EV membrane protein single molecule detection method based on droplet microfluidics and application thereof

Abstract

The invention discloses an EV membrane protein single molecule detection method based on droplet microfluidics and application thereof. The method comprises the following steps: firstly, constructing an Ab-oligo compound which is formed by sequentially connecting an antibody, Linker, Linker connection DNA, RNA capable of being specifically cut by RNA enzyme and template DNA for starting signal amplification; then capturing the extracellular vesicles in the plasma by using the CD9 / CD63 / CD81 functionalized magnetic beads; the method comprises the following steps: adding an Ab-oligo compound and a sealing buffer solution into extracellular vesicles, incubating, removing the uncombined Ab-oligo compound, adding RNA enzyme for incubating and enzyme digestion, collecting template DNA for starting signal amplification, and performing microdroplet digital PCR analysis to realize single molecule analysis of EV membrane protein. According to the invention, the one-to-one correspondence relationship between the antibody and oligonucleotide is established through Linker for the first time, single protein information is converted into single nucleic acid chain information, and EV membrane protein single molecule detection is realized. The method is simple to operate, can qualitatively and quantitatively detect the single EV membrane protein, and is beneficial to further research on the relationship between the EV membrane protein and diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and in particular relates to a single-molecule detection method for EV membrane proteins based on droplet microfluidics and its application. Background Art

[0002] Extracellular vesicles (EVs) are nanovesicles with a lipid bilayer structure that are secreted by cells. They carry signaling molecules derived from the parent cell, such as proteins, DNA, RNA, and lipids, and can be stably present in various bodily fluids, including blood, urine, and saliva. Functional nucleic acids and active proteins in EVs are two widely studied types of biomarkers, with membrane protein markers carried by EVs being particularly representative. These membrane proteins, as the most direct functional molecules, are reportedly involved in various stages of cancer development and progression, and have the potential to be used for early cancer diagnosis. For example, GPC1 can be used for the early diagnosis of pancreatic cancer with a sensitivity and specificity of 100%, while a marker panel composed of four EV membrane proteins has an accuracy rate exceeding 96% for colorectal cancer detection.

[0003] In recent years, EV membrane protein detection technologies have evolved from traditional Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) to innovative, interdisciplinary biosensor technologies based on optics, electrochemistry, and microfluidics, significantly improving the diagnostic performance of tumor EV protein markers. However, most of these technologies still rely on batch testing. Because tumor tissue secretes relatively few EVs in the early stages, their membrane proteins are often masked by a large number of normal proteins, resulting in levels far below the limit of detection (LOD) of batch detection technologies, hindering early cancer diagnosis. Therefore, the development of more precise single-molecule analytical techniques for EV membrane protein quantification is expected to facilitate early cancer diagnosis.

[0004] Droplet microfluidics is based on the principle of immiscibility between oil and aqueous phases. With the assistance of surfactants, the continuous aqueous phase is segmented into discrete microdroplets by the oil phase. These droplets can be used to encapsulate single particles (such as cells, bacteria, viruses, etc.) or single molecules. Subsequently, signal amplification within the droplets enables high-throughput, robust, and absolutely quantitative single-molecule digital analysis. Currently, the most widely used technologies in this field are single-cell sequencing and single-molecule nucleic acid analysis based on droplet digital PCR (ddPCR). In recent years, several studies have reported novel methods for EV detection based on droplet fluidics. For example, a dual-targeting aptamer recognition and activation in situ ligation system on the exosome membrane has successfully achieved quantification of tumor-derived exosome-derived PD-L1, thereby successfully distinguishing cancer patients. Ko et al. developed an immuno-ddPCR amplification method for multiplexed analysis of EV proteins. In our previous work, we developed a droplet digital ExoELISA (Liu C, XuX, Li B, Situ B, Pan W, Hu Y, An T, Yao S, Zheng L. Single-Exosome-CountingImmunoassays for Cancer Diagnostics. NanoLetters, 2018, 18(7): 4226-4232.) and a droplet digital immuno-PCR for digital quantification of specific membrane protein expression in single EVs (Liu C#, Lin H#, Guo J,Yang C, Chen J, Pan W, Cui B, Feng J, Zhang Y, Li B, Yao S,Zheng L. Profilingof single-vesicle surface proteins via droplet digital immuno-PCR for multi-subpopulation extracellular vesicles counting towards cancer diagnostics. Chem. Eng. J. 2023, 471: 144364.).Furthermore, we have developed a quadruple ddPCR technology to enable digital detection of EV-derived mRNA (Liu C, Li B, Lin H, Yang C, Guo J, Cui B, Pan W, Feng J, Luo T, Chu F, Xu X, Zheng L, Yao S. Multiplexed analysis of small extracellular vesicle-derived mRNAs by droplet digital PCR and machine learning improves breast cancer diagnosis. Biosensors and Bioelectronics. 2021, 194, 113615.). However, these methods focus on analyzing single EVs or single molecules of nucleic acid carried by EVs. Studies have reported that high-throughput sequencing based on droplet encoding can simultaneously achieve EV enumeration and single-molecule quantification of specific membrane proteins, but this method has the disadvantages of cumbersome operation and complex workflow. Because structurally intact EVs are a collection of membrane proteins, analysis of individual EV membrane proteins is difficult, and thus single-molecule analysis of EV membrane proteins remains challenging. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a single-molecule detection method for EV membrane proteins based on droplet microfluidics.

