A reagent, quantitative detection reagent and method for identifying and capturing extracellular vesicles, and their applications.

Abstract

This invention provides a reagent, quantitative detection reagent, method, and application for identifying and capturing extracellular vesicles (EVs), relating to the field of extracellular vesicle technology. The invention provides a reagent for identifying and capturing EVs and, based on this, constructs a detection method capable of simultaneously capturing and quantifying EVs. This invention uses aptamers loaded on a carrier as recognition elements and amplifies the fluorescence signal through a hybridization chain reaction for the enrichment and quantitative detection of EVs. Furthermore, the detection method provided by this invention exhibits high selectivity, demonstrating high sensitivity, specificity, and stability for MCF-7-derived EVs, and holds promise for application in conjunction with other detection or imaging techniques for the early diagnosis, prognosis, and disease monitoring of diseases such as tumors.

Description

Technical Field

[0001] This invention belongs to the field of extracellular vesicle technology, specifically relating to a reagent, quantitative detection reagent and method for identifying and capturing extracellular vesicles, and their applications. Background Technology

[0002] Extracellular vesicles (EVs) are tiny, double-membrane vesicles produced and secreted by cells within or shed from their cell membranes. EVs are abundant in animal and plant fluids, such as animal serum, plasma, saliva, urine, emulsions, and amniotic fluid. Carrying a large amount of DNA, RNA, proteins, and other biomolecules inherited from parent cells, EVs can regulate anti-tumor immune responses, initiate the formation of pre-metastatic ecological environments, and monitor tumor progression and metastasis. Therefore, EVs isolated from body fluids hold promise as promising non-invasive cancer biomarkers for non-invasive early diagnosis, prognosis, and disease monitoring. Targeting specific proteins highly expressed on the surface of extracellular vesicles can serve as a detection method for cancer-related extracellular vesicles. Due to the large contact area between the aptamer structure and the target substance, aptamers exhibit higher affinity and specificity compared to protein antibodies.

[0003] Currently, quantitative techniques for extracellular vesicles mainly include immunoaffinity chromatography (IAC), asymmetrical-flowfield flowfractionation (AF4), nanoparticle tracking analysis (NTA), dynamic light scattering (DSL), surface plasmon resonance (SPR) detection, and flow cytometry. However, each method has its limitations, such as the need for EV pretreatment and poor reproducibility. Recently, Chinese patent CN110106233A proposed using digital PCR for the quantitative detection of extracellular vesicles / exosomes, but magnetic beads themselves cannot capture a large number of extracellular vesicles / exosomes, resulting in low detection results. Therefore, it is necessary to develop new methods for identifying and capturing extracellular vesicles, as well as methods for quantitative detection of extracellular vesicles. Summary of the Invention

[0004] The purpose of this invention is to provide a reagent, quantitative detection reagent, method, and application for identifying and capturing extracellular vesicles. The aptamer is modified onto monodisperse polystyrene microspheres (PS) to achieve specific identification and capture of EVs. Then, the signal is amplified by hybridization chain reaction (HCR) to increase detection sensitivity.

[0005] The present invention provides a reagent for specifically recognizing and capturing extracellular vesicles, comprising an aptamer modified on polystyrene, the aptamer being designed based on specific membrane protein receptors on extracellular vesicles.

[0006] The present invention also provides a method for preparing the above-mentioned reagent, comprising the following steps: mixing carboxyl-modified polystyrene with EDC and NHS for a first incubation, centrifuging and discarding the supernatant, mixing the precipitate with MUC1-A for a second incubation, centrifuging and discarding the supernatant to obtain an aptamer modified on monodisperse polystyrene.

[0007] Preferably, the method for preparing the carboxyl-modified polystyrene includes: mixing deionized water, methanol, styrene and methacrylic acid, heating in an oil bath to 70°C under a protective atmosphere, adding potassium persulfate after holding at 70°C for 1 h, 4 h and 8 h respectively, stopping heating after reacting for 10 h, and obtaining carboxyl-modified polystyrene after cooling.

[0008] The present invention also provides a method for specifically identifying and capturing extracellular vesicles, comprising the following steps: mixing the above reagent with a suspension containing extracellular vesicles and incubating, then centrifuging and discarding the supernatant.

