogs and other animals.

[0039] In some embodiments of the present invention, the fluid sample containing EV particles includes cells secreted or obtained by at least one ultrasonic disruption of cell fragments and living cells.

[0040] In some embodiments of the present invention, the fluid sample containing EV particles includes a solution containing EV particles obtained by differential refrigerated centrifugation, density gradient centrifugation, polymer-based precipitation, ultrafiltration, size exclusion chromatography, immunoseparation and the like; preferably, the fluid sample containing EV particles includes a solution containing EV particles obtained by differential refrigerated centrifugation, the centrifugal force of the differential refrigerated centrifugation is 2000g~200000g, and the time is 30min~200min; preferably, the centrifugal force of the differential refrigerated centrifugation is 15000g~200000g, and the time is 30min~150min.

[0041] In some embodiments of the present invention, the device for analyzing extracellular vesicle glycans in a fluid sample includes a detection kit, a magnetic separation system, a detection system, and the like.

[0042] The third aspect of the present invention provides use of the method described in the first aspect of the present invention and / or the device described in the second aspect of the present invention in analyzing glycans in a single extracellular vesicle.

[0043] The beneficial effects of the present invention are: This paper presents for the first time a simplified and rapid analytical strategy based on polydisperse microdroplets for the detection of glycans on single EVs.

[0044] The present invention overcomes the limitation that the traditional EV glycan analysis method can only analyze EV glycans as a whole, and can identify and analyze glycan components at the level of a single EV, providing glycan information on the heterogeneity of individual vesicles in EV samples, while having higher sensitivity and lower detection limits. The entire test process does not require special experimental instruments, devices, reagents, and does not involve any microfluidic chips and microfluidic equipment. It is simple to operate, convenient and fast, and has low detection costs and good economic efficiency.

[0045] The method of the present invention uses nano-sized magnetic particles as the manipulation medium for EVs, making the capture and glycan labeling process of EVs easy to operate. More importantly, the present invention uses nano-sized magnetic particles to replace the micron-sized microbeads used in conventional micro-droplet detection methods, and utilizes the excellent monodispersity of nanoparticles in the solution to help achieve the effect of directly dispersing a single EV particle into a single droplet, eliminating the limitations of magnetic beads in the micro-droplet detection method and improving the utilization efficiency of micro-droplets.

[0046] The entire detection process of the present invention can be completed on a centrifuge tube and a commercial glass slide. The preparation of polymer droplets can be achieved by simple oscillation, and all droplets are generated in parallel without the need for microfluidic chips and devices. The signal acquisition of the method of the present invention uses a conventional fluorescence microscope, and does not require high-end and expensive microscopic imaging devices such as super-resolution microscopes and total internal reflection microscopes. The method of the present invention has good repeatability, low sample demand, and the sample operation volume can be freely adjusted. The present invention is not limited to the number of droplets analyzed and can be freely adjusted according to demand. The number of target particles collected in each microscope image field of view is greater than 1,000, and the number of image acquisitions is also not limited. The detection limit of the method of the present invention is significantly lower than that of holistic analysis methods such as traditional ELISA methods, and the detection performance of EV glycans is more sensitive. Moreover, the EV glycan information obtained by the method of the present invention is more specific and refined, which can fully reflect the heterogeneity of glycan characteristics between different EV particles. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 A single EV glycan analysis workflow based on polydisperse droplets.

[0048] Figure 2 Characterization of functionalized magnetic nanoparticles (MNP@PDA): (A) particle concentration and size distribution; (B) SEM image of MNP@PDA particles.

[0049] Figure 3 (A) is the TEM morphology of EV particles; (B) is the NTA concentration and size distribution of EV particles.

[0050] Figure 4 Polydisperse droplets (A) prepared in Example 3 and their droplet diameter distribution (B).

[0051] Figure 5 The capture of EVs by functionalized magnetic nanoparticles and their dispersion in droplets.