[0006] Another object of the present invention is to provide an application of the above-mentioned EV membrane protein single-molecule detection method based on droplet microfluidics.

[0007] The purpose of the present invention is achieved through the following technical solution: a method for single-molecule detection of EV membrane proteins based on droplet microfluidics, comprising the following steps: (1) Preparation of Ab-oligo complex: After connecting the oligonucleotide chain and the linker, it is connected to the antibody to obtain the Ab-oligo complex; the oligonucleotide chain is obtained by sequentially connecting the linker DNA for connecting to the linker, the RNA that can be specifically cut by RNase, and the template DNA for initiating signal amplification; (2) Obtaining template DNA specific for extracellular vesicle membrane proteins: A. Capture extracellular vesicles in plasma using CD9 / CD63 / CD81 functionalized magnetic beads; B. Add Ab-oligo complexes and blocking buffer to the extracellular vesicles obtained in step A, incubate to identify the bound Ab-oligo complexes, and remove unbound Ab-oligo complexes; C. Add RNase for incubation and enzyme digestion. The template DNA used to initiate signal amplification is released from the extracellular vesicles bound to the magnetic beads, and the template DNA used to initiate signal amplification is collected; (3) Droplet digital PCR analysis: The template DNA obtained in step (2) is injected into the droplet microfluidic chip for droplet generation, signal amplification, droplet reading, and data analysis to achieve single-molecule analysis of EV membrane proteins.

[0008] The sequence of the linker DNA in step (1) is as follows: 5'- AAGTATT / ACCAGAAA -3'; wherein the slash represents the position where dRep specifically recognizes, cuts and connects single-stranded DNA.

[0009] The sequence of the RNA that can be specifically cleaved by RNase described in step (1) is as follows: 5'-GCUGUG-3'.

[0010] The template DNA used to initiate signal amplification in step (1) can be arbitrarily designed, preferably without linker DNA and easy to amplify; the preferred sequence is one of the nucleic acids shown below: 5'-TGTTGTAAGGGCCCGTGACTATGTCGAAGCGACCCGGCGATATAATCATTTCCACGCCCGTC-3'; 5'-CATAGGAGAAACTGAGATGCCAACTGTGATGAATGGGCTTATGGTTTGGTGCATTGAAAATGGAACCTCGCCA-3'.

[0011] The antibody described in step (1) is preferably at least one of an EGFR antibody and a MUC1 antibody.

[0012] The CD9 / CD63 / CD81 functionalized magnetic beads described in step (2) are preferably prepared by the following method: 1) Incubate Sulfo-NHS-LC-Biotin with CD9 antibody, CD63 antibody, and CD81 antibody to obtain biotinylated antibodies; 2) The biotinylated antibody was coupled to streptavidin magnetic beads to obtain CD9 / CD63 / CD81 functionalized magnetic beads.

[0013] The biotinylated antibodies described in step 1) can be mixed first and then biotinylated, or the antibodies can be biotinylated separately and then mixed.

[0014] The amount of Sulfo-NHS-LC-Biotin used in step 1) is preferably calculated based on 2.4-2.7 μg antibody to 0.7 nmol; more preferably, 2.6-2.7 μg antibody to 0.7 nmol.

[0015] The incubation operation in step 1) is preferably as follows: first incubate at 20-30°C for 20-40 minutes, then transfer to 2-8°C for incubation for 12-20 hours; more preferably as follows: first incubate at 24-26°C for 25-35 minutes, then transfer to 4°C for incubation for 12-16 hours.

[0016] The coupling operation described in step 2) is preferably as follows: dilute the biotin antibody with PBS, add the streptavidin magnetic beads washed with PBS and incubate; after the incubation, wash with PBS and then block with bovine serum albumin; after blocking, wash with PBS and then resuspend with PBS.

[0017] The amount of magnetic beads used in step 2) is preferably calculated based on 2.4-2.7 μg of antibody to 1 mg of magnetic beads.

[0018] The capture described in step (2) is achieved by mixing CD9 / CD63 / CD81 functionalized magnetic beads and plasma and then incubating them.

[0019] The incubation time is preferably 30 to 90 minutes, more preferably 60 minutes.

[0020] The amount of CD9 / CD63 / CD81 functionalized magnetic beads used in step (2) is preferably calculated based on 10 to 30 μg of magnetic beads per 50 μL of plasma; more preferably, it is calculated based on 20 μg of magnetic beads per 50 μL of plasma.

[0021] The dosage of the Ab-oligo complex in step (2) is preferably 0.10 to 0.5 fmol of Ab-oligo complex per 50 μL of plasma; more preferably 0.15 to 0.3 fmol of Ab-oligo complex per 50 μL of plasma.

[0022] The blocking buffer in step (2) preferably has the following composition: 1× PBS containing 5% w / v BSA, 0.05% w / v dextran sulfate, and 0.2 mg / mL salmon sperm DNA. w / v corresponds to g / mL.

[0023] The amount of blocking buffer used in step (2) is preferably such that the volume of the reaction system is the same as the volume of the plasma.

[0024] The incubation time in step (2) B is preferably 30 to 90 minutes, more preferably 60 minutes.

[0025] The step of removing unbound Ab-oligo complexes in step (2) is preferably as follows: after obtaining the magnetic beads by magnetic force, washing with washing buffer.

[0026] The RNase described in step (2) C is preferably RNase A / T1; more preferably 5000 U / mL RNase A / T1.