[0009] Preferably, the reagent further includes BSA-blocking sites before mixing.

[0010] The present invention also provides a reagent for quantitative detection of extracellular vesicles, comprising a reagent for specifically recognizing and capturing extracellular vesicles, an aptamer CD63-A, a hairpin probe H2, and a luminescent hairpin probe H1-Cy5, wherein the nucleotide sequence of CD63-A is shown in SEQ ID No. 2, the nucleotide sequence of H1 is shown in SEQ ID No. 3, and the nucleotide sequence of H2 is shown in SEQ ID No. 4;

[0011] The reagent that specifically recognizes and captures extracellular vesicles includes an aptamer MUC1-A modified on polystyrene, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0012] The present invention also provides a method for quantitative detection of extracellular vesicles for non-diagnostic purposes, comprising the following steps: mixing the reagent that specifically recognizes and captures extracellular vesicles in the above reagent with a suspension containing extracellular vesicles and incubating, then centrifuging and discarding the supernatant;

[0013] The precipitate was mixed with aptamer CD63-A and incubated for a third time. After centrifugation, the supernatant was discarded.

[0014] The precipitate was incubated for the fourth time with a mixture of annealed hairpin probe H2 and luminescent hairpin probe H1-Cy5, and the supernatant was discarded by centrifugation.

[0015] The precipitate was dispersed in PBS solution, and the number of extracellular vesicles was calculated based on fluorescence changes.

[0016] Preferably, in the mixture of annealed hairpin probe H2 and luminescent hairpin probe H1-Cy5, the concentrations of both hairpin probe H2 and luminescent hairpin probe H1-Cy5 are 1 μM.

[0017] Preferably, the linear relationship between the fluorescence change ratio and the number of extracellular vesicles is: Y = 2.16·lg[EV] - 6.41, R 2 =0.9984, where Y = I F / I F0 -1, I F I represents the fluorescence intensity of the precipitate dispersed in PBS solution. F0 The fluorescence intensity is shown in the PBS blank control group.

[0018] The present invention also provides the application of the above-mentioned reagents for specifically recognizing and capturing extracellular vesicles or for quantitatively detecting extracellular vesicles in the preparation of tools for early detection, prognosis and development of tumors.

[0019] Beneficial effects: This invention provides a reagent for specifically recognizing and capturing extracellular vesicles, and develops a method for specifically recognizing and capturing extracellular vesicles based on the reagent. MUC1-A is modified on a carboxylated PS surface by an amino group at one end, forming PS / MUC1-A. Then, because MUC1-A can specifically bind to the MUC1 receptor on EVs, the EVs are immobilized on the PS surface to achieve the purpose of capture.

[0020] This invention also provides a novel reagent and method for the quantitative detection of EVs. Figure 1 The aptamer is modified onto monodisperse polystyrene microspheres (PS) to specifically recognize and capture EVs. Then, hydroxyl radical refraction (HCR) is used to amplify the signal and increase detection sensitivity. Specifically, after successful EV capture, a solution of CD63-A and H1 and H2 is added to the system. One end of the CD63-A single strand specifically binds to the CD63 receptor on the EV, while the other end opens the fluorescent hairpin probe H1-Cy5. H1-Cy5 then opens the hairpin probe H2, resulting in an HCR reaction. After the H1-Cy5 hairpin opens, the Cy5 fluorescent group at the 3' end of the nucleotide chain moves away from the 5' quencher group, emitting fluorescence around 665 nm after excitation with 635 nm light. Finally, by detecting the change in fluorescence intensity, EVs can be detected.

[0021] Based on the above scheme, this invention obtains the EV content in the test solution according to the relationship between EV concentration and fluorescence intensity, constructing a detection system that can simultaneously capture and quantitatively detect EVs. Examples demonstrate that when the EV concentration is 1.7 × 10⁻⁶... 3 Up to 1.7×10 6 When the fluorescence change ratio is within the range of particulates / μL, it is greater than that of (I F / I F0 -1) and lg[EV] show a good linear relationship, with the formula Y = 2.16·lg[EV] - 6.41, and the correlation coefficient (R) 2 The value was 0.9984. The PS / Aptamer / HCR detection method constructed in this invention exhibits high sensitivity, specificity, and stability for MCF-7-derived extracellular vesicles, and is expected to be applied in conjunction with other detection or imaging methods for the early diagnosis, prognosis, and disease monitoring of diseases such as tumors. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The flowchart of the method (PS / Aptamer / HCR) for specifically identifying, capturing, and quantitatively detecting extracellular vesicles according to the present invention is shown below.