[0052] Figure 6Detection and analysis of single EV glycans in Example 5: (A) Fluorescence microscopy images of polydisperse droplets without EV and loaded with single EV (Bright field image, AF488 fluorescence image and Merge overlay image); (B) single EV glycan fluorescence intensity stacking distribution diagram and (C) statistical distribution diagram; the lectin used in this figure is SNL.

[0053] Figure 7 Representative polydisperse droplet fluorescence images of different EV concentrations, where the EV concentrations are 0 and 1×10 8 , 1×10 9 , 1×10 10 particles / mL.

[0054] Figure 8 This is the correlation between the single EV glycan detection method based on polydisperse droplets and the ELISA method; a total of seven lectins, including ConA, DBA, GSL-I, PHA-L, sWGA, DSL, and ECL, were used in this figure.

[0055] Fig. 9 The EV glycan information is collected by the single EV glycan detection method based on polydisperse droplets (the method of the present invention) and the ELISA method; each point of the data obtained by the method of the present invention in the figure represents the fluorescence intensity of a droplet, and a total of 22 different lectins are used. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0057] The overall analysis process of the present invention is as follows Figure 1 As shown, the technical concept is as follows: Step 1: EV immobilization. Functionalized magnetic nanoparticles (MNP@PDA) were mixed with EV solution to capture EVs in the solution and obtain EV-loaded magnetic nanoparticles (denoted as MNP@PDA-EV).

[0058] Step 2: Preparation of nanocomplexes for enzyme-linked immunosorbent assay. First, the MNP@PDA-EV was surface-blocked. Biotin-coupled lectins were used to specifically bind to the surface polysaccharides of EVs in the nanocomplexes, and then the streptavidin-coupled enzyme molecules were bound to the lectins to obtain nanocomplexes for enzyme-linked immunosorbent assay. During the entire preparation process, all processes involving the separation of the solid phase (magnetic nanoparticles) from the liquid phase were performed using magnetic separation with the assistance of magnets.

[0059] Step 3: Preparation of polydisperse droplets. The prepared ELISA nanocomplex is uniformly dispersed in the substrate solution, quickly mixed with the oil phase and fully shaken until the solution is milky white, thereby obtaining polydisperse droplets encapsulating the nanocomplex and the substrate solution.

[0060] Step 4: Droplet imaging and image acquisition. A certain volume of the polydisperse droplets prepared in step 3 is drawn into the sample pool so that the droplets are dispersed in a single layer in the sample pool. Under light-shielding conditions, the enzyme molecules in the nanocomplex of the ELISA react with the substrate to produce a fluorescent signal. The droplets in the sample pool are imaged using a fluorescence microscope. Image processing and data analysis. After quality screening and processing of the collected images, the relevant data of the droplets in the image are extracted and collected, and statistics and analysis are performed.

[0061] Example 1 Preparation of functionalized magnetic nanoparticles (MNP@PDA) First, the solvothermal method was used to synthesize the magnetic core. 1.6 g of ferric chloride, 3 g of sodium acetate, 0.6 g of sodium citrate, and 1 ml of water were dissolved in 50 ml of ethylene glycol, mixed thoroughly, and reacted at 180 ° C for 12 hours to obtain magnetic nanoparticles, recorded as MNP. Then, polydopamine was used to coat the surface of MNP to achieve functionalization. That is, with the assistance of ultrasonic dispersion, 3 mg of MNP particles were dispersed in 100 ml of water, and then 100 mg of dopamine and 1 ml of 28% ammonia water were added in turn. After thorough mixing, the reaction was allowed to stand at room temperature for 4 hours, and finally the core-shell structure functionalized magnetic nanoparticles were obtained, recorded as MNP@PDA.

[0062] Nanoparticle tracking analysis (NTA) was used to characterize the particle concentration and particle size distribution of MNP@PDA. Figure 2 As shown in Figure A, the average particle size of MNP@PDA particles is about 150 nanometers. The morphology of MNP@PDA was characterized by scanning electron microscopy (SEM). Figure 2 As can be seen in B, MNP@PDA has a relatively smooth surface and its size is well matched with the NTA test.

