WO2024001798A1 - 一种基于核酸适体的细胞外囊泡荧光极化检测方法及应用 - Google Patents

一种基于核酸适体的细胞外囊泡荧光极化检测方法及应用 Download PDF

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WO2024001798A1
WO2024001798A1 PCT/CN2023/100289 CN2023100289W WO2024001798A1 WO 2024001798 A1 WO2024001798 A1 WO 2024001798A1 CN 2023100289 W CN2023100289 W CN 2023100289W WO 2024001798 A1 WO2024001798 A1 WO 2024001798A1
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evs
cancer
nucleic acid
detection method
aptamer
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French (fr)
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段维
徐长城
范强岳
向颂喜
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上海万何圆生物科技有限公司
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Priority to EP23794226.3A priority Critical patent/EP4321872A1/en
Publication of WO2024001798A1 publication Critical patent/WO2024001798A1/zh

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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
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    • G—PHYSICS
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6445—Measuring fluorescence polarisation
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • the invention relates to the technical field of C12N15/115 (aptamers that bind specifically and with high affinity to target molecules), and in particular to a nucleic acid aptamer-based extracellular vesicle fluorescence polarization detection method and its application.
  • NTA nanoparticle tracking analysis technology
  • EV extracellular vesicle
  • ELISA enzyme-linked immunosorbent assay
  • lab-on-a-chip technologies such as microfluidics and micromechanical chips require advanced micromachining technology and precision instruments to read signals and analyze data, limiting the widespread application of these technologies in EV research in the laboratory and clinical fields.
  • High-sensitivity flow cytometry is not only used to detect and quantify EVs but also to characterize individual EVs with the potential for high-throughput and multiplex analysis.
  • fluorescence polarization has been used in clinical settings and high-throughput analysis (i.e., drug discovery).
  • the rapid development of this technology can be attributed to its good reproducibility, high degree of autonomy, adaptability to small sample volumes (approximately 10 ⁇ L), and the lack of separation of free and bound ligands in homogeneous analysis.
  • Heterogeneous techniques for separating unbound and bound species are not only more laborious but also suffer from inaccurate quantification of actual biomolecular interactions.
  • fluorescence-based homogeneous assays such as fluorescence resonance energy transfer (FRET), time-resolved fluorescence (TRF) or time-resolved FRET (TR-FRET) require multiple labels rather than the single label used in fluorescence polarization.
  • fluorescence polarization as a ratiometric measurement method, eliminates artifacts triggered by internal filtering effects, reduces the impact of the analytical environment, and is not affected by absorbance or color quenching of other compounds.
  • FP detection methods In the development of fluorescence polarization (FP) detection methods, it is crucial to select detection substances with molecular weights less than 15 kDa. Based on this situation, when lipophilic dyes such as PKH26 and PKH67, as well as carbocyanine dyes (DiI, DiO) are used as ligands, although their molecular weight is very small ( ⁇ 1kDa), they themselves have inherent limitations, such as with Additional effects include non-specific binding of non-EV particles, low labeling efficiency, and dye aggregation. Although the antibody specifically binds the EV marker, its large size (approximately 150 kDa) may not be suitable for FP. In contrast, aptamers (chemical antibodies) are much smaller in size ( ⁇ 15kDa) and more stable in chemical synthesis, so they are ideal detection substances for EV detection based on FP.
  • lipophilic dyes such as PKH26 and PKH67, as well as carbocyanine dyes (DiI,
  • CD63 aptamer was used to directly detect A549 cell line or EVs in human plasma without prior isolation of EVs. Although the applicant has confirmed that the sensitivity of this experimental method to CD63-positive EVs is much higher than that of other soluble CD63 proteins, regardless of its use in cancer diagnosis, in the extraction In EVs or human plasma, the probes used will interact with soluble proteins and/or protein aggregates in EVs, resulting in positive detection results where the detected targets are soluble proteins or membrane fragments rather than derived from cancer cells. Secreted extracellular vesicles, because the LOD of the FP detection method of aptamers can reach 86aM or 28aM.
  • This application provides a nucleic acid aptamer-based fluorescence polarization detection method for extracellular vesicles (fluorescence polarization using aptamers for the detection of extracellular nanovesicles, FluPADE). Thanks to the high specificity of the antibodies and aptamers and the small size of the aptamers, this method can efficiently and accurately detect cancer-derived EVs in cell culture media or human plasma.