[0024] Figure 2 Characterization diagrams of polystyrene microspheres (PS) and extracellular vesicles (EVs) are shown. In the figure, A: TEM image of polystyrene microspheres; B: particle size analysis diagram of polystyrene microspheres; C: microscopic image of polystyrene microspheres; D: TEM image of extracellular vesicles; E: particle size distribution diagram of extracellular vesicles; F: UV absorption spectrum of polystyrene microspheres before and after modification with aptamers.

[0025] Figure 3Figure 1 shows the feasibility analysis results of using PS / Aptamer / HCR to detect EVs. A: Polyacrylamide gel electrophoresis results of the HCR reaction products (lane 1: CD63-A; lane 2: H1-Cy5; lane 3: H2; lane 4: CD63-A+H1-Cy5+H2); B: Fluorescence spectra of the system without MUC1-A (purple-red) and with MUC1-A (black); C: Fluorescence spectra of the system without CD63-A (blue) and with CD63-A (black); D: Fluorescence spectra of the system without EVs (green) and with EVs (black); E: Fluorescence spectra after different probes participate in the HCR reaction; F: Bright-field and fluorescence images taken by confocal microscopy under four different detection conditions, scale bar 20 μm.

[0026] Figure 4 Figure 1 shows the results of the optimization experiments for the PS / Aptamer / HCR detection system. Figure A shows the relationship between fluorescence intensity at 665 nm and HCR reaction time (0, 15, 30, 45, 60, 75, and 90 minutes), with error bars representing the standard deviation of the three experiments. Figure B shows the fluorescence spectra with different concentrations of H1-Cy5 and H2. Figure C shows the fluorescence spectra with different concentrations of MUC1-A. Figure D shows the fluorescence spectra with different concentrations of CD63-A.

[0027] Figure 5 The figure shows the results of the sensitivity and specificity study of the PS / Aptamer / HCR system for detecting EVs. In the figure, A represents the fluorescence spectra of different concentrations of MCF-7-derived EVs, with values ​​from a to h being 0, 1.7 × 10⁻⁶ ... and 1.7 × 10⁻⁶, respectively. 3 8.5×10 3 1.7×10 4 8.5×10 4 1.7×10 5 8.5×10 5 1.7×10 6 Particals / μL; B: Relative fluorescence intensity (I0): ... F / I F0 -1) and the concentration of MCF-7 derived EVs (from 1.7 × 10⁻¹) 3 Up to 1.7×10 6 Linear relationship of logarithm (lg[EV]) of particals / μL; C: Fluorescence spectra of MCF-7EVs and HepG2EVs detected under the same conditions. Detailed Implementation

[0028] The present invention provides a reagent for specifically recognizing and capturing extracellular vesicles, comprising an aptamer modified on polystyrene, the aptamer being designed based on specific membrane protein receptors on extracellular vesicles.

[0029] In this invention, MUC1 and CD63 are both specific membrane protein receptors highly expressed on the surface of EVs. For these two specific membrane protein receptors, two nucleic acid aptamers that can recognize them can be designed respectively. For example, in the embodiments of this invention, two aptamers, MUC1-A and CD63-A, were designed for extracellular vesicles of the MCF-7 cell line. MUC1-A is modified on polystyrene. The nucleotide sequence of MUC1-A is shown in SEQ ID No. 1. The italicized parts in Table 1 are complementary fragments of CD63-A and H1, and the straight lines and underlined parts under the wavy lines represent complementary parts of H1 and H2.