[0027] The amount of the RNase used in step (2) C is preferably 0.05 μL or more; more preferably 0.05 to 2 μL.

[0028] The time for the enzymatic digestion in step (2) C is preferably more than 30 seconds; more preferably 30 seconds to 60 minutes.

[0029] The amplification system described in step (3) is preferably as follows: a final concentration of 1× ddPCR universal probe mixture, a final concentration of 900 nM forward primer, a final concentration of 900 nM reverse primer, a final concentration of 300 nM probe, and a template chain, wherein the amount of the template chain is 1 / 10 to 1 / 9 of the system volume.

[0030] The amplification conditions described in step (3) are preferably as follows: initial denaturation at 94°C for 5 minutes; 45 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 2 minutes, and extension; and finally, slow cooling to 4°C.

[0031] The above-mentioned droplet microfluidics-based EV membrane protein single-molecule detection method can be used for qualitative and quantitative analysis of single membrane proteins on extracellular vesicles, can be used to study the composition of membrane proteins on extracellular vesicles, and further analyze the relationship between membrane proteins on extracellular vesicles and diseases.

[0032] Application of the above-mentioned droplet microfluidics-based EV membrane protein single-molecule detection method in gastric cancer detection.

[0033] Experimental design principles such as Figure 1 As shown. First, we designed an oligonucleotide chain (Oligonucleotide template), which contains three functional regions: a linker connection sequence (Connected DNA, used for connection with the linker), an RNA enzyme (RNaseA / T1) specific cleavage sequence, and a DNA template sequence (TemplateDNA) for initiating signal amplification. The bifunctional linker is used to achieve a one-to-one connection between the antibody (Antibody) and the oligonucleotide chain, thus constructing an Ab-oligo complex. The linker not only retains the antibody activity, but also releases the DNA template chain in the oligonucleotide chain through enzymatic cleavage (such as Figure 1To more comprehensively capture EVs in plasma, we prepared functionalized magnetic beads coated with three tetraspanins (CD9 / CD63 / CD81) that can be directly incubated with plasma to capture EVs. Subsequently, the constructed Ab-oligo complex was added to specifically recognize specific membrane proteins on EVs. After washing to remove excess Ab-oligo complexes, RNase A / T1 was added to cleave the RNA sites in the Ab-oligo that recognized specific EV membrane proteins (as shown in Figure 1). Figure 1 Then, the released DNA template strands are collected and injected into a droplet microfluidic chip, where droplet generation, signal amplification, droplet reading, and data analysis are performed to achieve single-molecule analysis of EV membrane proteins (e.g. Figure 1 (as shown in C in the figure).

[0034] The present invention has the following advantages and effects compared to the prior art: (1) This invention establishes a one-to-one correspondence between antibodies and oligonucleotides through a linker for the first time, converting single protein information into single nucleic acid chain information, thus enabling single-molecule detection of EV membrane proteins. This invention is simple to operate and can perform qualitative and quantitative detection of single EV membrane proteins.

[0035] (2) The method provided by the present invention is conducive to further studying the relationship between EV membrane proteins and diseases.

[0036] (3) The method provided by the present invention is applied to the detection of plasma samples from gastric cancer patients, which greatly improves the sensitivity of early gastric cancer diagnosis and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the single-molecule detection method for EV membrane proteins based on droplet microfluidics; A is a schematic diagram of the process of obtaining the Ab-oligo complex; B is a schematic diagram of the process of using two Ab-oligo complexes to recognize EVs in plasma captured by CD9 / CD63 / CD81-functionalized magnetic beads and RNaseA / T1 enzyme cleavage to obtain the DNA template chain; C is a schematic diagram of the analysis of the DNA template chain by droplet digital PCR (ddPCR).

[0038] Figure 2The following are the results of characterization of plasma EVs and verification of the feasibility of functionalized magnetic beads in capturing EVs; A is the TEM image of plasma-derived EVs; B is the size distribution and concentration of diluted plasma-derived EVs determined by NTA; C is the expression of specific protein markers (CD9, CD63, TSG101 and calnexin) in EV samples analyzed by WB; D is the naked functionalized microspheres (Negative Control), functionalized microspheres that capture plasma EVs (Plasma-EV), and functionalized microspheres that capture EVs in plasma (Plasma); E is the result of verifying the activity of functionalized magnetic beads using flow cytometry.

[0039] Figure 3 This is a diagram showing the feasibility of Ab-oligo complexes capturing specific EV membrane proteins. A verifies the feasibility of linking the Linker to the oligonucleotide chain Ts3, and B shows the activity verification result of the Ab-oligo complex.

[0040] Figure 4 The figure shows the feasibility and condition optimization results of RNase A / T1 verified by flow cytometry. A shows the feasibility verification results of enzyme digestion, B shows the enzyme digestion results with different dosages of RNase A / T1, and C shows the enzyme digestion results with different incubation times.

[0041] Figure 5 It is a feasibility result diagram of single-molecule analysis of EV membrane proteins; among them, A is the one-dimensional fluorescence image of the DNA template chain before and after washing in the CY5 channel; B is the one-dimensional fluorescence image after the DNA template chain is recovered; C is the removal rate and recovery rate of the DNA template chain, and the error bars represent the standard deviation of three repeated experiments; D is the feasibility result of using CD63 as a target to verify the use of EV membrane proteins for single-molecule analysis.