  • the first aspect of the present invention provides a nucleic acid aptamer-based extracellular vesicle fluorescence polarization detection method, as shown in Figure 1, including the following steps:
  • nucleic acid aptamers adapted to EV markers or cancer cell markers for incubation, and the nucleic acid aptamers are fluorescently labeled;
  • step S3 Perform fluorescence polarization detection on the product of step S2 to achieve qualitative and quantitative analysis of EVs secreted by cancer cells. No cleaning is required during the operation.
  • EVs secreted by cancer cells are actually the inducement for fluorescence polarization signals to be displayed when EVs are captured and detected using various biomarkers.
  • the present invention uses three different nucleic acid aptamers in FluPADE detection to prove that the detection method can be optimized by exploring multiple aptamers targeting EV surface biomarkers and exploring the size specifications of the aptamers.
  • the present invention uses EVs secreted by cancer cells derived from three types of solid tumor cell lines, specifically colorectal cancer (HT29), breast cancer (SKBR3) and hepatocellular carcinoma (HepG2), to demonstrate the universal applicability of this method. sex. Therefore, the sensitivity, ease of operation, and functionalization of the FluPADE test determine that this aspect can be well applied to clinical research in many oncology disciplines.
  • EVs derived from MDA-MB-231 with HER2 gene knocked out had the largest size (142.3 ⁇ 10.3nm), while EVs from the HT29 cell line had the smallest size (98.7 ⁇ 8.5nm).
  • the body sizes of the five EVs all fit within the size range of small EVs (size ⁇ 200nm).
  • the EV marker includes at least one of CD9, CD63, and CD81.
  • the cancer cell markers include EpCAM and/or HER2.
  • EVs should contain at least one transmembrane protein, one cytoplasmic protein, and no negative regulatory proteins. Therefore, this application demonstrates through immunoblotting experiments that CD63 (a transmembrane protein) and The presence of Alix, a cytosolic protein, and the absence of negative regulatory proteins (calnexin, an endoplasmic reticulum marker) were demonstrated by other cancer markers (EpCAM and HER2). As shown in Figure 3C , CD63 and Alix appeared in all EVs, while calnexin did not appear in any of the five EVs.
  • EpCAM was found in EVs derived from HT29, SKBR3 and HepG2, but no expression of EpCAM appeared in EVs derived from HEK293.
  • HER2 gene expression was only found in EVs derived from HT29, SKBR3 and HepG2, but not in MDA-MB-231-derived EVs with the HER2 gene knocked out (Fig. 3C).
  • EV surface proteins are direct binding targets in the present invention. Therefore, in addition to characterizing proteins through blotting, the present invention also uses magnetic bead flow cytometry to characterize several biomarkers of EVs from five sources, including three three EV marker proteins (CD9, CD63 and CD81) and two cancer cell markers (EpCAM and HER2).
  • streptavidin-coated 10 ⁇ m magnetic beads (Merck, Cat No.: LSKMAGT02) were treated with 10 times the volume of binding buffer (0.22 ⁇ m filtered, phosphate buffer containing 5% bovine serum albumin). ) and blocked with 10 volumes of binding buffer for 1 hour at room temperature.
  • the supernatant was discarded, and the blocked magnetic beads were resuspended in binding buffer to isolate EVs.
  • Use sufficient washing buffer (0.22 ⁇ m filtered, composed of phosphate buffer containing 0.1% Tween 20) to wash the antibody-coated magnetic beads 3 times, then add 100 ⁇ L of 5 ⁇ 10 8 EV and 100 ⁇ L of binding buffer and incubate overnight at 4°C.
  • the EV-magnetic bead complex was washed three times with sufficient wash buffer.
  • APC-anti-human CD81 (Thermo Fisher Scientific, Cat No.: 17-0819-42) or fluorescently labeled anti-human CD81 antibody (BioLegend, Cat No.: 349504) was used To detect a mixture of anti-CD9 antibody (BioLegend, Cat No.: 312112) and anti-CD63 antibody (BioLegend, Cat No.: 353017) that captures EVs.
  • Alexa -anti-human EpCAM antibody (50nM, R&D Systems, CatNo.: FAB9601R100UG) was used to detect EV-captured anti-CD9 antibody (BioLegend, CatNo.:312112), anti-CD63 antibody (BioLegend, CatNo.:353017) and anti-CD81 antibody (BioLegend, CatNo.:349514).