[0030] Table 1 DNA oligonucleotide sequences

[0031]

[0032]

[0033] The polystyrene described in this invention is preferably carboxyl-modified polystyrene. The preparation method of the carboxyl-modified polystyrene includes: mixing deionized water, methanol, styrene (St) and methacrylic acid (MAA), heating in an oil bath to 70°C under a protective atmosphere, adding potassium persulfate (KPS) after holding at 70°C for 1 h, 4 h and 8 h respectively, stopping heating after reacting for 10 h, and obtaining carboxyl-modified polystyrene after cooling.

[0034] The present invention also provides a method for preparing the above-mentioned reagent, comprising the following steps: mixing carboxyl-modified polystyrene with EDC and NHS for a first incubation, centrifuging and discarding the supernatant, mixing the precipitate with MUC1-A for a second incubation, centrifuging and discarding the supernatant to obtain an aptamer modified on monodisperse polystyrene.

[0035] The preparation method of carboxyl-modified polystyrene described in this invention is preferably the same as described above, and will not be repeated here. In the embodiments of this invention, preferably, 100 μL of EDC (0.25 mM) and 100 μL of NHS (0.45 mM) are added to 500 μL of carboxyl-modified PS (20 μg / mL), incubated at 37°C for 1 hour, centrifuged at 8000 rpm for 10 minutes, and the supernatant is discarded to remove free EDC and NHS; 100 μL of MUCI-A solution (2 μM) is added and incubated at 37°C for 2 hours, centrifuged at 8000 rpm for 10 minutes, and the supernatant is discarded to remove unmodified MUCI-A.

[0036] The present invention also provides a method for specifically identifying and capturing extracellular vesicles, comprising the following steps: mixing the above reagent with a suspension containing extracellular vesicles and incubating, then centrifuging and discarding the supernatant.

[0037] In this invention, it is preferable to mix the aptamer modified on monodisperse polystyrene with a suspension containing extracellular vesicles and then incubate it. In the examples, it is preferable to add 100 μL of EV suspension of various concentrations to the precipitate after removing unmodified MUC1-A and incubate it at 37°C for 2 hours, centrifuge at 8000 rpm for 10 minutes, and discard the supernatant. Before capturing EVs, this invention preferably further includes adding 500 μL of 5% BSA solution to the precipitate after removing unmodified MUC1-A, incubating it on a shaker at 37°C for 1.5 hours to block the sites, and washing three times with PBS after blocking (centrifuging at 8000 rpm for 10 minutes each time), and discarding the supernatant.

[0038] The present invention also provides a reagent for quantitative detection of extracellular vesicles, comprising a reagent for specifically recognizing and capturing extracellular vesicles, an aptamer CD63-A, a hairpin probe H2, and a luminescent hairpin probe H1-Cy5, wherein the nucleotide sequence of CD63-A is shown in SEQ ID No. 2, the nucleotide sequence of H1 is shown in SEQ ID No. 3, and the nucleotide sequence of H2 is shown in SEQ ID No. 4;

[0039] The reagent that specifically recognizes and captures extracellular vesicles includes an aptamer MUC1-A modified on polystyrene, the nucleotide sequence of which is shown in SEQ ID No. 1.

[0040] The nucleotide sequences of the reagents described in this invention are listed in Table 1. They were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and purified by HPLC.

[0041] The present invention also provides a method for quantitative detection of extracellular vesicles for non-diagnostic purposes, comprising the following steps: mixing the above reagent with a suspension containing extracellular vesicles and incubating, then centrifuging and discarding the supernatant;

[0042] The precipitate was mixed with aptamer CD63-A and incubated for a third time. After centrifugation, the supernatant was discarded.

[0043] The precipitate was incubated for the fourth time with a mixture of annealed hairpin probe H2 and luminescent hairpin probe H1-Cy5, and the supernatant was discarded by centrifugation.

[0044] The precipitate was dispersed in PBS solution, and the number of extracellular vesicles was calculated based on fluorescence changes.

[0045] The capture operation of EVs described in this invention is preferably the same as described above, and will not be repeated here. After capturing EVs, 100 μL of CD63-A solution (1.5 μM) is added, and the mixture is incubated at 37°C for 2 hours. The mixture is then centrifuged at 8000 rpm for 10 minutes, and the supernatant is discarded. Sixth, 500 μL of annealed hairpin probe mixture (H1-Cy5 and H2 concentrations are both 1 μM) is added, and the mixture is incubated at 37°C for 2 hours. The mixture is then centrifuged at 8000 rpm for 10 minutes, and the supernatant is discarded. The final product is dispersed in 200 μL of PBS solution and stored at 4°C for later use.