[0042] Figure 6 Figure 1 is a performance evaluation of the detection method; A is a fluorescent droplet image of the DNA template chain stock solution; B is a fluorescent droplet image of the DNA template chain diluted 10 times; C is a fluorescent droplet image of the DNA template chain diluted 100 times; D is a fluorescent droplet image of the DNA template chain diluted 1000 times; E is a fluorescent droplet image of the negative control; F is a linear relationship diagram between the expected value and the measured value, and the error bars represent the standard deviation of three repeated experiments; G is a schematic diagram comparing the principles of ELISA and droplet microfluidics-based detection methods; H is the detection results of MUC1 expression levels on EVs by ELISA and droplet microfluidics detection methods; I is the detection results of EGFR expression levels on EVs by ELISA and droplet microfluidics detection methods; in the figure, the scale bar of the image is 400 μm, **** P <0.0001, ns indicates no statistically significant difference.

[0043] Figure 7 : These are the results of the diagnostic performance evaluation of EGFR and MUC1 in droplet analysis; wherein, A is a heat map of the concentrations of EGFR and MUC1 on EVs, B is a scatter plot of the concentrations of EGFR on EVs, C is a scatter plot of the concentrations of MUC1 on EVs, D is the ROC analysis result of EGFR and MUC1 in healthy donors using the method of the present invention, E is the ROC analysis result of CEA and CA199 in healthy donors using the conventional method, F is the ROC analysis result of EGFR and MUC1 in patients with benign gastric cancer, and G is the ROC analysis result of CEA and CA199 in patients with benign gastric cancer; ****P<0.0001, ns indicates no significance. DETAILED DESCRIPTION

[0044] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0045] Example 1 1. Materials and Methods 1.1 Materials 1.1.1 Research subjects The use of clinical plasma samples in this study was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. Plasma samples from healthy individuals and patients with benign gastric disease were collected from the Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University (Guangzhou, China), and plasma samples from patients with gastric cancer (GC) were collected from the Department of General Surgery, Nanfang Hospital. The diagnosis of patients with benign gastric disease and GC was confirmed by pathological examination of tissue biopsies.

[0046] 1.1.2 Main reagents and consumables Table 1 Main reagents and consumables

[0047] Table 2 Summary of instrument names and manufacturers

[0048] Table 3 Nucleic acid sequences required for the experiment

[0049] The above DNA sequences were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0050] 1.2 Methods 1.2.1 Collection and storage of plasma samples Venous blood samples were collected using EDTA tubes and centrifuged twice at 2,500 g for 15 minutes at room temperature to remove excess blood cells. Plasma was aliquoted and stored at −80°C for subsequent analysis.

[0051] 1.2.2 Extraction of plasma EVs In this study, pooled plasma from healthy individuals was diluted 1:2 with phosphate-buffered saline (PBS) and centrifuged at 3,000 g for 20 minutes to remove cellular debris. Subsequently, the supernatant was centrifuged at 16,000 g for 30 minutes to remove microvesicles. The resulting supernatant was further subjected to ultracentrifugation (UC) at 135,000 g for 70 minutes, repeated twice, to isolate EVs. The resulting EVs were resuspended in 100 μL of PBS, and all centrifugation steps were performed at 4°C. The EVs were stored at -80°C for subsequent analysis.

[0052] 1.2.3 Characterization of plasma EVs EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (WB). In TEM analysis, EVs samples were placed on a copper mesh slide, fixed for 5 minutes, stained with phosphotungstic acid for 5 minutes, and then washed with deionized water. After the samples were dried at room temperature, they were observed using TEM. In NTA analysis of EVs concentration and size distribution, EVs samples were diluted to approximately 10 9 Particles / mL were measured to achieve optimal counting efficiency. Particle size distribution data were captured and analyzed using NTA 3.4 analysis software. Western blotting was used to detect protein markers in plasma EVs, with gastric cancer cell Mkn28 cells used as a control. EV samples were mixed with 5× SDS loading buffer for protein denaturation. The denatured protein samples were loaded onto polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked with rapid blocking buffer. The membranes were incubated overnight at 4°C with primary antibodies against CD9, CD63, TSG101, and calnexin, followed by further incubation with corresponding goat anti-rabbit enzyme-linked secondary antibodies. Finally, the samples were processed using an enhanced chemiluminescence (ECL) kit and imaged.

[0053] 1.2.4 ddPCR Experimental Process All oligonucleotides were synthesized and provided by Sangon Biotech and dissolved in 1× TE buffer. Details of the oligonucleotide sequences and their final concentrations are provided in Table 3. The final volume of the PCR amplification system was 20 μL, consisting of: 10 μL of 2× ddPCR universal probe mix, 1.8 μL of forward primer, 1.8 μL of reverse primer, 0.6 μL of 10× probe, 3.8 μL of nuclease-free water, and 2 μL of template strand reaction (template strand 1 used forward primer 1, reverse primer 1, and probe 1-CY5; template strand 2 used forward primer 2, reverse primer 2, and probe 2-FAM). The reaction mixture, along with the droplet generation oil, was loaded onto the ddPCR droplet generation chip for droplet generation. The generated droplets were transferred to an eight-tube strip and amplified on a thermal cycler for DNA target sequence amplification. Based on an annealing temperature gradient optimization experiment, the final thermal cycling conditions were as follows: initial denaturation at 94°C for 5 minutes; 45 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 2 minutes, and extension; and finally, slow cooling to 4°C. The amplified droplets were injected into a droplet analysis chip, and the experimental results were read and analyzed using the ExoStar Droplet Reader.