  • PE-anti-human HER2 antibody (50nM, BioLegend, Cat No.:324405) was used to detect EV-captured anti-CD9 antibody (BioLegend, Cat No.:312112), anti-CD63 antibody (BioLegend, Cat No.: 353017) and anti-CD81 antibody (BioLegend, Cat No.: 349514).
  • test tube containing the antibody and EV-immobilized magnetic beads was placed in a HulaMixer sample mixer (Thermo Fisher Scientific, Cat No: 15920D) for 30 minutes at room temperature.
  • EV-coated magnetic beads were washed three times with sufficient wash buffer on a magnet holder before being analyzed by flow cytometry (10,000 samples analyzed per experiment).
  • the corresponding median fluorescence intensity (MFI) and fluorescence histogram were recorded using a BDFACS-Canto TM II flow cytometer, and then analyzed using FlowJo TM (v10.6.2).
  • Figure 3D summarizes the profile of EV surface biomarkers from 5 cell sources.
  • Figure 3E shows a histogram of EV surface marker protein expression analyzed by flow cytometry. As shown in the figure, EVs from all sources have expression of marker proteins (CD9, CD63 and CD81) except for the absence of CD81 expression in EVs derived from HepG2. As far as cancer cell markers are concerned, they are derived from HT29, EVs from HepG2 and SKBR3EV expressed EpCAM, but EVs derived from HEK293 did not express EpCAM. These results analyzed by flow cytometry (Figure 3D, Figure 3E) were completely consistent with the results of Western blot analysis (Figure 3C).
  • HER2 protein expression was present in EVs derived from HT29, SKBR3 and HepG2, but not in EVs derived from MDA-MB-231 where the HER2 gene was knocked out.
  • this application pioneered the research on a fluorescence polarization analysis method based on nucleic acid aptamers.
  • the purpose of the present invention is to develop an assay capable of selectively and sensitively detecting cancer-derived EVs. Therefore, we used two different antibodies simultaneously in one experiment to capture EVs through the interaction of the antibodies with EV markers or cancer cell markers. For example, biotinylated anti-human EpCAM antibodies were used to capture HT29-derived EVs, and biotinylated anti-human CD9/CD81 antibodies were used to immobilize SKBR3-derived EVs in 96-well plates coated with black streptavidin. First, the present invention demonstrates that biotinylated antibodies and streptavidin-coated microplates can indeed ensure the isolation of EVs.
  • the S1 step is performed in a microwell plate, specifically using microwell capture antibodies coated with streptavidin, and then EVs are immobilized.
  • FIG. 4A Fluorescently labeled anti-human CD63 antibodies were used to detect captured cancer EVs (Figure 4A).
  • Figure 4B shows the fluorescence of anti-human CD63 antibodies in HT29-derived EVs and SKBR3-derived EVs with/without Triton X-100 treatment.
  • Strength FI
  • the CD63 signal of EVs captured by anti-EpCAM antibody was significantly higher (p ⁇ 0.0001) than that of samples treated with Triton -IgG antibody (BioLegend, Cat No.: 400104) coated the well plate, and no EVs were captured in this sample.
  • the antibody in the S1 step, when the EV is derived from HT29, the antibody is a biotinylated anti-human EpCAM antibody, and the concentration of the antibody is 2.0-15.0 ⁇ g/mL; further preferably 8.0 ⁇ g/mL.
  • the antibody in the S1 step, when the EV is derived from SKBR3, the antibody is a biotinylated anti-human CD9/CD81 antibody (mass ratio 1:1), and the concentration of the antibody is 2.0-15.0 ⁇ g/mL; further preferably 8.0 ⁇ g/mL.
  • the CD63 signal showed an increasing trend in the range of antibody dosage from 2.0 ⁇ g/mL to 8.0 ⁇ g/mL.
  • the antibody concentration exceeded 8.0 ⁇ g/mL, the CD63 signal did not change significantly. Therefore, the optimal concentration for capturing EVs using biotinylated anti-EpCAM antibodies or biotinylated CD9/CD81 antibodies is 8.0 ⁇ g/mL.
  • the capturing antibody time is 0.1-1.5 hours; the fixing time is 4-20 hours, and the fixing temperature is 4°C.
  • FP is a homologous gene technology that can truly quantify the interaction between ligand and target; it should be noted that FP characterization results rely on the binding affinity during the interaction process.