[0063] Example 2 Isolation and purification of EVs Continuous differential refrigerated centrifugation was used to separate and purify EVs. The A549 cell culture medium solution containing EV particles was first centrifuged at 2500g for 1 hour, and then the supernatant was centrifuged at 20000g for 1 hour. The supernatant was then further centrifuged at 167000g for 2 hours. Finally, the extracellular vesicle precipitate at the bottom of the centrifuge tube was collected and recorded as EV for subsequent glycan analysis. Among them, g represents the acceleration due to gravity.

[0064] The morphology of the EV samples collected above was characterized by transmission electron microscopy (TEM). Figure 3 A shows a typical vesicle structure. The particle concentration and particle size distribution of the EV sample were characterized by a nanoparticle tracking analyzer. Figure 3 As shown in B, the average size of EV particles is approximately 130 nm.

[0065] Example 3 Immobilization of EVs and Identification and Labeling of EV Surface Polysaccharides Take 5 μl of MNP@PDA particle stock solution (1 mg / ml) and 50 μl of PBS solution in a 0.6 ml centrifuge tube, rinse the nanoparticles and remove the washing solution after magnetic separation. Take 50 μl of EV sample and incubate with MNP@PDA particles for 30 minutes to allow MNP@PDA particles to fully capture EVs in the solution to obtain MNP@PDA-EV complexes.

[0066] After magnetic separation of the complex, the liquid phase was removed, and then 100 μL of 1% bovine serum albumin solution was added and the surface of the MNP was blocked at room temperature for 1 hour. After removing the blocking solution, 50 μL of 5 μg / mL biotin-modified SNL solution was added and the mixture was allowed to stand at room temperature for 30 minutes to ensure that SNL effectively recognized the sialic acid sugars on EVs. After magnetic separation, 50 μL of 4 μg / mL streptavidin-coupled β-galactosidase solution was added to the particles and incubated for 30 minutes. Based on the affinity of biotin-streptavidin, the enzyme molecules were bound to SNL, thereby achieving enzyme labeling of EV glycans. After magnetic separation to remove excess solution, 100 μL of PBS was added to the centrifuge tube to wash the particles, and the mixture was repeated three times. The washed particles were redispersed in 30 μL of PBS solution and transferred to a 1.5 mL centrifuge tube for use.

[0067] Operation time: (1) In step 1, the capture time of EV by MNP@PDA particles is ≥ 10 minutes, and 30 minutes is used in this embodiment; (2) In step 2, the surface sealing treatment time of MNP@PDA-EV particles is ≥ 15 minutes, and 60 minutes is used in this embodiment; (3) In step 2, the binding time of lectin modified with biotin and EV glycan structure is ≥ 10 minutes, and 30 minutes is used in this embodiment; (4) In step 2, the binding time of streptavidin-coupled enzyme molecule and lectin is ≥ 10 minutes, and 30 minutes is used in this embodiment.

[0068] This embodiment further tested concanavalin A agglutinin (ConA), lentil agglutinin (LCA), pea agglutinin (PSA), aurantiacus aurantium agglutinin (AAL), urea oleifera agglutinin I (UEA-I), wheat germ agglutinin (WGA), tomato agglutinin (LEL), potato agglutinin (STL), Datura stramonium agglutinin (DSL), succinylated wheat germ agglutinin (Succinylated There are 22 lectins in total, including Griffonia suffruticosa Lectin (GSL-I), Griffonia suffruticosa Lectin (GSL-WGA), Griffonia suffruticosa Lectin II (GSL-II), PHA-E, PHA-L, Ricinus communis Lectin I (RCA-I), Erythrina chinensis Lectin (ECL), Jacalin, Peanut lectin (PNA), Dolichos biflorus Lectin (DBA), Soybean lectin (SBA), Vicia villosum Lectin (VVL), Griffonia suffruticosa Lectin I (GSL-I), and Sambucus nigra Lectin (SNL).