[0054] 1.2.5 Preparation of functionalized magnetic beads Sulfo-NHS-LC-Biotin was dissolved in PBS to a concentration of 10 mM and further diluted 100-fold with PBS to a final concentration of 0.1 mM. Subsequently, 7 μL of Sulfo-NHS-LC-Biotin was added to 1.65 μL of 0.53 mg / mL CD9 antibody, 1.65 μL of 0.53 mg / mL CD63 antibody, and 1.65 μL of 0.53 mg / mL CD81 antibody. The mixture was incubated at room temperature (25°C) for 30 minutes and then transferred to 4°C for overnight incubation (16 hours) to obtain biotinylated antibodies. The biotinylated antibody was then conjugated to streptavidin-coated magnetic beads as follows: 100 μL of 10 mg / mL streptavidin-coated magnetic beads was aspirated and washed twice with 100 μL PBS. 100 μL PBS was added to the biotinylated antibody solution, and the magnetic beads were incubated with 100 μL of the antibody solution at room temperature for 50 minutes. The beads were then washed three times with 100 μL PBS, blocked with 100 μL 1% bovine serum albumin (BSA) for 30 minutes, washed three times again with 100 μL PBS, and finally resuspended in 100 μL PBS. Functionalized polystyrene microspheres (CD9 / CD63 / CD81 functionalized microspheres conjugated to biotinylated antibodies) were prepared using the same method for subsequent scanning electron microscopy (SEM) analysis. All functionalized magnetic beads and microspheres were stored at 4°C until further use.

[0055] 1.2.6 Scanning Electron Microscope (SEM) Analysis To 150 μL of PBS, 50 μL of healthy human plasma pretreated as described in 1.2.1 and 50 μL of diluted plasma-derived EVs were added, followed by 2 μL of 10 mg / mL CD9 / CD63 / CD81-functionalized microspheres. The samples were incubated at room temperature for 1 hour to obtain two samples. The samples were then washed twice with 50 μL of PBS and resuspended in 50 μL of PBS. The samples were further fixed with 2% v / v paraformaldehyde and 2.5% v / v glutaraldehyde for 2 hours. Dehydration was performed using a gradient of ethanol (50%, 70%, 80%, 90%, and 100%), followed by rinsing with PBS after each step to remove residual ethanol. Ethanol was then replaced with isoamyl acetate, and residual water was further removed. The samples were then freeze-dried overnight, and the microspheres were sputtered with gold to enhance conductivity for SEM imaging using an Apero 2S HiVac system.

[0056] 1.2.7 Flow cytometry feasibility verification of functionalized magnetic beads After preparing CD9 / CD81-functionalized magnetic beads according to step 1.2.5, add 0.5 μL of FITC-fluorescent anti-CD63 antibody to 2 μL of functionalized magnetic beads. One group was added with PBS to a final volume of 50 μL as a negative control; the other group was added with diluted plasma-derived EVs to a final volume of 50 μL as a positive control. All mixtures were incubated at room temperature for 1 hour and then washed three times with 50 μL of PBS. Finally, the cells were analyzed by flow cytometry.

[0057] 1.2.8 Verification of Linker-Oligo Coupling To the reaction system, 2 μL of an oligonucleotide labeled with a fluorescent group (FAM) and carrying a quencher group at the other end (100 μM, Ts3) was added, along with 10 μL of 2× ssDNA buffer (100 mM HEPES, 300 mM NaCl, 2 mM MgCl2, 2 mM MnCl2, pH 8.0), and 4.5 μL of ultrapure water. The negative control group was supplemented with 3.5 μL of ultrapure water, while the experimental group was supplemented with 3.5 μL of Linker (1 mg / mL). The mixture was incubated at 37°C for 1 hour. After incubation, 5 μL of Linker-Ts3-FAM was diluted 12-fold and analyzed using a fluorescence spectrophotometer.

[0058] According to the above method, Linker-Ts4-FAM was prepared.

[0059] 1.2.9 Activity Verification of Ab-oligo Complex After constructing the Ab-oligo complex, we further validated its activity. Previous studies have shown that the S9.6 antibody (Ab S9.6) specifically recognizes DNA-RNA hybrids. Therefore, we modified magnetic beads with DNA-RNA hybrids and added the constructed S9.6-oligo complex with a fluorescent moiety (Ab S9.6-Ts4-FAM). The specific steps were as follows: 5 μL of 6.4 μmol / L Linker-Ts4-FAM and 3 μL of 10.7 μmol / L anti-DNA-RNA hybrid S9.6 antibody were added to a test tube and incubated at 4°C for 1 hour to form the Ab-oligo complex (S9.6-Ts4-FAM). The complex was then diluted to 30 μL with PBS and stored at 4°C until use. Meanwhile, DNA-RNA hybrid double-stranded modified magnetic beads were prepared: 2.5 μL of DNA 1 (10 μM) and 2.5 μL of RNA 1 (20 μM) were added to 5 μL of 10 mg / mL streptavidin magnetic beads. The beads were incubated at room temperature for 1 hour, washed three times with 100 μL of PBS, and stored at 4°C for subsequent experiments. Prior to analysis, 2 μL of 4 μmol / L S9.6-Ts4-FAM and 46 μL of PBS were added to the tubes. One group was treated with 2 μL of bare magnetic beads as a negative control, while the other group was treated with 2 μL of DNA-RNA hybrid double-stranded modified magnetic beads. After incubation at room temperature for 1 hour, the samples were washed twice with 100 μL of PBS and analyzed by flow cytometry.

