Application of plasma small extracellular vesicle miRNA markers, detection method and detection reagent for expression amount

Abstract

The application provides an application of a plasma small extracellular vesicle miRNA marker, a detection method and a detection reagent of an expression amount, the application of the plasma small extracellular vesicle miRNA marker in preparation of a reagent for detecting an expression amount of a lung cancer-derived small extracellular vesicle, and the plasma small extracellular vesicle miRNA marker is a combination of miR-21-5p, miR-375-3p and miR-451a with nucleotide sequences such as SEQ.No.1-3.The application can realize efficient capture, mild release and in-situ synchronous detection of internal miRNA of small extracellular vesicles.Methodological evaluation shows that the detection limit is low, the recovery rate is good, and the application can provide a molecular basis with high specificity and clinical relevance for lung cancer screening and auxiliary diagnosis.

Description

Technical Field

[0001] This invention relates to the field of molecular detection, specifically to the application of plasma small cell extracellular vesicle miRNA markers, methods for detecting their expression levels, and detection reagents. Background Technology

[0002] Small extracellular vesicles (sEVs) are considered an ideal entry point for liquid biopsy due to their abundant content in body fluids, high stability, and rich content of tumor-specific molecular targets. Screening for tumor-specific biomarkers can provide highly specific molecular targets for lung cancer screening and auxiliary diagnosis, thereby improving the sensitivity and specificity of screening and supporting early and accurate diagnosis. Studies have shown that small RNAs in sEVs, including microRNAs (miRNAs), are considered potential biomarkers for various cancers. However, current detection methods typically use RT-qPCR for quantitative detection. While this technology can reliably confirm the expression trends of candidate miRNAs in plasma sEVs from lung cancer patients and healthy controls, it can only detect one target at a time, resulting in low detection efficiency. Furthermore, the sEVs must first be lysed, which can degrade miRNAs and affect detection accuracy.

[0003] Patent application CN114107503A discloses a composite locked nucleic acid magnetic bead probe for detecting miRNA markers, its construction method, and a diagnostic reagent containing this composite probe. The composite locked nucleic acid magnetic bead probe contains three probes within a single magnetic bead: an LNA probe-486-5p probe labeled with the FAM fluorescent group, an LNA probe-21 probe labeled with the Cy5 fluorescent group, and an LNA probe-210 probe labeled with the TAMRA fluorescent group. This method immobilizes the modified fluorescently labeled DNA probe molecules onto the magnetic beads via biotin-streptavidin linkage. The DNA probe sequence matches the target miRNA and hybridizes to form a double strand. Under the action of DSN enzyme, the DNA probe in the hybrid double strand is hydrolyzed into fragments, and the fluorescent group molecules are suspended in the reaction solution, while the miRNA remains intact and hybridizes again with the DNA probe, undergoing DSN digestion. This process is repeated, resulting in a significant amplification of the number of fluorescent groups in the final system. Sensitive detection of target miRNA concentration can be achieved by testing the signal intensity of fluorescent groups in the supernatant. This method allows for convenient and ultrasensitive simultaneous detection of three different lung cancer biomarker miRNAs in body fluids, enabling accurate diagnosis of lung cancer. However, this method detects free lung cancer biomarker miRNAs in body fluids, which are susceptible to degradation and interference from body fluids. Furthermore, this method utilizes magnetic beads to capture probes and performs DSN enzyme cycling amplification, which carries the risk of cross-hybridization of multiple probes and can lead to high background signals due to unreacted probes becoming directly free. Summary of the Invention

[0004] To improve the detection effect and accuracy of lung cancer screening and auxiliary diagnosis, the technical solution adopted in this invention is: the application of a plasma small extracellular vesicle miRNA marker in the preparation of a reagent for detecting the expression level of lung cancer-derived small extracellular vesicles, wherein the plasma small extracellular vesicle miRNA marker is a combination of miR-21-5p, miR-375-3p and miR-451a with nucleotide sequences such as SEQ.No.1-3.

[0005] Based on the above, the small extracellular vesicles are derived from lung squamous cell carcinoma cells, lung adenocarcinoma cells, and small cell lung cancer cells.

[0006] The present invention also provides a detection reagent for detecting the expression level of lung cancer-derived small extracellular vesicles, including a reagent for targeting and capturing plasma small extracellular vesicles, free biotin, and Lip@AuNFs@DSN; The reagent for targeting and capturing small extracellular vesicles in plasma includes: streptavidin-modified magnetic beads and three dethiobiotin-labeled nucleic acid aptamers (Apt) coupled to the surface of the streptavidin-modified magnetic beads. EpCAM Apt PD-L1Apt EGFR Three nucleic acid aptamers Apt EpCAM Apt PD-L1 Apt EGFR The nucleotide sequences are shown in SEQ.No.8-10, and the three nucleic acid aptamers correspond to EpCAM, PD-L1 and EGFR, which target small extracellular vesicles, respectively; The free biotin is used to competitively replace the desulfobiotin aptamer-sEVs complex bound to the surface of the streptavidin-modified magnetic beads and release intact plasma microcellular extracellular vesicles. Lip@AuNFs@DSN is a multifunctional liposome containing gold nanoparticle flares and a DSN enzyme encapsulated with three fluorescently labeled DNA probes. It comprises: liposomes and gold nanoparticle flares and a DSN enzyme encapsulated within the liposomes; wherein the liposomes are cationic liposomes prepared by thin-film hydration using 1,2-dioleoyl-3-trimethylammonium propane, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol, and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, serving to provide a release site for miRNA within sEVs after membrane fusion and to encapsulate the reaction system; the gold nanoparticles... The particle flare comprises gold nanoparticles and three DNA probes cDNA21, cDNA375, and cDNA451, such as SEQ. No. 12-14, coupled to the surface of the gold nanoparticles. DNA probe cDNA21 is labeled with the FAM fluorescent group, DNA probe cDNA375 is labeled with the TAMRA fluorescent group, and DNA probe cDNA451 is labeled with the Cy5 fluorescent group. The DSN enzyme is a double-stranded specific nuclease used to cleave lung cancer-derived small cell extracellular vesicle miRNA and the corresponding DNA probe to form a DNA-RNA double strand.

[0007] Based on the above, the reagent for targeting and capturing small extracellular vesicles is prepared through the following steps: First, three dethiobiotin-labeled nucleic acid aptamers are synthesized, and the three nucleic acid aptamers Apt... EpCAM Apt PD-L1 Apt EGFR The reagent for targeting and capturing small extracellular vesicles was prepared by targeting EpCAM, PD-L1, and EGFR of small extracellular vesicles, respectively, and then using the affinity between streptavidin magnetic beads and dethiobiotin to couple the three nucleic acid aptamers to the surface of streptavidin magnetic beads.