[0069] Example 4 Preparation of polydisperse droplets After magnetic separation of the particle suspension in the 1.5 ml centrifuge tube in Example 3, all the water phase was removed, and 20 μl of substrate solution (fluorescein di-β-D-pyranogalactoside with a concentration of 50 μmol / ml) was added, and the magnetic nanoparticle complex and the substrate solution were evenly mixed. Then, 200 μl of oil phase (a mixture of mineral oil, 0.1% by mass of Triton X-100 and 3% by mass of EM90) was immediately added to the centrifuge tube, and the mixture was shaken for 20 seconds to fully emulsify the oil and water phases in the centrifuge tube to form polydisperse droplets of water in oil. The reaction time of the enzyme and the substrate was recorded from the time when the magnetic nanoparticle complex and the substrate solution were mixed.

[0070] The operation time in this step is: the contact time between the oil phase and the water phase is ≥5 seconds, preferably 10 to 30 seconds in this embodiment; the reaction time after the enzyme contacts the substrate is ≥5 minutes, preferably 30 minutes in this embodiment.

[0071] Example 5 Droplet Imaging, Image Collection, and Data Analysis Take 5 microliters of the prepared polydisperse droplets in a sample pool with a length of 25 mm, a width of 10 mm, and a depth of 20 microns, and gently cover the liquid surface with a cover glass to seal the slide. The droplets are dispersed into a single layer in the sample pool by capillary action. The carrier at the bottom of the sample pool is a standard-grade slide with a thickness of 1.0±0.2 mm, and the top is an ultra-white glass cover glass with a thickness of 0.17±0.01 mm. Let it stand under light-shielding conditions, and when the enzyme and substrate are in contact for 30 minutes, use a wide-field fluorescence microscope to capture images of the droplets in the sample pool. Use a 10x objective lens to identify and locate the droplets in the bright field, and collect the fluorescence channel image of the droplets at the same time. Collect 300 to 500 pictures of 35 mm × 20 mm for each sample.

[0072] In this embodiment, a commercial standard glass slide with a thickness of 1.0±0.2 mm and a commercial ultra-clear glass cover glass with a thickness of 0.17±0.01 mm are used. The depth of the sample pool is 20 microns.

[0073] The collected images are quickly screened for quality. After confirming the high-quality images, the data extraction stage is entered. Zen Blue 3.5 is used to process the collected images, identify the droplets in the images, and automatically analyze and extract the droplet-related data (such as the statistical number of droplets, size parameters, fluorescence intensity values, etc.). At the same time, a csv file is generated for subsequent data analysis and processing.

[0074] The results are as follows: Figure 4 A is a bright field image of the polydisperse droplets prepared by the method of the present invention, and the distribution of the droplet diameters is statistically analyzed ( Figure 4 In Figure B), it can be seen that the diameter of the obtained microdroplets is continuously distributed within 1-54 μm, which is consistent with the characteristics of polydisperse droplets, among which the droplets of 2-25 μm can reach 98.8% of the total number of droplets. In this embodiment, microdroplets of 5-20 μm are preferably used for subsequent glycan feature information analysis.

[0075] Scanning electron microscopy (SEM) was used to characterize the EV-capturing nanoparticles. Figure 5 As can be seen in Figure A, the nanoparticles (black arrow) successfully captured the EVs (red arrow). In the method of the present invention, the nanoparticles that capture EVs are uniformly dispersed into polydisperse droplets. Figure 5 From the bright field image of the polydisperse droplets in Figure B, it can be seen that the polydisperse droplets are all wrapped with nanoparticles (red arrows).