[0060] 1.2.10 Enzyme Digestion Feasibility Verification and Condition Optimization To verify the feasibility of enzymatic digestion of the template strand, 1 μL of 10 μM FAM-labeled biotinylated template strand (Ts5-FAM) was incubated with 2 μL of streptavidin magnetic beads in 50 μL of PBS at room temperature for 1 hour. The beads were then washed twice with 50 μL of PBS. One tube served as a positive control, while the other tube was incubated with 1 μL of RNase A / T1 (5000 U / mL) and incubated for another hour at room temperature. After incubation, both tubes were washed twice with PBS as experimental controls. A negative control consisted of 1 μL of 10 μM unlabeled biotinylated template strand (Ts6) mixed with 2 μL of streptavidin magnetic beads. All samples were analyzed by flow cytometry. In addition, we evaluated the effects of varying RNaseA / T1 volumes (2 μL, 1.5 μL, 1 μL, 0.5 μL, 0.25 μL, 0.1 μL, and 0.05 μL) and incubation times (60 minutes, 40 minutes, 20 minutes, 10 minutes, 5 minutes, 3 minutes, 2 minutes, 1 minute, and 30 seconds) on the digestion conditions. When evaluating different RNaseA / T1 volumes, the incubation time was 1 hour; when evaluating the incubation time, 1 μL of RNaseA / T1 was used.

[0061] 1.2.11 Feasibility Verification of Single-Molecule Detection Methods Because this method has single-molecule sensitivity, any excess Ab-oligo complexes in the solution or nonspecific adsorption of DNA template strands released by enzymatic cleavage may affect the subsequent quantification of EV membrane proteins. To evaluate the accuracy of this method, we verified the efficiency of washing to remove excess Ab-oligo complexes and recovering DNA template strands released by enzymatic cleavage. The specific steps are as follows: 2 μL of CD9 / CD81 magnetic beads were added to the experimental group, along with 43 μL of blocking buffer (1× PBS containing 5% BSA, 0.05% dextran sulfate, and 0.2 mg / mL salmon sperm DNA, the same below) and 5 μL of CD63 complex (CD63 antibody-Ts1, 120 μM). After incubation for 1 hour, the solution was discarded and the beads were washed twice with wash buffer 1 and twice with wash buffer 2, using 50 μL each wash. 0.1 μL of RNase A / T1 was added and the volume was made up to 50 μL with PBS. After incubation at room temperature for 20 minutes, the supernatant was collected and 2 μL was used for ddPCR analysis. To the positive control group, 45 μL PBS and 5 μL complex (CD63 antibody-Ts1, 120 μM) were added, and 2 μL was taken for ddPCR detection.

[0062] Before recovery: add 43 μL PBS and 5 μL 10 pm Ts1 nucleic acid chain, react for 1 hour, collect the supernatant, and take 2 μL for ddPCR detection.

[0063] After recovery: add 2 μL CD9 / CD81 magnetic beads to the experimental group, add 43 μL blocking buffer, react for 1 hour (block the magnetic beads), wash twice with 50 μL PBS, add 43 μL PBS and 5 μL 10 pm Ts1 nucleic acid chain to the magnetic beads, react for 1 hour, collect the supernatant, and take 2 μL for ddPCR detection.

[0064] Wash buffer 1 consisted of PBS containing 5% BSA and 0.05% dextran sulfate, and wash buffer 2 consisted of PBS containing 0.1% salmon sperm.

[0065] 1.2.12 Flow of this experimental method To simultaneously detect epidermal growth factor receptor (EGFR) and mucin 1 (MUC1), 2 μL of 10 mg / mL CD9 / CD63 / CD81-functionalized magnetic beads was added to 50 μL of plasma. The mixture was incubated on a rotary mixer for 1 hour at room temperature to capture and enrich EVs. The EV-captured beads were then recovered by magnetic attraction on a magnetic stand. The sample was then washed twice with 50 μL of PBS to remove residual plasma. EGFR-oligo complex (using Ts2) and MUC1-oligo complex (using Ts1) were prepared as previously described (i.e., using EGFR and MUC1 antibodies in steps 1.2.8 and 1.2.9). The final concentration was adjusted to 60 pM in PBS. 2.5 μL of EGFR-oligo complex and 2.5 μL of MUC1-oligo complex were added to the sample, and the mixture was brought up to 50 μL with blocking buffer. The mixture was then incubated at room temperature for 1 hour. After incubation, wash twice with wash buffer 1 and then twice with wash buffer 2, each time using 50 μL. Then, add 0.1 μL RNase A / T1 and make up to 50 μL with PBS. Incubate at room temperature for 20 minutes, then collect the supernatant for ddPCR analysis.

[0066] 1.2.13 Exploration of ddPCR platform detection sensitivity and linear detection range 2 µL of DNA template chains of different concentrations (10 pm, 1 pm, 100 fm, 10 fm) were added to the ddPCR platform detection system for reaction. After the reaction, the positive droplets produced by the system were analyzed and counted using a fluorescence microscope. The test was repeated three times for each concentration of DNA template chain sample, and the test results were recorded. The linear range and detection limit of the ddPCR platform for detecting DNA template chains were calculated and analyzed to explore the detection sensitivity of the ddPCR system.