[0008] Based on the above, the Lip@AuNFs@DSN is prepared by the following steps: first, gold nanoparticles are prepared using the sodium citrate reduction method; then, three fluorescently labeled DNA probes are coupled to the surface of the gold nanoparticles to obtain gold nanoflares; finally, the gold nanoflares and DSN enzyme are dissolved in a reaction buffer for preparing cationic liposomes by the thin-film hydration method, and cationic liposomes are prepared by the thin-film hydration method using the reaction buffer to obtain the Lip@AuNFs@DSN.

[0009] Based on the above, the biotin concentration is 100 mmol / L and the DSN enzyme dosage is 1.0 U.

[0010] Based on the above, the detection limit of the detection reagent for miR-21-5p in sEVs membrane is 2.7 × 10⁻⁶. 2 The detection limit for miR-375-3p in sEVs membranes was 4.0 × 10⁻⁶ particles / µL. 2 The detection limit for miR-451a in sEVs membrane particles / µL is 1.45×10⁻⁶. 3 particles / µL.

[0011] The present invention also provides an application of the above-mentioned detection reagent in detecting the expression level of extracellular vesicles in lung cancer-derived small cells.

[0012] The present invention also provides a method for detecting the expression level of extracellular vesicles in lung cancer-derived small cells using the above-mentioned detection reagent, comprising the following steps: The plasma small extracellular vesicles were captured using the reagent for targeted capture of plasma small extracellular vesicles. After magnetic separation and purification, the SAMA-sEVs complex was obtained. The SAMA-sEVs complex was mixed with the free biotin and incubated by shaking to elute and release the small extracellular vesicles from the SAMA-sEVs complex, thereby obtaining purified target sEVs. The purified target sEVs were mixed with the Lip@AuNFs@DSN to form a film fusion, and the intensity of each fluorescence channel was measured simultaneously.

[0013] This invention has outstanding substantive features and significant progress compared to the prior art. Specifically, the application of plasma small extracellular vesicle miRNA markers, the method for detecting expression levels, and the detection reagents provided by this invention firstly identify three miRNAs—miR-21-5p, miR-375-3p, and miR-451a—as core markers through multi-omics screening combined with clinical validation. Then, a system for efficient isolation and in situ synchronous detection of tumor-derived sEVs was established, realizing efficient capture, gentle release, and in situ synchronous detection of internal miRNAs in sEVs.

[0014] Specifically, this invention selects low-abundance miRNAs within small extracellular vesicles (sEVs) as the detection target. Because the miRNAs are encapsulated within sEVs, their structure is intact and highly stable. Simultaneously, an aptamer-magnetic bead specific capture method is used, followed by gentle elution via biotin exchange, resulting in high capture efficiency and complete release of the miRNAs after elution. During fluorescence detection, after membrane fusion, the miRNAs inside the sEVs are released into the liposome lumen, and the detection reagent enters the shared reaction space formed by the fusion, thereby initiating target recognition and signal generation. In other words, this invention employs liposome membrane fusion probe delivery + DSN enzyme cascade amplification. The liposomes fuse with the sEV membrane, reacting in situ within the lumen. Utilizing the liposome encapsulation of the reaction system, the quenching-recovery mechanism significantly reduces background. Furthermore, the liposomes are loaded with different fluorescently labeled probes, achieving crosstalk-free detection using three-color fluorescence. Therefore, methodological evaluation shows that the detection reagents and methods provided by this invention have low detection limits, good recoveries (96.5%–114.6%), and are highly correlated with qRT-PCR (r≥0.982).

[0015] Meanwhile, testing of 450 clinical samples confirmed that miR-21-5p and miR-451a were significantly elevated in the lung cancer group, while miR-375-3p was significantly decreased. Furthermore, miR-375-3p was relatively highly expressed in small cell lung cancer and advanced patients. This provides molecular evidence with both high specificity and clinical relevance for lung cancer screening and auxiliary diagnosis, and provides reliable technical support for the accurate detection of low-abundance biomarkers, thus strongly promoting the clinical translation of sEVs liquid biopsy technology. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the SAMA system used for the separation and release of SEVs.

[0017] Figure 2 This is a schematic diagram of the principle of in situ synchronous detection of three miRNAs in sEVs based on membrane fusion and DSN enzyme.

[0018] Figure 3 The characterization results show that the SAMA system can specifically capture sEVs. Figure 3 A is a scanning electron microscope image of SA@MBs at a scale of 100 nm; Figure 3 B is a scanning electron microscope image of SAMA at a scale of 100 nm; Figure 3 C is a scanning electron microscope image of the SA@MBs-Apt complex capturing the target sEVs at a scale of 100 nm.

[0019] Figure 4To characterize the competitive release of sEVs from biotin eluent using laser confocal fluorescence microscopy. The figures show fluorescence, bright field, and superimposed images of SAMA magnetic beads at different treatment stages; After isolation: Samples after SAMA magnetic beads captured PKH67-labeled sEVs; After Release: Magnetic bead samples after treatment with biotin eluent and magnetic separation; Control: Background control group where SAMA magnetic beads were incubated with only PKH67 dye; Fluorescent: PKH67 green fluorescence channel; Bright: Bright field channel; Merge: Superimposed image of fluorescence and bright field.

[0020] Figure 5 To characterize the integrity and bioactivity of sEVs after release from the biotin eluent. Figure 5 A shows the morphology of sEVs after release as observed by TEM. Figure 5 B represents the effect of sEVs on the proliferation of BEAS-2B cells as detected by CCK-8 assay (n=6).

[0021] Figure 6 The TEM characterization results show the membrane fusion between Lip@AuNFs@DSN and sEVs.

[0022] Figure 7 The scratch width and migration distance of cells in each group at different time points are shown. Figure 7 A is the original image from the cell scratch assay; Figure 7 B represents the quantitative result of the migration distance.

[0023] Figure 8 To verify the cleavage activity of DSN enzyme on miRNA-cDNA hybrid double strands and its signal cycling amplification effect. Figure 8 Lane A represents the polyacrylamide gel electrophoresis characterization of the cleavage activity of DSN enzyme on miRNA-cDNA hybrid double strands; Lane 1: Marker; Lane 2: 500 nmol / L miRNA + 500 nmol / L cDNA; Lane 3: 500 nmol / L miRNA + 500 nmol / L cDNA + 1 UDSN enzyme; Lane 4: 500 nmol / L miRNA; Lane 5: 500 nmol / L cDNA; Figure 8 B represents the cyclic amplification of the DSN enzyme signal using fluorescence spectroscopy.

[0024] Figure 9 The fluorescence spectra of three fluorescent probes are shown. Figure 9 A represents cDNA21-FAM; Figure 9 B represents cDNA375-TAMRA; Figure 9 C stands for cDNA451-Cy5.