[0076] When the method of the present invention is used to detect single EV glycans, a fluorescence microscope is used to image the polydisperse droplets, and a bright field image, a fluorescence image, and a superimposed image thereof of the polydisperse droplets can be obtained at the same time ( Figure 6In the middle (A), it can be seen that when the droplets do not contain EVs, the droplets have no obvious fluorescence signal ( Figure 6 A, blank group); when the droplets contained EVs, the polydisperse droplets had obvious green fluorescence signals in the fluorescence image ( Figure 6 Middle A, EV group).

[0077] The droplet data extracted from the image were statistically analyzed to obtain the fluorescence intensity accumulation distribution diagram of the microdroplets without EV and loaded with a single EV ( Figure 6 B) and statistical distribution diagram ( Figure 6 (C) SNL was used as a representative lectin to collect the corresponding glycan signals in single EVs. Figure 6 As can be seen in Figure B, the fluorescence signal intensity distribution range of droplets without EVs is mainly concentrated between 164 and 5687, while the fluorescence signal distribution range of droplets carrying a single EV is between 282 and 15862, which is much larger than the signal intensity of droplets without EVs. This indicates that the significant change in droplet fluorescence intensity comes from EVs captured by the nanoparticles in the droplets. Figure 6 Figure C counts the number of droplets distributed in different fluorescence intervals. It can be seen that the distribution range of the fluorescence signal of the droplets without EVs and the fluorescence signal of the droplets loaded with EVs is almost non-overlapping, indicating that the method of the present invention can effectively detect and collect glycan information on single EV particles. The present invention preferably uses the average fluorescence intensity value of the droplets without EVs plus three times the standard deviation as the threshold for subsequent analysis. Figure 6 As shown in B, 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 a single EV contained therein. The different fluorescence values ​​in different droplets just show that the expression of the same type of glycans in different EV particles is different. This shows that the method of the present invention can reveal the heterogeneity of glycan expression on different EV particles, and this ability is not available in the holistic analysis method, because the holistic analysis method can only give a single overall value for the glycan expression of all EV particles.

[0078] like Figure 7 As shown in the figure, the fluorescence images of polydisperse droplets detected by the method of the present invention at different concentrations of EVs are shown. When the EV concentration is 0, no obvious signal appears in the droplets in the fluorescence image; when the EV concentration is 1×10 8 , 1×10 9 , 1×10 10 particles / mL, it can be seen that as the EV concentration increases, the number of obvious fluorescent droplets in the collected images also increases.

[0079] like Figure 8As shown in the figure, the fluorescence values ​​of the single microdroplets obtained by the method of the present invention were summed and averaged, and then the EV glycan signals corresponding to different lectins obtained by ELISA were matched one by one. The R 2 The value is 0.95, which shows that the method of the present invention has a good correlation with the gold standard ELISA method.

[0080] like Fig. 9 As shown, the method of the present invention is suitable for the detection of different types of glycans. The glycan profile of the A549 EV sample collected by the method of the present invention using 22 different lectins targeting glycan structures. The differences in different glycan structures within the same EV sample are shown; the heterogeneity of glycan expression between different single EVs is also shown. 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 invention comes from a single EV.

[0081] Figure 8 and Fig. 9 The ELISA method used in the experiment was as follows: 50 μL of EV sample was placed in a 96-well ELISA plate. After incubation for 30 minutes, the EV solution was removed and 100 μL of 1% BSA solution was added to block the ELISA plate. After blocking for 1 hour, the blocking solution was removed and 50 μL of lectin solution modified with biotin was added at a concentration of 5 μg / mL. Incubate for 30 minutes to allow the lectin molecules to target and bind to the glycan structure on the EV. Then remove the lectin supernatant, add 50 μL of β-galactosidase solution coupled with streptavidin at a concentration of 4 μg / mL, 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 and then wash the ELISA plate with 100 μL of PBS solution to remove free enzyme molecules that are not bound to the lectin molecules. Finally, 50 μL of substrate solution (fluorescein di-β-D-galactopyranoside solution with a concentration of 50 μmol / mL) was added, and after 30 minutes of reaction, the fluorescence signal was collected using an enzyme reader.