[0046] In this embodiment of the invention, the above-mentioned EV capture and detection operation was performed using MCF-7 derived EVs, and the fluorescence change ratio (I F / I F0 -1) shows a linear relationship with the logarithm of EV concentration (lg[EV]), at 1.7 × 10 3 Up to 1.7×10 6 Within the linear range of particals / μL, I F / I F0 -1 and lg[EV] show a good linear relationship, with the formula Y = 2.16·lg[EV] - 6.41, and the correlation coefficient (R) 2 The value is 0.9984, I F I represents the fluorescence intensity of different experimental groups. F0 The fluorescence intensity is the value of the PBS blank control.

[0047] The present invention also provides the application of the above-mentioned reagents for specifically recognizing and capturing extracellular vesicles or for quantitatively detecting extracellular vesicles in the preparation of tools for early detection, prognosis and development of tumors.

[0048] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a reagent, quantitative detection reagent, method, and application for identifying and capturing extracellular vesicles provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0049] In the embodiments of the present invention, unless otherwise specified, the materials, reagents, instruments and experimental methods used are all common solutions in the art.

[0050] Experimental instruments involved in the embodiments of this invention:

[0051] In this experiment, the morphology of the nanomaterials was characterized using transmission electron microscopy (JEM-2100, JEOL). The particle size was measured using a Zeta-Size Nano instrument (Zen 3600, Malvern Instruments Ltd.). Ultraviolet (UV)-visible spectra were recorded using a Cary 50 UV-vis-NIR spectrophotometer. Absorbance measurements for BCA protein experiments were performed using a BioTek Epoch full-wavelength microplate reader. Fluorescence data were detected and recorded using an F-4600 fluorescence spectrophotometer (Hitachi). Cell fluorescence images were acquired using a laser confocal microscope (Nikon C2plus, Germany).

[0052] Experimental reagents involved in the embodiments of this invention:

[0053] Styrene (St), methacrylic acid (MAA), potassium persulfate (KPS), methanol, and glacial acetic acid were purchased from Sinopharm Chemical Reagent Co., Ltd. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), acrylamide (AM), bisacrylamide (Bis), tris(hydroxymethyl)aminomethane (Tris), and ethylenediaminetetraacetic acid (EDTA) were purchased from Aladdin Reagent (Shanghai) Co., Ltd. A rapid extraction kit for extracellular vesicles from cell culture supernatant was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. DNA loading buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. All water used in this invention was sterilized ultrapure water. All oligonucleotides used in this invention were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) and purified by HPLC; their sequences are shown in Table 1. All DNA sequences were dissolved and diluted to a final concentration of 10 μM with PBS buffer and stored at 4°C until use. All MCF-7 breast cancer cells used in the experiment were obtained from Wuhan Pronosei Life Sciences Co., Ltd.

[0054] Example 1

[0055] I. Experimental Procedure

[0056] 1. Preparation of carboxylated polystyrene

[0057] Add 150 mL of deionized water and 50 mL of methanol to a 500 mL four-necked flask equipped with a condenser, magnetic stirrer, and thermometer. Then add 30 g of St and 1 g of MAA. Maintain a stirring rate of 300 rpm and heat in an oil bath to 70 °C under nitrogen purging. After one hour, dissolve 0.5 g of KPS in 30 mL of deionized water and add 10 mL of the prepared APS solution to the flask. Then, add 10 mL of the APS solution at 4 h and 8 h of heating, respectively, while maintaining stirring. After reacting for 10 h, stop heating and cool to obtain a white emulsion. Wash the white emulsion three times with PBS (centrifuged at 8000 rpm for 10 minutes each time), discard the supernatant, and redisperse the precipitate in 5 mL of PBS. Store at 4 °C for later use.

[0058] 2. Cell Culture

[0059] In this experiment, the MCF-7 and HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. During cell culture, the incubator temperature was maintained at 37°C and the CO2 concentration at 5%.