[0025] Figure 10 The mutual interference of three fluorescent probes was investigated. Figure 10 A represents cDNA21-FAM; Figure 10 B represents cDNA375-TAMRA; Figure 10 C represents cDNA451-Cy5; the curves in the figure correspond to the fluorescence intensity response at different excitation wavelengths (Ex); the vertical axis represents fluorescence intensity (FL, unit: au), and the horizontal axis represents wavelength (Wavelength, unit: nm).

[0026] Figure 11 Comparison of fluorescence signals from different experimental groups. Figure 11 A represents the fluorescence intensity of FAM-cDNA21 under different experimental conditions; Figure 11 B represents the fluorescence intensity of TAMRA-cDNA375 under different experimental conditions; Figure 11 C represents the fluorescence intensity of Cy5-451 under different experimental conditions.

[0027] Figure 12 The capture and release efficiencies of SAMA under different reaction parameters are shown. Figure 12 A represents the sEVs capture efficiency curves corresponding to different amounts of SAMA magnetic beads; Figure 12 B represents the sEVs capture efficiency curves corresponding to different SAMA incubation times; Figure 12 C represents the sEVs release efficiency curves corresponding to different biotin concentrations; Figure 12 D represents the sEVs release efficiency curves corresponding to different elution times.

[0028] Figure 13 The effect of different reaction conditions on the increase of optical signal. Figure 13 A represents the dosage of different DSN enzymes; Figure 13 B represents different reaction temperatures; Figure 13 C represents different Lip@AuNFs@DSN concentrations; Figure 13 D represents the different incubation times for Lip@AuNFs@DSN and sEVs.

[0029] Figure 14 This is a standard curve for the simultaneous detection of three miRNAs within the membrane of sEVs mediated by membrane fusion. Figure 14 A is miR-21-5p; Figure 14 B is miR-375-3p; Figure 14 C is miR-451a.

[0030] Figure 15 To characterize and investigate the specificity of MVs. Figure 15 A represents the TEM characterization of MVs; Figure 15 B represents the NTA of MVs; Figure 15C represents the specificity assessment of the SAMA system.

[0031] Figure 16 To investigate the specificity of the Lip@AuNFs@DSN system.

[0032] Figure 17 This section compares the SAMA system and the differential centrifugation method. Figure 17 A represents a comparison of the time used; Figure 17 B represents a yield comparison; Figure 17 C represents the comparison of capture efficiency.

[0033] Figure 18 The comparison is between the established method and RT-qPCR. The horizontal axis represents the relative expression level of RT-qPCR, and the vertical axis represents the difference in fluorescence signal intensity (FL-FL0) measured by the established method, i.e., the fluorescence intensity of the target channel (FL) minus the background fluorescence intensity (FL0).

[0034] Figure 19 This is a heatmap showing the expression of three miRNAs in plasma sEVs in three population groups. Figure 19 A is miR-21-5p; Figure 19 B is miR-375-3p; Figure 19 C represents miR-451a; NC represents normal controls; BLD represents benign lung disease; LC represents lung cancer patients.

[0035] Figure 20 The expression levels of three miRNAs in plasma sEVs were compared between the lung cancer group and the control group. Figure 20 A is miR-21-5p; Figure 20 B is miR-375-3p; Figure 20 C represents miR-451a; x-axis: NC: normal control, BLD: benign lung disease, LC: lung cancer patient. Detailed Implementation

[0036] Specifically, this invention constructs a targeted separation system based on the coupling of streptavidin-coated magnetic beads (SA@MBs) and aptamers (Apt), abbreviated as SAMA (SA@MBs-Apt), which aims to achieve efficient and specific capture and gentle release of sEVs.

[0037] like Figure 1 As shown, its core working principle is as follows: (1) Targeted capture and magnetic separation purification: Three dethiobiotin-labeled nucleic acid aptamers were synthesized, targeting EpCAM, PD-L1 and EGFR respectively, and named Apt respectively. EpCAMApt PD-L1 and Apt EGFR The aforementioned aptamers were coupled to the surface of SA@MBs via the high affinity of streptavidin-desulfobiotin to construct a functionalized capture system. This system utilizes the specific recognition of EpCAM, PD-L1, and EGFR on the sEVs membrane surface by the aptamers to achieve precise capture of target sEVs. The magnetic bead-sEVs complex was rapidly separated under an applied magnetic field, and unbound and non-specifically adsorbed components were removed by washing to obtain high-purity target sEVs.

[0038] (2) Mild competitive elution release: Utilizing the ultra-high affinity of free biotin for streptavidin (stronger than that of desulfurized biotin and streptavidin, approximately 10 times that of desulfurized biotin). 4 By adding an excess of free biotin to competitively replace the desulfurized biotin aptamer-sEVs complex bound to the surface of magnetic beads, intact sEVs are gently released under neutral pH 7.4 conditions, thus preserving their morphology and biological activity to the greatest extent.

[0039] On the other hand, this invention utilizes a high-sensitivity detection strategy based on membrane fusion delivery and DSN signal amplification for the in situ simultaneous quantitative detection of three low-abundance miRNAs within sEVs. This method integrates miRNA release, specific recognition, and signal amplification within the same reaction space by constructing multifunctional cationic liposomes, such as... Figure 2 As shown, the specific principle is as follows: (1) Construction and membrane fusion delivery of multifunctional liposomes: Multifunctional liposomes (Lip@AuNFs@DSN) loaded with fluorescently labeled DNA probes and DSN enzyme were prepared. The three DNA probes targeted different miRNAs (labeled with FAM, TAMRA, and Cy5 fluorescent groups, respectively); the initial fluorescence of the cDNA probe coupled to the surface of the gold nanoparticles was effectively suppressed by the quenching effect of the adjacent gold nanoparticles. When Lip@AuNFs@DSN fused with sEVs, the miRNA inside the sEVs was released into the liposome cavity, and the detection reagent entered the common reaction space formed by the fusion, thereby initiating target recognition and signal generation.

[0040] (2) DSN enzyme-triggered signal conversion and cyclic amplification: The released miRNA hybridizes with the corresponding DNA probe to form a DNA-RNA double strand. The DSN enzyme specifically cleaves the DNA strand in the double strand, allowing the fluorescent group to escape the quenching effect of AuNFs and restoring the fluorescence signal. After cleavage, the miRNA remains intact and can rehybridize with new probes, initiating multiple rounds of cyclic cleavage to achieve cascade amplification of the signal of a single miRNA molecule.

[0041] (3) Multiplexing detection: After the three cDNA probes are cleaved by DSN enzyme, they release three distinguishable fluorescent signals. By simultaneously measuring the intensity of each fluorescent channel, the in situ synchronous quantitative analysis of the three miRNAs can be achieved.