  • this case first determined the binding affinity of these ligands to the EVs used in the present invention. All nucleic acid aptamers are initially synthesized from linear oligonucleotides, which are folded into a suitable 3D structure before use, and the folding scheme adopted is determined through rigorous regulation.
  • the nucleic acid aptamer includes CD63-BP, HER2-HApt, HER2-2A At least one.
  • the nucleic acid aptamer is folded before use.
  • the specific steps include: diluting the nucleic acid aptamer to the target concentration with phosphate buffer added with 0.5-2.0mM MgCl2 , and then diluting it at 90 Denature at -98°C for 2-10 minutes, incubate on ice or room temperature for 5-20 minutes, and then fold at 35-38°C for another 10-30 minutes.
  • the nucleic acid aptamer is diluted to the target concentration with phosphate buffer added with 1.0mMMgCl2 , then denatured at 95°C for 5 minutes, and incubated on ice for 10 minutes. , then fold again at 37°C for 15 minutes.
  • phosphate buffer added with 1.0mMgCl2 dilute the nucleic acid aptamer to the target concentration with phosphate buffer added with 1.0mMgCl2 , then denature it at 95°C for 5 minutes, incubate on ice for 15 minutes, and then fold at 37°C for another 15 minutes.
  • the nucleic acid aptamer was diluted with phosphate buffer, and 2.5mMgCl 2 was added, then denatured at 95°C for 5 min, incubated at room temperature for 10 minutes, and then refolded at 37°C for at least 15 minutes.
  • the K D for SKBR3 source EV are 2.92nM and 77.28nM respectively.
  • the concentration of the probe substance in a FP assay should not exceed twice the binding affinity of the probe substance to its target to avoid stoichiometric titration of the probe substance.
  • the present invention compares the FP response at three different concentrations of the detection substance, including concentrations equal to K D , twice K D and 5 nM.
  • concentrations equal to K D twice K D and 5 nM.
  • the three concentrations showed no EV (only free ligand) and EV ( The ⁇ FP value of the partially bound ligand) sample tends to change negatively, indicating that both antibodies cannot be detected by FP.
  • the fluorescently labeled CD63-BP aptamer and fluorescently labeled HER2-HApt aptamer exhibited positive ⁇ FP values at three concentrations ( Figure 5C, 5F).
  • this application demonstrates that in the detection of three different concentrations of CD63 and HER2 aptamers, immobilized HT29 and SKBR3 source EVs can be clearly detected by FP, but antibodies that bind to the same target protein on the EV surface cannot be detected. .
  • the target concentration of the nucleic acid aptamer is 1-8 nM; even more preferably, it is 5 nM.
  • the parallel/perpendicular fluorescence intensity ratio of the CD63-BP aptamer increased from 6.9 at 1 nM. ⁇ 0.7 or 7.6 ⁇ 3.7 increased to 341.2 ⁇ 2.1 or 419.0 ⁇ 5.9 at 50nM, and the parallel/perpendicular fluorescence intensity ratio of the HER2-HApt aptamer increased from 8.6 ⁇ 0.8 or 10.6 ⁇ 2.1 at 1nM to 303.5 ⁇ 2.3 at 50nM. Or 402.3 ⁇ 6.4.
  • the parallel/perpendicular fluorescence intensity ratio of HER2-2A aptamers ranged from 8.8 ⁇ 0.6 or 11.4 ⁇ 1.9 at 1 nM to 313.3 ⁇ 5.7 or 467.4 ⁇ 6.4 at 50 nM.
  • the optimal concentration of ligand in FluPADE detection should be the lowest concentration possible. At this concentration, the parallel and vertical fluorescence intensity of the background contributes minimally to the final FP, so that the FP signal mainly comes from aptamers rather than background noise.
  • the concentration of three nucleic acid aptamers is 5 nM
  • the signal-to-noise ratio of parallel and vertical fluorescence intensities is higher than 30 ( Figure 6B). Based on these ratios, the parallel and perpendicular fluorescence intensities of the detected aptamers dominate the final FP signal.
  • the concentration of 5 nM is much lower than the K D of the nucleic acid aptamer, ensuring the reliability of FP signal detection.
  • the FP signal remained unchanged when the aptamer concentration reached 5 nM and above ( Figure 6B). Therefore, the optimal concentration of the three fluorescently labeled nucleic acid aptamers is preferably 5 nM.
  • the S2 step specifically includes adding 60-140 ⁇ L of buffer containing fluorescently labeled nucleic acid aptamers to the product obtained in the S1 step, and placing the microwell plate on a shaker at room temperature in the dark for 0.5 -2 hours.