[0067] 1.2.14 ELISA test process Plasma samples from 15 healthy individuals and 15 GC patients were collected and processed as described above. EGFR levels in 100 μL of plasma were assayed using an EGFR-ELISA kit. The following steps were performed: Standards were serially diluted in sample diluent; plasma samples were added to the diluent to a final volume of 200 μL. Standards and diluted samples were added to a microplate and incubated at 37°C for 90 minutes. The liquid in the wells was discarded, and 100 μL of biotinylated antibody working solution was added, followed by incubation at 37°C for 1 hour. Each well was washed three times with 350 μL of wash buffer, followed by the addition of 100 μL of HRP conjugate working solution, and incubation at 37°C for 30 minutes. After five washes, 90 μL of TMB chromogenic substrate was added, and the cells were incubated at 37°C in the dark for 15 minutes. Finally, 50 μL of stop solution was added, and absorbance was measured at 450 nm. MUC1 detection followed the same procedure.

[0068] 2. Results 2.1 Characterization of plasma EVs and feasibility verification of EV capture using functionalized magnetic beads Figure 2 As shown, it can be seen that extracellular vesicles mainly distributed in the range of 50 to 200 nm were obtained (see Figure 2 Extracellular vesicles (Plasma-EVs) obtained from healthy human plasma expressed CD9, CD63, and TSG101, and gastric cancer cell Mkn28 expressed CD63, TSG101, and Calnexin (see Figure 2 Functionalized microspheres can effectively capture EVs in plasma (see Figure 2 D in the figure); Flow cytometry was used to verify the activity of the functionalized magnetic beads. Figure 2 The blue upper picture of E is the PBS negative control group, and the red lower picture is the diluted EVs positive control group. Since the CD9 / CD81 functionalized magnetic beads capture EVs, the FITC fluorescent Anti-CD63 antibody recognizes EVs and binds to them, showing fluorescence.

[0069] 2.2 Effect of oligonucleotide Ts3 binding to linker Figure 3 As shown in A: Compared with the negative control Ts3, the experimental group Ts3+Linker showed a significant fluorescence signal after the addition of Linker, proving that Linker can successfully bind to the oligonucleotide chain.

[0070] 2.3 Flow cytometric detection results of Ab-oligo complex capturing S9.6 antibody Figure 3 As shown in Figure B: Compared with the negative control, the positive group showed a significant fluorescence signal, proving that the Ab-oligo complex maintained functional activity and could be used for subsequent EV membrane protein identification.

[0071] 2.4 RNase A / T1 digestion feasibility results Figure 4 As shown: RNaseA / T1 can effectively cut the oligonucleotide chain (see Figure 4 A in the figure); RNase A / T1 should be used in an amount of 0.05 μL or more and the enzyme digestion time should be more than 30 s to achieve effective cleavage (see Figure 4 B and C in ).

[0072] 2.5 The feasibility verification results of the method of the present invention are as follows Figure 5 As shown: The results showed that the removal rate of Ab-oligo complex and the recovery rate of DNA template chain were 99.89% ± 0.08185% and 102.9% ± 5.732%, respectively (see Figure 5 A large body of evidence indicates that CD63 is one of the most abundant protein markers in EVs. We further validated the feasibility of single-molecule analysis of EV membrane proteins using CD63 as a target. Compared with the negative control group, the expression of CD63 in plasma and plasma-derived EVs was significantly increased, demonstrating the feasibility of this detection system (see Figure 5 D in the figure).

[0073] 2.6 The dynamic range of this assay spans four orders of magnitude. Figure 6 The results of AF in the experiment showed that the concentration of DNA template chain showed a good linear relationship with the dilution factor, with an R² correlation coefficient of 0.989 and a detection range of 0.90-11265.78 copies / μL. In addition, we evaluated the limit of quantification (LOQ) of this method, and the results showed that its lowest limit of quantification was 18 copies / 20μL reaction system. Subsequently, we compared this method with ELISA, which is commonly used for EV protein quantification in clinical samples. Previous studies have confirmed that EGFR and MUC1 have good diagnostic potential for GC. Therefore, we divided the plasma samples of 15 healthy individuals and 15 GC patients into two groups and detected EGFR and MUC1 using this method and ELISA, respectively (see Figure 6 G in ). Figure 6 As shown in Figures H and I, there was no significant statistical difference in the two markers detected by ELISA between the healthy control group and the GC patient group, while the method provided by the present invention was able to significantly distinguish the two groups, indicating that it has excellent detection performance and potential application value in GC diagnosis.

[0074] 2.7 To evaluate the applicability of this method for GC diagnosis in clinical samples, we expanded the GC cohort to 90 subjects, including 15 healthy controls, 15 patients with benign gastric diseases, and 60 patients with malignant tumors (15 with stage I, 15 with stage II, and 30 with stage III-IV). Figure 7The heatmap in Figure A summarizes the expression levels of EGFR and MUC1 on EVs in the GC cohort determined by this method. Figure 7 Figures B and C show the concentration distribution of these two markers in healthy individuals, patients with benign diseases, and all GC patients. It is worth noting that there are significant differences in the marker concentrations between GC patients and healthy controls or benign disease groups.