[0042] The technical solution of the present invention will be further described in detail below through specific embodiments. 1. Main facts and materials.

[0043] All reagents and materials used in the experiment, as well as all nucleic acid sequences (including DNA probes, aptamers, and miRNA mimics), were purchased from Sangon Biotech Co., Ltd. The water used in the experiment was ultrapure water prepared using the Milli-Q ultrapure water system (Millipore, USA). The instruments and equipment used in the experiment were those commonly used in this field.

[0044] Table 1. Nucleic acid names and base sequences 2. Material preparation.

[0045] The preparation methods of the main buffer solutions and reagents involved in this invention are as follows: RPMI-1640 / DMEM complete medium: Mix 5 mL of inactivated fetal bovine serum with 45 mL of basal medium containing penicillin-streptomycin antibiotics. 1×PBS (0.01 mol / L, pH 7.4): Dissolve one packet of PBS powder in 2 L of Milli-Q ultrapure water, stir to dissolve, and autoclave. 1×PBST (pH 7.4): Slowly add 250 μL of Tween-20 to 500 mL of 1×PBS, and gently mix. 1×TBST: Adjust the volume of 10×TBST concentrate to 1 L with Milli-Q ultrapure water, and mix well. Hybridization buffer (pH 7.4): Dissolve 5.844 g of NaCl, 2.033 g of MgCl2·6H2O, and 2.423 g of Tris in 950 mL of Milli-Q water, adjust the pH to 7.4 with 0.1 mol / L HCl, and adjust the volume to 1 L. Autoclave. MES buffer (0.01 mol / L, pH 6.0): Dissolve 2.0 g of MES in 1 L of Milli-Q water, and adjust the pH to 6.0 with KOH. 10 mmol / L Tris(2-carboxyethyl)phosphine buffer (TCEP): Weigh 2.8665 g of TCEP, dissolve it in Milli-Q ultrapure water, and bring the volume to 1 L; aliquot and store at 4 ℃ for short-term storage, or freeze at -20 ℃.

[0046] Cancer cell source: The lung adenocarcinoma cell lines (A549, H1650), lung squamous cell carcinoma cell lines (H520, H226), small cell lung cancer cell line (H446), and human bronchial epithelial cells (BEAS-2B) used in this invention are all derived from the Cell Bank of the Chinese Academy of Sciences.

[0047] Preparation of streptavidin-aptamer-based magnetic beads for capturing tumor-derived sEVs: To capture tumor-derived sEVs, the nucleic acid aptamers AptEpCAM, AptPD-L1, and AptEGFR were conjugated to the surface of SA@MBs using a dethiobiotin-streptavidin system to construct the specific capture probe SAMA. The specific steps included: taking 600 μL of SA@MBs (2 mg / mL), magnetically separating the beads, and washing three times with PBST. Adding 1 mL of an aptamer solution containing 400 nmol / L AptEpCAM / AptPD-L1 / AptEGFR, and incubating with shaking at room temperature for 1 h. Removing unbound aptamers with magnetic separation, washing three times with PBST, resuspending in PBS, and storing at 4 ℃ protected from light for use within one week.

[0048] Synthesis of gold nanoparticles: Gold nanoparticles (AuNPs) were prepared by sodium citrate reduction: 10 mL of 100 mmol / L chloroauric acid was dissolved in 90 mL of Milli-Q water by magnetic stirring to a final concentration of 1 mmol / L. A round-bottom flask connected to a condenser was heated to stable reflux in an oil bath at 120 °C. 10 mL of 38.8 mmol / L sodium citrate was quickly added, and the reaction was sealed. The solution color changed from pale yellow to colorless to grayish-black to wine red (about 1 min), indicating the formation of AuNPs. The reaction was continued for 15-20 min, then heating was stopped, and the mixture was allowed to cool naturally to room temperature with stirring. The solution was filtered through a 0.22 μm filter and stored at 4 °C protected from light. The absorbance at 520 nm was measured in the range of 450-700 nm using a UV-Vis spectrophotometer, and the molar extinction coefficient ε = 2.7 × 10⁻⁶ was used. 8 L·mol -1 ·cm -1 Calculate the concentration.

[0049] Synthesis of gold nanoflares: 60 μL of 10 μmol / L cDNA was mixed with 2 μL of 0.2 mol / L TCEP and incubated at a constant temperature for 30 min to reduce thiol groups. 300 μL of AuNPs was added, mixed at room temperature, and allowed to stand for 5 min; then incubated at -20 ℃ in the dark for 2 h. The mixture was thawed at 4 ℃ for 30 min, with 15 μL of 2.5 mol / L NaCl added every 30 min to a final concentration of 500 mmol / L. The mixture was incubated overnight at 4 ℃ in the dark to allow for complete cDNA assembly. The mixture was centrifuged at 12000 ×g for 15 min, and the supernatant was discarded; the mixture was resuspended in PBS and centrifuged again, followed by two washes. The mixture was resuspended in 300 μL of PBS and stored at 4 ℃ in the dark.

[0050] Synthesis of cationic liposomes: Cationic liposomes (Lips) were prepared by thin-film hydration: 6.0 mg of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 4.9 mg of cholesterol, 3.9 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG), and 6.3 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were weighed and dissolved in 8 mL of chloroform and 2 mL of methanol. After sonication for 10 min, the solution was transferred to a 250 mL round-bottom flask. The organic solvent was removed by rotary evaporation under reduced pressure at 37 °C (150 r / min) to form a lipid film. Further evaporation under reduced pressure for 30 min was then carried out to remove residual solvent. Add 2 mL of reaction buffer, wash the membrane by rotating at 45 r / min under normal pressure, and hydrate at 37 ℃ for 2 h. Disperse the liposomes by squeezing them back and forth 10 times using a liposome extruder into 2 mL of reaction buffer, and store at 4 ℃ for later use. Dissolve the pre-prepared AuNFs and DSN enzyme in the reaction buffer, replacing the blank buffer in the hydration step above, and perform hydration and squeezing in the same manner to obtain cationic liposomes (Lip@AuNFs@DSN) encapsulated with AuNFs and DSN enzyme. The remaining steps are the same as for the preparation of blank liposomes. 3. Effect verification.