[0060] 3. Extraction of EVs

[0061] Extracellular vesicles were extracted and isolated from MCF-7 cell culture supernatant using a rapid extracellular vesicle extraction kit (Yisheng Biotechnology (Shanghai) Co., Ltd.). The extraction procedure was performed according to the kit's instructions. Before extracting the extracellular vesicles, the regular fetal bovine serum in the cell culture medium was replaced with fetal bovine serum without extracellular vesicles, and the medium was passaged three times consecutively before the extraction experiment.

[0062] 4. Detection system for capturing and detecting EVs

[0063] First, add 100 μL of EDC (0.25 mM) and 100 μL of NHS (0.45 mM) to 500 μL of carboxyl-modified PS (20 μg / mL) and incubate at 37 °C for 1 hour. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant to remove free EDC and NHS. Second, add 100 μL of MUCI-A solution (2 μM) and incubate at 37 °C for 2 hours. Centrifuge at 8000 rpm for 10 minutes, discard the supernatant to remove unmodified MUCI-A. Third, add 500 μL of BSA solution (5%) and incubate on a shaker at 37 °C for 1.5 hours to block the sites. After blocking, wash three times with PBS (centrifuge at 8000 rpm for 10 minutes each time), discard the supernatant. Fourth, add 100 μL of EV suspension of various concentrations and incubate at 37 °C for 2 hours. Centrifuge at 8000 rpm for 10 minutes and discard the supernatant. Fifth, add 100 μL of CD63-A solution (1.5 μM) and incubate at 37°C for 2 hours. Centrifuge at 8000 rpm for 10 minutes and discard the supernatant. Sixth, add 500 μL of annealed hairpin probe mixture (H1-Cy5 and H2 concentrations are both 1 μM) and incubate at 37°C for 2 hours. Centrifuge at 8000 rpm for 10 minutes and discard the supernatant. The final product is dispersed in 200 μL of PBS solution and stored at 4°C for later use.

[0064] Annealing: Place the primers in a PCR instrument, heat to 95°C and hold for 5 minutes. Then, slowly cool to 25°C at a rate of 3°C per minute and hold for 20 minutes.

[0065] 5. Polyacrylamide gel electrophoresis

[0066] DNA samples were subjected to 10% polyacrylamide gel electrophoresis (PAGE) at a fixed potential of 90V using 1×TAE buffer as the electrophoresis buffer. During sample pretreatment, the ratio of DNA solution to loading buffer was 5:1. After electrophoresis, the gel was stained with Super Red dye for 1 hour, followed by imaging on a gel imaging system.

[0067] 6. Fluorescence spectroscopy measurement

[0068] Fluorescence measurements were performed on samples after the HCR reaction to investigate the feasibility and specificity of the proposed fluorescence detection system. All samples were prepared according to the previously mentioned steps, and each sample was diluted to 200 μL with PBS buffer for subsequent detection. Then, using an F-4600 fluorescence spectrophotometer with a xenon lamp as the excitation source and 635 nm as the excitation wavelength, the fluorescence spectra from 650 nm to 750 nm were recorded.

[0069] II. Experimental Results

[0070] 1. Preparation and characterization of PS and EVs

[0071] PS was characterized using transmission electron microscopy (TEM) and a DLS particle size analyzer. The results are as follows: Figure 2 As shown in Figures A and B, the prepared PS exhibits a uniform spherical morphology with PS particles distributed at approximately 2 μm, consistent with expectations. The morphology of the PS, as captured by an optical microscope, is shown below. Figure 2 As shown in Figure C, the spherical nanoparticles of PS exhibit good uniformity and dispersibility.

[0072] The ultraviolet absorption spectra of the nanoparticles before and after modification with the aptamers were recorded using an ultraviolet spectrophotometer. The results are as follows: Figure 2 As shown in Figure F, the nanoparticles (black) showed no absorption peak at 260 nm before incubation with the aptamer. However, after incubation with the aptamer for 2 hours (red), a significant ultraviolet absorption peak appeared at 260 nm, demonstrating the successful modification of the MUC1-A aptamer onto the nanoparticles.