  • the signal of fluorescence polarization in the S3 step is read by a multifunctional disk analyzer; the multifunctional disk analyzer is configured with a 475-490nm excitation filter and a 520-nm excitation filter. 565nm emission filter.
  • the present invention explores the culture time of three kinds of nucleic acid aptamers and fixed EVs, and optimizes the FluPADE detection scheme.
  • the ⁇ FP of the CD63-BP aptamer significantly increased from 7.5 ⁇ 0.4 mP at 0.5 h to 10.8 ⁇ 0.3 mP at 1.0 h (p ⁇ 0.05), and ⁇ FP no longer increased after 1.5 h.
  • the optimal culture time of HER2-HApt aptamer was 1.0 hours, and the ⁇ FP peak value was 8.7 ⁇ 0.4mP ( Figure 7B).
  • the HER2-2A aptamer reached a ⁇ FP peak of 5.8 ⁇ 0.5 mP at 1.5 h (Fig. 7C).
  • EVs were fixed in a 96-well plate using 8 ⁇ g/mL capture antibody for more than 16 hours at 4°C. After washing, 100 ⁇ L of one of the 5.0 nM fluorescently labeled aptamers (CD63-BP aptamer: PBS plus 1.0mM MgCl 2 ; HER2-HApt aptamer: PBS plus 5.0mM MgCl 2 ; HER2-2A aptamer: PBS plus 2.5mM MgCl 2 ). The microplate was placed on a shaker (Thermoline Scientific, model: TL400) and incubated at room temperature in the dark for 1.5 hours.
  • a shaker Thermoline Scientific, model: TL400
  • the FP signal was read out by the multifunctional disk analyzer CLARIOstar Plus (BMG Labtech) using a 485nm excitation filter and a 535nm emission filter to measure the FP value. All FP values are expressed in millipolarization (mP) units. The FP value of each sample is calculated from the average of 3 different wells. The FP value of each well is the average of 3 measurements of the well. Control wells (containing only free aptamer) were prepared using the same method, except that only PBS was added to the wells.
  • a control group was set up in the experiment, in which fixed EVs were treated with 1% Triton X-100 (polyethylene glycol octylphenyl ether) at room temperature for 15 minutes to ensure no intact EV remains.
  • Triton X-100 polyethylene glycol octylphenyl ether
  • For single-factor control samples of free EVs and proteins add 100 ⁇ L of 50 nM EpCAM protein into the wells, and fix EVs from cells that do not express EpCAM or HER2 as a control group for further specificity control. Process at room temperature for 15 minutes to ensure no intact EV remains.
  • For single-factor control samples of free EVs and proteins add 100 ⁇ L of 50 nM EpCAM protein into the wells, and fix EVs from cells that do not express EpCAM or HER2 as a control group for further specificity control.
  • the buffer includes synthetic buffer or human plasma.
  • the synthetic buffer is PBS buffer.
  • the human plasma is derived from a blood donor whose blood type is any one of type A, type B, type AB, type O, Rh+, and Rh-;
  • the human plasma comes from blood donors aged 0-120 years old;
  • the human plasma is derived from healthy people or non-healthy people; further preferably, the human plasma is derived from non-healthy people, and the non-healthy people are preferably tumor patients.
  • the EVs to be detected are suspended in human plasma rather than PBS; based on this, to simulate liquid biopsy conditions, cell line-derived EVs are added to the human plasma.
  • cell line-derived EVs were diluted at 1:10 before isolating EVs. (v/v) ratio was added to human plasma, and 8 ⁇ g/mL of the required antibody was used to capture cell line-derived EVs in human plasma. Control wells were treated similarly except that cell line-derived EVs were replaced with the same volume of PBS.
  • HT29-derived EVs were immobilized with EpCAM-adapted antibodies in addition to isotype-matched negative controls using anti-IgG antibodies.
  • fluorescently labeled CD63-BP aptamer was used to detect the presence of captured EVs, and it was found that the ⁇ FP (fluorescence polarization change) of the negative control aptamer was significantly lower than that of HT29 source EVs ( ⁇ FP was 10.1 ⁇ 0.7mP, p ⁇ 0.0001)
  • the negative control aptamer includes a scrambled nucleotide sequence that does not bind to CD63, Triton X-100 that dissolves EVs, and HEK293-derived EVs that do not express EpCAM.