[0051] Feasibility verification of sEV capture: To systematically evaluate the capture efficiency and specificity of the SAMA system for target sEVs, scanning electron microscopy was used for characterization. The specific operational steps are as follows: Streptavidin-modified aptamer-functionalized magnetic beads were washed twice with 1×PBST, and magnetic separation was performed using a magnetic rack to remove unbound components and impurities from the surface. 30 μL of SAMA magnetic beads (2 mg / mL) were mixed with 200 μL of sEV suspension (20 μL stock solution + 180 μL 1×PBS), and incubated at 37 ℃ and 600 r / min for 30 min with shaking to ensure sufficient binding of the aptamer to the target on the sEV surface. The bound magnetic beads were collected by magnetic separation, washed three times with PBST, and the supernatant was discarded to remove non-specific proteins and free sEVs. The purified SAMA-sEV complex was resuspended in PBS buffer for later use. 10 μL of SA@MBs suspension and the complex of SA@MBs-Apt and sEVs incubated were respectively added to a silicon wafer and allowed to stand at room temperature for 10 min. Fixation was performed with 2.5% glutaraldehyde at room temperature for 15 min, followed by washing three times with PBS; dehydration was carried out in a gradient of 30%–100% ethanol (10 min per stage). After vacuum drying, gold was sputtered onto the wafer (~5 nm), and the morphology was observed by SEM at an accelerating voltage of 5 kV.

[0052] Test results as follows Figure 3 As shown, from Figure 3 As can be seen from the SEM images, the SA@MBs are approximately spherical with a diameter of about 300 nm, exhibiting good dispersion. The magnetic core is located at the center, surrounded by a polymer coating layer. Spherical particles of 30–250 nm are visible attached to the surface of the SAMA-sEVs composite, suggesting that SAMA effectively captures the target sEVs.

[0053] Feasibility study of sEV release: To verify whether the SAMA system can efficiently and completely release sEVs after capture, a biotin-competitive elution method was used to recover sEVs, and the elution effect was characterized by CLSM and NTA techniques. Biotin-mediated release of sEVs: The captured sEVs-SAMA magnetic bead complex was mixed with 100 μL of elution buffer (containing 100 mmol / L biotin, solvent 0.1 mol / L PBS, pH 7.4), and incubated with gentle shaking at 200 r / min for 30 min at room temperature. After magnetic separation, the supernatant sample was stored at -80 ℃ for subsequent analysis. Laser confocal fluorescence microscopy characterization: 200 μL of sEVs suspension (1×10⁻⁶) was used... 7Add PKH67 membrane dye to a final concentration of 2 μmol / L (particles / μL), incubate at 4 ℃ in the dark for 10 min, and remove free PKH67 dye by ultrafiltration. After PKH67 labeling of sEVs, three experimental groups were set up: capture group (SAMA magnetic beads + sEVs, incubated at 37 ℃ and 600 r / min for 30 min, and the complex was retained after magnetic separation and washing), elution group (the above complex + 100 mmol / L biotin, incubated at 37 ℃ and 200 r / min for 30 min, and the supernatant and magnetic bead precipitate were collected separately after magnetic separation), and control group (PKH67 dye + SAMA magnetic beads). Samples from each group were added to confocal culture dishes, and green fluorescence (λex=490 nm, λem=504 nm) and bright-field images were acquired by CLSM. The co-localization changes of sEVs and magnetic beads before and after elution were analyzed by superposition.

[0054] PKH67 green fluorescently labeled sEVs were used, and the fluorescence signal distribution at different stages was observed using CLSM to visually verify the competitive release effect of the biotin eluent. Results are as follows: Figure 4 As shown, after SAMA magnetic beads captured sEVs, the green fluorescence signal and the bright-field signal of the magnetic beads were completely colocalized, indicating that the sEVs were stably bound to the surface of the magnetic beads. After treatment with biotin eluent and magnetic separation, only a very weak green fluorescence remained on the magnetic beads, with no obvious fluorescence colocalization with the magnetic beads, indicating that biotin can efficiently release the sEVs on the surface of the magnetic beads through competitive binding. The control group, which was incubated with SAMA magnetic beads only with PKH67 dye, showed no obvious green fluorescence signal, eliminating background interference from non-specific fluorescence adsorption of the magnetic beads. The results confirm that SAMA captures sEVs through the specific interaction between the desulfurized biotinylated aptamer and the sEVs, and that the biotin eluent can efficiently release the captured sEVs through a competitive binding mechanism.

[0055] Integrity and bioactivity of released sEVs: CCK-8 assay: BEAS-2B cells were trypsin-digested and resuspended in complete culture medium to 2.0 × 10⁻⁶. 4 Cells / mL, seeded in 96-well plates (100 μL / well, approximately 2.0 × 10⁻⁶ cells / mL). 3 Cells / well, outer wells with PBS added), cultured at 37 ℃, 5% CO2. After adhesion, change the medium; experimental groups were treated with high (1×10⁻⁶) PBS. 6 particles / μL), medium (1×10 4 particles / μL), low (1×10⁻⁶) 2 After release of sEVs at a concentration of particles / μL, the control group was given an equal volume of PBS. The cultures were continued and detected at 0, 24, 48, and 72 h (6 replicates). Before detection, 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated at 37 ℃ in the dark for 2 h. OD was then measured using a microplate reader.450 (Reference wavelength 630 nm), with OD 450 The value reflects cell proliferation activity. The test results are as follows: Figure 5 As shown in the figure. TEM revealed that biotin-competitively released sEVs were round or oval membranous vesicles with clear bimembrane boundaries, and a diameter of 30–200 nm, conforming to the definition of ISEVs. No membrane rupture or leakage of contents was observed. CCK-8 assay showed that the cell proliferation rates of high, medium, and low concentration sEVs treatment groups were significantly higher than those of the control group within 0–72 h (P<0.05), showing a concentration-dependent effect.

[0056] Membrane fusion characterization results: Transmission electron microscopy characterization of membrane fusion morphology features: such as Figure 6 The image shown is a TEM image of the fusion of Lip@AuNFs@DSN and sEVs membranes. The fusion product is a vesicle-like composite structure formed by the fusion of Lip@AuNFs@DSN and sEVs membranes, with continuous boundaries and preserved membrane phase characteristics. The composite contains six discretely and uniformly distributed spherical AuNFs, without aggregation or leakage, indicating that the membrane fusion did not disrupt the AuNFs encapsulation state. Morphologically, this confirms successful membrane fusion and stable internal encapsulation.

[0057] Figure 7 The scratch width and migration distance of cells in each group at different time points are shown. Figure 7 A is the original image from the cell scratch assay; Figure 7 B represents the quantitative results of migration distance. Scratch assays showed that the migration distance of the sEVs-treated group was significantly greater than that of the control group at both 12 h and 24 h (12 h: 315 μm vs 185 μm, P<0.01; 24 h: 182 μm vs 95 μm, P<0.05), indicating that after release, sEVs retain their intact membrane structure, cellular uptake capacity, and can effectively promote cell migration, preserving their natural biological activity.