[0073] Meanwhile, the morphology of the extracted extracellular vesicles was characterized. Figure 2 Transmission electron microscopy (TEM) images show successful extraction of extracellular vesicles. Simultaneously, particle size distribution results (D) Figure 2 The results also showed that the extracted extracellular vesicles had a particle size distribution of around 200 nm, which was in line with expectations.

[0074] 2. Feasibility analysis of EV testing

[0075] First, the feasibility of the HCR reaction was verified by polyacrylamide gel electrophoresis. The results are as follows: Figure 3 As shown in Figure A, the four lanes from left to right are the products obtained after CD63-A, H1, H2, and CD63-A+H1+H2 react for 45 minutes, respectively. The top band in the fourth lane is the product after the HCR reaction, proving that the presence of CD63-A will open H1 and thus initiate the HCR reaction.

[0076] Fluorescence detection experiments were used to investigate the necessity of five detection components in the detection of EVs: MUC1-A aptamer, CD63-A aptamer, EVs, H1-Cy5 hairpin probe, and H2 hairpin probe. Experimental groups with no MUC1-A, no CD63, no EVs, and all components were designed, and fluorescence spectra were measured for each. The results are shown below. Figure 3 As shown in B, C, and D, only the All group that simultaneously satisfies all five experimental elements can produce a significant fluorescence signal, thus enabling the detection of EVs.

[0077] Simultaneously, groups PBS, No H2, No H1-Cy5, and All were designed, and their fluorescence spectra were recorded. Experimental results are as follows: Figure 3 As shown in Figure E, the HCR reaction can only occur smoothly and produce a detectable fluorescence signal when both hairpin probes H1 and H2 are involved in the reaction system.

[0078] To further demonstrate the necessity of the presence of the three elements MUC1-A, CD63-A, and EVs in the fluorescence detection system, this invention also verified them using confocal microscopy. During verification, four control experimental groups were designed: No MUC1-A, No EVs, No CD63-A, and All. Confocal images of these four groups were captured under 635nm excitation light. The results are as follows: Figure 3 As shown in Figure F, PS microspheres only generate emission signals (All) when excited by excitation light, provided that all elements are present. In summary, these results demonstrate the feasibility of the PS / Aptamer / HCR detection method and the necessity of the detection component.

[0079] 3. Condition optimization experiment

[0080] To obtain the best detection performance of this detection system, the concentrations of the two aptamers of EVs, the concentrations of fluorescent probes H1 and H2, and the HCR reaction time were optimized.

[0081] First, the changes in fluorescence intensity were recorded from the time the probe was added (0 min) to 90 min of the HCR reaction, while keeping other conditions constant during the experiment (H1-Cy5 and H2 concentrations were both 1.5 μM; MUC1-A concentration was 1 μM; CD63-A concentration was 1 μM). The results are as follows: Figure 4 As shown in Figure A, the fluorescence intensity of the reaction system gradually increases with the increase of HCR reaction time, and reaches a plateau after 45 minutes. This means that the HCR reaction time in this system should be controlled at around 45 minutes.

[0082] Next, the optimal concentrations of hairpin probes H1-Cy5 and H2 were investigated. Keeping other conditions constant (HCR reaction time 45 min; MUC1-A concentration 1 μM; CD63-A concentration 1 μM), only the concentrations of H1-Cy5 and H2 were changed before performing the HCR reaction. The fluorescence signals of different experimental groups were then recorded, and the results are shown below. Figure 4 As shown in Figure B, the fluorescence signal of the system is strongest when the added probe concentration is 1 μM. In other words, the optimal probe concentration required for this detection method is 1 μM.

[0083] Next, the optimal concentrations of the two aptamers, MUC1-A and CD63-A, in this detection system were investigated. Specifically, keeping other conditions constant (HCR reaction time 45 min; H1-Cy5 and H2 concentrations both 1 μM; CD63-A concentration 1 μM), only the concentration of added MUC1-A was changed, and the subsequent reaction was performed. The fluorescence signals of different experimental groups were then recorded. The results are as follows: Figure 4 As shown in Figure C, the optimal concentration of MUC1-A is approximately 2 μM.