[0075] Subsequently, we performed receiver operating characteristic (ROC) curve analysis to evaluate the diagnostic performance of EGFR and MUC1 in distinguishing GC patients from healthy donors or patients with benign diseases and compared them with traditional markers CEA and CA199 (see Figure 7 The results showed that combined EGFR and MUC1 detection had the highest AUC value of 0.9989 [95% confidence interval (CI): 0.9952 - 1.000] for distinguishing healthy controls from GC patients, with a sensitivity of 98.33% (95% CI: 91.14% - 99.91%) and a specificity of 100% (95% CI: 79.61% - 100%). Similarly, combined EGFR and MUC1 detection had the highest AUC value of 0.9944 (95% CI: 0.9823 - 1.000) for distinguishing patients with benign diseases from GC patients, with a sensitivity of 98.33% (95% CI: 91.14% - 99.91%) and a specificity of 100% (95% CI: 79.61% - 100%). We found that the diagnostic performance of these two markers was significantly better than that of traditional markers in distinguishing GC patients from healthy donors or patients with benign diseases, indicating that this detection method can be further applied to the accurate diagnosis of GC.

[0076] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for single-molecule detection of EV membrane proteins based on droplet microfluidics, characterized in that The steps include: (1) Preparation of Ab-oligo complex: After connecting the oligonucleotide chain and the linker, it is connected to the antibody to obtain the Ab-oligo complex; the oligonucleotide chain is obtained by sequentially connecting the linker DNA for connecting to the linker, the RNA that can be specifically cut by RNase, and the template DNA for initiating signal amplification; (2) Obtaining template DNA specific for extracellular vesicle membrane proteins: A. Capture extracellular vesicles in plasma using CD9 / CD63 / CD81 functionalized magnetic beads; B. Add Ab-oligo complexes and blocking buffer to the extracellular vesicles obtained in step A, incubate to identify the bound Ab-oligo complexes, and remove unbound Ab-oligo complexes; C. Add RNase for incubation and enzyme digestion. The template DNA used to initiate signal amplification is released from the extracellular vesicles bound to the magnetic beads, and the template DNA used to initiate signal amplification is collected; (3) Droplet digital PCR analysis: The template DNA obtained in step (2) is injected into the droplet microfluidic chip for droplet generation, signal amplification, droplet reading, and data analysis to achieve single-molecule analysis of EV membrane proteins.

2. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The sequence of the linker DNA in step (1) is as follows: 5'- AAGTATT / ACCAGAAA -3'; The sequence of the RNA that can be specifically cleaved by RNase described in step (1) is as follows: 5'-GCUGUG-3'; The template DNA used to initiate signal amplification in step (1) is template DNA-1 with a nucleotide sequence as shown in SEQ ID NO.1 or template DNA-2 with a nucleotide sequence as shown in SEQ ID NO.2; The antibody described in step (1) is at least one of an EGFR antibody and a MUC1 antibody.

3. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The CD9 / CD63 / CD81 functionalized magnetic beads described in step (2) were prepared by the following method: 1) Incubate Sulfo-NHS-LC-Biotin with CD9 antibody, CD63 antibody, and CD81 antibody to obtain biotinylated antibodies; 2) The biotinylated antibody was coupled to streptavidin magnetic beads to obtain CD9 / CD63 / CD81 functionalized magnetic beads.

4. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 3, characterized in that: The dosage of Sulfo-NHS-LC-Biotin described in step 1) is calculated as 2.4-2.7 μg antibody to 0.7 nmol; The incubation operation described in step 1) is as follows: first incubate at 20-30°C for 20-40 minutes, then transfer to 2-8°C and incubate for 12-20 hours; The coupling operation described in step 2) is as follows: dilute the biotin antibody with PBS, add the streptavidin magnetic beads that have been washed with PBS, and incubate; after the incubation, wash with PBS, and then block with bovine serum albumin; after blocking, wash with PBS, and then resuspend in PBS; The amount of magnetic beads described in step 2) is calculated based on 2.4-2.7 μg antibody to 1 mg magnetic beads.

5. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The dosage of CD9 / CD63 / CD81 functionalized magnetic beads described in step (2) is calculated based on 10-30 μg of magnetic beads per 50 μL of plasma; The dosage of the Ab-oligo complex described in step (2) is 0.10 to 0.5 fmol Ab-oligo complex per 50 μL of plasma; The amount of RNase used in step (2) C is 0.05 μL or more.

6. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The blocking buffer described in step (2) was composed of the following: 1× PBS containing 5% w / v BSA, 0.05% w / v dextran sulfate, and 0.2 mg / mL salmon sperm DNA; The RNase described in step (2) C is RNase A / T1.

7. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The incubation time in step (2) B is 30 to 90 minutes; The steps for removing unbound Ab-oligo complexes described in step (2) are as follows: after obtaining the magnetic beads by magnetic force, washing with washing buffer; The enzyme digestion time in step (2) C is more than 30 seconds.

8. The method for single-molecule detection of EV membrane proteins based on droplet microfluidics according to claim 1, characterized in that: The amplification system described in step (3) is as follows: a final concentration of 1× ddPCR universal probe mixture, a final concentration of 900 nM forward primer, a final concentration of 900 nM reverse primer, a final concentration of 300 nM probe, and a template chain, where the amount of the template chain is 1 / 10 to 1 / 9 of the system volume; The amplification conditions described in step (3) were as follows: initial denaturation at 94°C for 5 minutes; 45 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 2 minutes, and extension; and finally, slow cooling to 4°C.

9. The droplet microfluidics-based EV membrane protein single-molecule detection method according to any one of claims 1 to 8 can be used to study the composition of membrane proteins on extracellular vesicles.

10. Application of the EV membrane protein single-molecule detection method based on droplet microfluidics according to any one of claims 1 to 8 in gastric cancer detection.

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