[0058] Verification of the cleavage and signal amplification effects of double-stranded specific nucleases: Polyacrylamide gel electrophoresis was used to verify the cleavage effect of DSN enzyme and to analyze the feasibility of DSN enzyme signal amplification. Figure 8 As shown in the figure, gel electrophoresis revealed that channel 2 miRNA hybridized with cDNA to form a double-stranded band; after adding DSN enzyme to channel 3, the hybridization band completely disappeared, and the brightness of the single-stranded cDNA band significantly decreased, indicating that DSN enzyme specifically cleaves the cDNA strand in the hybrid double strand; channel 4 miRNA single strand and channel 5 cDNA single strand served as controls. The results confirm that DSN enzyme can specifically cleave miRNA-cDNA hybrid double strands.

[0059] Fluorescence spectroscopy showed extremely low fluorescence in the AuNPs group; slightly higher fluorescence in the AuNFs group, but still low; limited increase in fluorescence in the AuNFs+miRNA group; no difference between the AuNFs+DSN group and the AuNFs group, indicating that DSN enzyme acting alone on cDNA single strands does not interfere with fluorescence; the fluorescence in the AuNFs+miRNA+DSN group was significantly higher than that of the other groups. These results indicate that after miRNA hybridizes with the cDNA probe, it is cleaved by DSN enzyme, causing the fluorescent group to move away from the AuNPs surface, thus relieving quenching and amplifying the signal.

[0060] 4. Feasibility verification of in situ synchronous detection of three miRNAs in the sEVs membrane.

[0061] Spectroscopic characteristics of three fluorescent probes: To clarify the optical properties of three fluorescently labeled cDNA probes—cDNA21-FAM, cDNA375-TAMRA, and cDNA451-Cy5—their excitation and emission spectra were characterized using a fluorescence spectrometer. The results are as follows: Figure 9 As shown, the maximum excitation wavelength of cDNA21-FAM is approximately 490 nm, and the maximum emission wavelength is approximately 521 nm, which perfectly matches the characteristic spectrum of the FAM fluorophore. The maximum excitation wavelength of cDNA375-TAMRA is approximately 557 nm, and the maximum emission wavelength is approximately 585 nm, consistent with the typical optical response characteristics of the TAMRA fluorophore. The maximum excitation wavelength of cDNA451-Cy5 is approximately 649 nm, and the maximum emission wavelength is approximately 670 nm, corresponding to the characteristic spectral range of the Cy5 fluorophore. These spectral results define the core optical parameters of the three fluorescent probes, providing crucial information for selecting excitation and detection conditions in subsequent experiments.

[0062] Interference between fluorescent probes: such as Figure 10 As shown in the fluorescence spectrum, the FAM channel shows a signal only in the green curve, while the TAMRA and Cy5 channels show no response; the TAMRA channel shows a signal only in the orange curve; and the Cy5 channel shows a signal only in the red curve. The excitation and emission peaks of the three probes are separated, with no cross-interference, meeting the requirements for multi-channel synchronous detection.

[0063] Feasibility verification of simultaneous multi-target detection: such as Figure 11 As shown in the fluorescence spectra, group 1 (Lip@AuNFs@DSN+sEVs) exhibited significant fluorescence enhancement in all three channels; group 2 (without DSN enzyme) showed no effective signal; group 3 (without AuNFs) had high background and no specificity; and group 4 (without sEVs) maintained baseline fluorescence. These results indicate that the Lip@AuNFs@DSN system can specifically respond to the three target miRNAs within sEVs, with no crosstalk between channels, meeting the requirements for simultaneous detection of multiple targets.

[0064] Optimization of detection system conditions: Optimization of key parameters for capture and release efficiency: like Figure 12 As shown, within the magnetic bead dosage range of 10–40 μL, the capture efficiency increased with increasing dosage, reaching a peak of 93.3 ± 0.5% at 30 μL, with no significant improvement upon further increases, thus the optimal dosage was determined to be 30 μL. Within the incubation time range of 15–60 min, the capture efficiency increased from 89% to 92.5% from 15–30 min, reaching a plateau from 30–45 min, thus the optimal time was determined to be 30 min. Within the biotin concentration range of 25–125 mmol / L, the release efficiency increased from 38.2 ± 2.8% to 65.9 ± 1.7% from 25–100 mmol / L, reaching a plateau at 100 mmol / L, thus the optimal concentration was determined to be 100 mmol / L. Within the elution time range of 10–40 min, the release efficiency increased from 36.4 ± 1.8% to 65.9 ± 1.1% from 10–30 min, reaching a plateau after 30 min, thus the optimal time was determined to be 30 min.

[0065] Optimization of key parameters for in situ synchronous detection of three miRNAs within the sEV membrane: such as Figure 13 As shown, the key conditions of the detection system were optimized using a single-factor experimental method, with ΔFL = FL − FL0 as the evaluation index. In the optimization of DSN enzyme dosage, the fluorescence signal increased in a dose-dependent manner with increasing enzyme concentration. When the enzyme dosage reached 1.0 U, the fluorescence intensity entered a plateau phase; therefore, 1.0 U was determined as the standard dosage for subsequent experiments. The fluorescence response of the system first increased and then decreased with increasing temperature, reaching a peak at 37 ℃, which is the optimal temperature for the DSN enzyme to exert its highest catalytic activity. The sEVs concentration was 1 × 10⁻⁶. 7 When the particle / μL concentration of Lip@AuNFs@DSN increases to 10×10, 7 The fluorescence signal tends to stabilize after particles / μL, and this concentration can meet the target miRNA's full response. The fluorescence signal gradually increases with the extension of reaction time, reaching a maximum value at 120 min. Therefore, 120 min is determined to be the optimal reaction time.

[0066] Establishment of standard curve: The sEVs standard was serially diluted to 1.0 × 10⁻⁶ using PBS. 4 ~5.0×10 8 Particles / μL series concentrations. Detection was performed under optimal conditions. A standard curve was established using linear regression with the logarithm of sEVs concentration on the x-axis and net fluorescence intensity on the y-axis, and the regression equation and R value were recorded. 2 .

[0067] Under optimized conditions, the Lip@AuNFs@DSN system supports 1.0×104 ~5.0×10 7 The detection performance of the three target miRNAs in sEVs within the particle / μL range was good, and ΔFL showed a linear relationship with lgCsEVs. Figure 14 As shown. Where, miR-21-5p: Y=17292.63X1—19076.63, R 2 =0.9993.

[0068] miR-375-3p: Y=15184.12X2—12247.81, R 2 =0.9977;miR-451a: Y=12893.67X3—17850.23, R 2 =0.9992. Y represents ΔFL, and X1, X2, and X3 are the common logarithms of the three miRNA concentrations, respectively. The system demonstrates good quantitative detection capability over a dynamic range of approximately four orders of magnitude, meeting the needs for simultaneous analysis of miRNAs with varying abundances in complex samples. 5. Methodological evaluation.