[0084] Similarly, keeping other conditions constant (HCR reaction time 45 min; H1-Cy5 and H2 concentrations both 1 μM; MUC1 concentration 2 μM), only the CD63-A concentration was changed, and the changes in fluorescence signal were compared. The results are as follows: Figure 4 As shown in Figure D, the optimal concentration of CD63-A to be added to the detection system is approximately 1.5 μM.

[0085] 4. To study the sensitivity and specificity of extracellular vesicle detection.

[0086] To investigate the ability of this detection method to detect EVs, after capturing and enriching MCF-7-derived EVs at different concentrations using a PS / Atamer, the same concentrations of H1-Cy5 and H2 were added to the reaction system and incubated for 45 minutes. The fluorescence signal intensity generated after the HCR reaction was then recorded. The results are as follows: Figure 5 As shown in Figure A, as the concentration of EVs added to the system gradually increases, the fluorescence signal value corresponding to the fluorescence spectrum ah also becomes stronger.

[0087] By statistically analyzing the data, the fluorescence change ratio (I) was recorded. F / I F0 -1) linear relationship with the logarithm of EV concentration (lg[EV]). The results are as follows: Figure 5 As shown in B, at 1.7 × 10 3 Up to 1.7×10 6 Within the linear range of particals / μL, I F / I F0 -1 and lg[EV] show a good linear relationship, with the formula Y = 2.16·lg[EV] - 6.41, and the correlation coefficient (R) 2 The value is 0.9984.

[0088] To evaluate the specificity of the detection method, fluorescence detection was performed on MCF-7-derived EVs and HepG2-derived EVs of the same concentration under the same experimental conditions. The results are as follows: Figure 5As shown in Figure C, the fluorescence signal of HepG2-derived EVs was about 3.04 times lower than that of MCF-7-derived EVs, which demonstrates the high selectivity of the detection method of the present invention.

[0089] To evaluate the reproducibility of this method, five reproducibility tests were conducted on EVs secreted by MCF-7 at the same concentration. The relative standard deviation (RSD) of the five sets of results was 1.64%, confirming that the method described in this invention has good reproducibility. In summary, the PS / Aptamer / HCR detection system has excellent sensitivity, specificity, and stability for the detection of extracellular vesicles.

[0090] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A reagent for quantitative detection of extracellular vesicles, characterized in that, The invention includes reagents for specifically recognizing and capturing extracellular vesicles, aptamer CD63-A, hairpin probe H2, and luminescent hairpin probe H1-Cy5, wherein the nucleotide sequence of CD63-A is shown in SEQ ID No. 2, the nucleotide sequence of H1 is shown in SEQ ID No. 3, and the nucleotide sequence of H2 is shown in SEQ ID No.

4. The reagent that specifically recognizes and captures extracellular vesicles includes an aptamer MUC1-A modified on polystyrene, the nucleotide sequence of which is shown in SEQ ID No.

1.

2. A method for quantitative detection of extracellular vesicles for non-diagnostic purposes, characterized in that, The process includes the following steps: mixing the reagent that specifically recognizes and captures extracellular vesicles in the reagent of claim 1 with a suspension containing extracellular vesicles, incubating the mixture, centrifuging and discarding the supernatant; The precipitate was mixed with aptamer CD63-A and incubated for a third time. After centrifugation, the supernatant was discarded. The precipitate was incubated for the fourth time with a mixture of annealed hairpin probe H2 and luminescent hairpin probe H1-Cy5, and the supernatant was discarded by centrifugation. The precipitate was dispersed in PBS solution, and the number of extracellular vesicles was calculated based on fluorescence changes.

3. The method according to claim 2, characterized in that, In the mixture of annealed hairpin probe H2 and luminescent hairpin probe H1-Cy5, the concentrations of both hairpin probe H2 and luminescent hairpin probe H1-Cy5 were 1 μM.

4. The method according to claim 2, characterized in that, The linear relationship between the fluorescence change ratio and the number of extracellular vesicles is: Y = 2.16·lg[EV] - 6.41, R 2 =0.9984, where Y=I F / I F0 -1, I F I represents the fluorescence intensity of the precipitate dispersed in PBS solution. F0 The fluorescence intensity is shown in the PBS blank control group.

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