Claims

1. A method for analyzing glycans in single extracellular vesicles in a fluid sample, characterized in that: The following steps are involved: S1: Mixing the fluid sample containing EV particles with functionalized magnetic nanoparticles to obtain MNP@poly-EV complex; S2: blocking treatment of MNP@poly-EV complex, followed by mixing with biotin-coupled lectin to obtain ELISA nanocomplex; S3: mixing the ELISA nanocomplex with a substrate solution and an oil phase liquid to prepare polydisperse droplets; S4: Optical signal detection.

2. The method according to claim 1, characterized in that: The mass ratio of EV particles to functionalized magnetic nanoparticles in the fluid sample containing EV particles described in S1 is (0.005~0.1):

1.

3. The method according to claim 1, characterized in that: In S2, after the sealing and mixing, the steps of solid-liquid separation and washing are also included.

4. The method according to claim 1, characterized in that: In S3, the method for preparing the polydisperse droplets includes one or more combinations of oscillation, stirring, blowing, impact, and ultrasound.

5. The method according to claim 4, characterized in that: The oil phase liquid includes a surfactant and an oily liquid; Preferably, the surfactant comprises a combination of one or more of Triton X-100, Tween 20, Span 80, EM90, DC5200, 5225C, SF1328, and BM-12; Preferably, the oily liquid includes at least one of fluorinated oil, mineral oil, silicone oil, esters, and liquid alkanes.

6. The method according to claim 5, characterized in that: The volume ratio of the oil phase liquid to the water phase components is (1~50):

1.

7. The method according to claim 1, characterized in that: In S4, the optical signal detection includes the steps of image acquisition, processing and analysis.

8. A device for analyzing glycans in single extracellular vesicles in a fluid sample, characterized in that: include: Functionalized magnetic nanoparticles are used to capture extracellular vesicles in a fluid sample containing EV particles to obtain MNP@poly-EV complexes; A blocking solution, which is used to block the MNP@poly-EV complex to obtain a blocked MNP@poly-EV complex; A biotin-modified lectin, which is used to affinity label the closed MNP@poly-EV complex to obtain biotinylated EV; An enzyme molecule coupled with streptavidin, which is used to specifically bind to the biotinylated EV to obtain an enzyme-labeled magnetic complex; The enzyme molecule corresponds to a substrate, which is used to react with the enzyme-labeled magnetic complex; Oil phase liquid, which is used to prepare polydisperse droplets.

9. The device for analyzing glycans in single extracellular vesicles in a fluid sample according to claim 8, characterized in that: The device for analyzing glycans in a single extracellular vesicle comprises: an inlet unit configured to receive a fluid sample containing EV particles; A mixing unit, the mixing unit being configured with: Functionalized magnetic nanoparticles are used to capture extracellular vesicles in the fluid sample containing EV particles to obtain MNP@poly-EV complexes; A blocking solution, which is used to block the MNP@poly-EV complex to obtain a blocked MNP@poly-EV complex; A biotin-modified lectin, which is used to affinity label the blocked MNP@poly-EV complex to obtain biotinylated EV; An enzyme molecule coupled with streptavidin, which is used to specifically bind to the biotinylated EV to obtain an enzyme-labeled magnetic complex; The enzyme molecule corresponds to a substrate, which is used to react with the enzyme-labeled magnetic complex; An oil phase liquid, which is used to prepare polydisperse droplets; A magnetic separation unit, wherein the magnetic separation unit is configured to sequentially magnetically separate and obtain the MNP@poly-EV complex, the biotinylated EV, and the enzyme-labeled magnetic complex; A detection unit is configured to measure the fluorescence signal of the polydisperse droplets.

10. A method for analyzing glycans in a single extracellular vesicle as claimed in any one of claims 1 to 7, or use of the device for analyzing glycans in a single extracellular vesicle as claimed in any one of claims 8 to 9 in analyzing glycans in a single extracellular vesicle.

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