[0069] Limits of Detection: The fluorescence intensities at 520 nm, 584 nm, and 670 nm of three blank samples (excluding sEVs) were measured using the established method. The X+3SD values ​​were substituted into the standard curve to obtain the limits of detection for miR-21-5p, miR-375-3p, and miR-451a within the sEVs membrane, which were 2.7 × 10⁻⁶. 2 4.0×10 2 and 1.45×10 3 particles / µL.

[0070] Precision and spiked recovery: sEVs were diluted to 5.0 × 10⁻⁶ with sEVs-free FBS. 4 1.0×10 6 5.0×10 7 The recoveries of particles / μL, miR-21-5p, miR-375-3p and miR-451a spiked were 96.53%–107.50%, with RSD < 7.14%, which met the requirements for biological sample analysis, indicating that the method has good repeatability and precision.

[0071] Specificity assessment: Specificity assessment of the SAMA system MVs are vesicle subtypes that naturally coexist with sEVs in body fluids. They share similar membrane structures and partially overlap in particle size range, and are used as a negative control. Figure 15As shown, MVs exhibit a typical membrane vesicle structure, with a round or elliptical shape, clear membrane edges, and a particle size of approximately 200–500 nm, consistent with the morphological characteristics of MVs. NTA results show that their particle size distribution exhibits a multi-peak characteristic, with the main peak concentrated in the 300–400 nm range.

[0072] The results showed that the SAMA+sEVs group had the highest ΔFL, significantly higher than the SAMA+MVs group and the two magnetic bead control groups (SA@MBs+sEVs and SA@MBs+MVs). This indicates that the SAMA system can specifically identify and capture sEVs through aptamer-specificity, effectively distinguishing MVs interference with overlapping particle sizes, and exhibiting excellent separation specificity.

[0073] Specificity assessment of Lip@AuNFs@DSN: such as Figure 16 As shown, real-world sample matrices commonly contain interfering components such as homologous nucleic acids, miscellaneous miRNAs, and non-target membrane structures. Therefore, specificity verification of the detection method is a crucial prerequisite for ensuring accurate identification of target miRNAs in complex environments. When AuNFs@DSN, as the core recognition unit, is co-incubated with non-target nucleic acids (miRNA-122, miRNA-141, miRNA-155, Random1), the fluorescence of each channel remains at background levels. Only when interacting with target miRNAs does the corresponding channel fluorescence significantly increase, confirming the excellent sequence discrimination capability of the cDNA probe. When Lip@AuNFs@DSN is co-incubated with non-target components (nucleic acids, membrane structures) or a single target miRNA, the fluorescence of the corresponding channel remains at background levels. Only when interacting with target sEVs does the corresponding channel fluorescence significantly increase, confirming that liposome encapsulation does not introduce non-specific binding, and the system is specifically adapted to complex matrix environments.

[0074] 6. Methodological comparison.

[0075] Comparison of the streptavidin magnetic bead-aptamer system and differential centrifugation: To verify the clinical application potential of the SAMA system, it was evaluated from three dimensions: time consumption, yield, and capture efficiency, using the classic ultracentrifugation method as a reference. The separation efficiency of SAMA and the A549 / H520 cell supernatant sEVs was compared. The concentration of sEVs separated by the two methods was determined using NTA technology, and the sEV yield per unit volume of cell supernatant was calculated to assess the yield. The total vesicle capture rate was calculated by detecting the change in PKH67 fluorescence intensity in the supernatant before and after capture, and the separation performance of the SAMA method was compared and evaluated.

[0076] like Figure 17As shown, the SAMA system requires only about 1 hour for the entire capture and release process, significantly shortening the sample processing cycle compared to ultracentrifugation (>5 hours). NTA quantitative analysis revealed that the concentration of sEVs recovered from the supernatant of A549 and H520 cells by the SAMA method was significantly higher than that by ultracentrifugation. Detection of fluorescence intensity changes in PKH67-labeled vesicles confirmed that the SAMA method's capture efficiency for sEVs was significantly superior to that of ultracentrifugation. In summary, the SAMA system demonstrates significant technical advantages in both time consumption and yield while maintaining separation purity.

[0077] Comparison of Lip@AuNFs@DSN system with RT-qPCR: To evaluate the quantitative accuracy of the constructed detection system, RT-qPCR, the gold standard for miRNA quantification, was used as a reference. The expression levels of miR-21-5p, miR-375-3p, and miR-451a in plasma sEVs were detected, and a correlation model was established between the fluorescence signal increment ΔFL = FL − FL0 and the relative expression level in RT-qPCR. Figure 18 As shown in the results, all three target miRNAs exhibited high linear correlations between this method and RT-qPCR: miR-21-5p correlation coefficient r = 0.988 (P < 0.001), miR-375-3p correlation coefficient r = 0.982 (P < 0.001), and miR-451a correlation coefficient r = 0.985 (P < 0.001). Compared with RT-qPCR, this system eliminates the need for sEV lysis and RNA extraction, allowing for the simultaneous detection of three target miRNAs with shorter detection time and simpler procedures. The high consistency between the two methods (r > 0.98) confirms that this method possesses quantitative accuracy and reliability comparable to the gold standard method for miRNA quantification in complex plasma samples, meeting the needs of clinical trace miRNA analysis.

[0078] 7. Testing of actual samples.

[0079] Plasma was removed from a -80 °C freezer and slowly thawed. The required volume of plasma sample was then taken, diluted 10-fold with PBS, and centrifuged at 2000 ×g for 10 min and 10000 ×g for 30 min, respectively. The supernatant was collected. Finally, large-sized interfering substances were removed from the centrifuged plasma sample using a 0.22 µm filter membrane. The sEVs in the plasma sample were captured and released, and the miR-21-5p, miR-375-3p, and miR-451a within the released sEVs membrane were detected and analyzed.

[0080] Analysis of clinical sample testing results: A total of 450 subjects were included in the actual sample testing, including lung cancer patients (n=150), patients with benign lung diseases (n=150), and normal controls (n=150). The pathological types and TNM stages of the lung cancer patients are shown in Table 2. The pathological types of lung cancer patients included squamous cell carcinoma, adenocarcinoma, and small cell lung cancer. Among the lung cancer patients selected in this invention, 53.33% had adenocarcinoma, 22.67% had squamous cell carcinoma, and 24.0% had small cell lung cancer. Furthermore, the sample distribution was balanced between stages I / II (52.67%) and III / IV (47.33%).

[0081] Table 2. Clinicopathological types and TNM staging of lung cancer patients

[0082] Analysis of miRNA expression levels in three groups of plasma sEVs: The established method was used to detect the expression levels of miR-21-5p, miR-375-3p, and miR-451a in the plasma sEVs of 450 subjects. Figure 19 As shown in Table 3, the three miRNAs exhibited differential expression patterns among the three groups, with significant differences between groups (all P < 0.0001).

[0083] Table 3. Description and analysis results of plasma samples.

[0084] like Figure 20 As shown in Table 4, pairwise comparisons revealed that miR-21-5p was significantly higher in the lung cancer group than in the benign lung disease group and the normal control group (all P < 0.001), and was also higher in the benign lung disease group than in the normal control group (P < 0.01). miR-375-3p was significantly higher in the normal control group than in the benign lung disease group and the lung cancer group (all P < 0.001), while there was no significant difference between the benign lung disease group and the lung cancer group (P > 0.05). miR-451a was significantly higher in both the lung cancer group and the benign lung disease group than in the normal control group (all P < 0.001), while there was no significant difference between the lung cancer group and the benign lung disease group.

[0085] Table 4. Results of pairwise comparison analysis of plasma samples

[0086] Therefore, the application of plasma small extracellular vesicle (sEV) miRNA biomarkers, the method for detecting their expression levels, and the detection reagents provided by this invention can achieve efficient capture, gentle release, and in-situ simultaneous detection of internal miRNAs from sEVs. This represents a breakthrough in the entire process from efficient sEV enrichment to multiplex miRNA detection. Methodological evaluation confirms that this technology system has a low detection limit, a recovery rate as high as 96.5%~104.8%, and good reproducibility (RSD<8%), providing reliable technical support for the accurate detection of low-abundance biomarkers and providing highly specific and clinically relevant molecular evidence for lung cancer screening and auxiliary diagnosis.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. The application of a plasma small extracellular vesicle miRNA marker in the preparation of a reagent for detecting the expression level of lung cancer-derived small extracellular vesicles, characterized in that: The plasma small cell extracellular vesicle miRNA markers are combinations of nucleotide sequences such as miR-21-5p, miR-375-3p, and miR-451a of SEQ.No.1-3.

2. The application according to claim 1, characterized in that: The small extracellular vesicles are derived from lung squamous cell carcinoma cells, lung adenocarcinoma cells, and small cell lung cancer cells.

3. A detection reagent for detecting the expression level of extracellular vesicles in lung cancer-derived small cells, characterized in that: Including reagents for targeted capture of small extracellular vesicles in plasma, free biotin, and Lip@AuNFs@DSN; The reagent for targeting and capturing small extracellular vesicles in plasma includes: streptavidin-modified magnetic beads and three dethiobiotin-labeled nucleic acid aptamers (Apt) coupled to the surface of the streptavidin-modified magnetic beads. EpCAM Apt PD-L1 Apt EGFR Three nucleic acid aptamers Apt EpCAM Apt PD-L1 Apt EGFR The nucleotide sequences are shown in SEQ.No.8-10. The three nucleic acid aptamers correspond to EpCAM, PD-L1 and EGFR, which target small extracellular vesicles, respectively. The free biotin is used to competitively replace the desulfobiotin aptamer-sEVs complex bound to the surface of the streptavidin-modified magnetic beads and release intact plasma microcellular extracellular vesicles. Lip@AuNFs@DSN is a multifunctional liposome containing gold nanoparticle flares and a DSN enzyme encapsulated with three fluorescently labeled DNA probes. It comprises: liposomes and gold nanoparticle flares and a DSN enzyme encapsulated within the liposomes; wherein the liposomes are cationic liposomes prepared by thin-film hydration using 1,2-dioleoyl-3-trimethylammonium propane, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol, and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, serving to provide a release site for miRNA within sEVs after membrane fusion and to encapsulate the reaction system; the gold nanoparticles... The particle flare comprises gold nanoparticles and three DNA probes cDNA21, cDNA375, and cDNA451, such as SEQ. No. 12-14, coupled to the surface of the gold nanoparticles. DNA probe cDNA21 is labeled with the FAM fluorescent group, DNA probe cDNA375 is labeled with the TAMRA fluorescent group, and DNA probe cDNA451 is labeled with the Cy5 fluorescent group. The DSN enzyme is a double-stranded specific nuclease used to cleave lung cancer-derived small cell extracellular vesicle miRNA and the corresponding DNA probe to form a DNA-RNA double strand.

4. The detection reagent for detecting the expression level of lung cancer-derived small cell extracellular vesicles according to claim 3, characterized in that: The reagent for targeting and capturing small extracellular vesicles is prepared through the following steps: First, three dethiobiotin-labeled nucleic acid aptamers are synthesized, and the three nucleic acid aptamers Apt... EpCAM Apt PD-L1 Apt EGFR The reagent for targeting and capturing small extracellular vesicles was prepared by targeting EpCAM, PD-L1, and EGFR of small extracellular vesicles, respectively, and then using the affinity between streptavidin magnetic beads and dethiobiotin to couple the three nucleic acid aptamers to the surface of streptavidin magnetic beads.

5. The detection reagent for detecting the expression level of extracellular vesicles in lung cancer-derived small cells according to claim 3 or 4, characterized in that: The Lip@AuNFs@DSN is prepared through the following steps: first, gold nanoparticles are prepared using the sodium citrate reduction method; then, three fluorescently labeled DNA probes are coupled to the surface of the gold nanoparticles to obtain gold nanoflares; finally, the gold nanoflares and DSN are dissolved in a reaction buffer for preparing cationic liposomes using the thin-film hydration method, and cationic liposomes are prepared using the reaction buffer via the thin-film hydration method to obtain the Lip@AuNFs@DSN.

6. The detection reagent for detecting the expression level of lung cancer-derived small cell extracellular vesicles according to claim 5, characterized in that: The biotin concentration is 100 mmol / L and the DSN enzyme dosage is 1.0 U.

7. The detection reagent for detecting the expression level of lung cancer-derived small cell extracellular vesicles according to claim 6, characterized in that: The detection limit of the assay reagent for miR-21-5p in the extracellular vesicles of plasma cells is 2.7 × 10⁻⁶. 2 The detection limit for miR-375-3p in sEVs membranes was 4.0 × 10⁻⁶ particles / µL. 2 The detection limit for miR-451a in sEVs membrane particles / µL is 1.45×10⁻⁶. 3 particles / µL.

8. The use of the detection reagent according to any one of claims 3 to 7 in detecting the expression level of extracellular vesicles in lung cancer-derived small cells.

9. A method for detecting the expression level of extracellular vesicles in lung cancer-derived small cells using the detection reagent according to any one of claims 3 to 7, comprising the following steps: The plasma small extracellular vesicles were captured using the reagent for targeted capture of plasma small extracellular vesicles. After magnetic separation and purification, the SAMA-sEVs complex was obtained. The SAMA-sEVs complex was mixed with the free biotin and incubated by shaking to elute and release the small extracellular vesicles from the SAMA-sEVs complex, thereby obtaining purified target sEVs. The purified target sEVs were mixed with the Lip@AuNFs@DSN to form a film fusion, and the intensity of each fluorescence channel was measured simultaneously.

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