  • the ⁇ FP levels of these negative controls were below the detection limit of this assay method.
  • the ⁇ FP of samples using isotype-matched control antibodies to capture EVs was -0.6 ⁇ 0.3 mP, which was completely consistent with the above results, further demonstrating the selectivity of this method for HT29 source EVs.
  • the present invention further sets up a negative control, adding only free EpCAM protein to the wells coated with anti-EpCAM. No EpCAM-positive EVs were added.
  • the ⁇ FP of the negative control was -0.5 ⁇ 0.4 mP, indicating that the positive ⁇ FP was not caused by the interaction between the fluorescently labeled CD63-BP aptamer and the free EpCAM protein immobilized in the microwell.
  • Figure 8A clearly demonstrates that this assay is selective and specific for EpCAM-positive HT29-derived EVs, and that the interaction between fluorescence, HT29-derived EVs, and antibody coating has minimal impact on the FP signal.
  • the present invention detects the specificity of the FP signal of the fluorescently labeled HER2 DNA aptamer to EVs captured by anti-CD9 and CD81 antibodies in the microwell plate.
  • the ⁇ FP of the HER2-HApt aptamer and HER2-2A aptamer derived from HER2-positive EVs in the experiment (8.7 ⁇ 0.4 mP and 5.8 ⁇ 0.5 mP, respectively) was significantly higher than that of the negative control sample.
  • Negative control samples included scrambled nucleotide sequence aptamers, EV-solubilizing detergents, isotype-matched control antibody coatings, and EVs derived from MDA-MB-231 cells with HER2 knockout (p ⁇ 0.0001, Figure 8B, Figure 8C). In fact, the ⁇ FP levels of these negative control samples were also below the detection limit of this assay (Figure 9).
  • EVs from cancer cell lines are used to prepare a mixture with a wide concentration range using buffer (F-PBS, phosphate buffered saline filtered through a 0.2 micron membrane) or human plasma to determine the test method.
  • buffer F-PBS, phosphate buffered saline filtered through a 0.2 micron membrane
  • LDR linear dynamic range
  • the LDR of total EVs in PBS was 5.0 ⁇ 10 8 -2.0 ⁇ 10 10 HT29 EVs/mL, which was wider than the LDR of EVs in human plasma (5.0 ⁇ 10 8 -1.0 ⁇ 10 10 ) ( Figure 9A and Figure 9B).
  • the FluPADE results of HER2-HApt aptamer showed that the LOD of SKBR3-derived EVs in PBS was low, 1.0 ⁇ 10 7 EVs/mL; while the LOD in human plasma was 3.0 ⁇ 10 7 SKBR EVs/mL.
  • the test results show that the LDR of EVs in PBS shifts downward, specifically 2.0 ⁇ 10 9 -2.0 ⁇ 10 10 SKBR3 EVs/mL; while the LDR of EVs in human plasma is wider, specifically 2.0 ⁇ 10 8 -1.0 ⁇ 10 10 SKBR3 EVs/mL ( Figure 9D and Figure 9F).
  • the FluPADE of the present invention detects HER2 in SKBR3-derived EVs in PBS and human plasma.
  • the LOD measured using the HER2-2A aptamer is lower than that using the HER2-HApt aptamer, but the LDR of the former is compared to the HER2-
  • the LDR of the HApt aptamer was narrower (Fig. 9C, E vs. Fig. 9D, F).
  • the same detection aptamers were used to analyze the same EVs in PBS or human plasma, and it was found that the difference in LODs and LDRs was caused by the total number of available biomarker proteins in the cell line-derived EVs in PBS and the EVs in human plasma. determined by the difference between quantities.
  • the amount of capture antibody-immobilized EVs in the microwell and the positive biomarker EVs in PBS or plasma is responsible for the difference in the final output of the fluorescence polarization signal. Therefore, the present invention investigated the contribution of EV marker protein amounts in human plasma.
  • the method for isolating EVs derived from human cell lines using antibodies as described above was used to detect EVs derived from human plasma.
  • EVs were isolated, and then fixed EVs from human plasma were incubated with 100 ⁇ L of 50 nM antibodies, including fluorescently labeled anti-EpCAM antibodies (the same anti-EpCAM antibodies used when fixing captured EVs), fluorescently labeled Anti-CD63 antibody (anti-CD63 antibody used when fixing EVs), PE-conjugated anti-CD9 antibody (anti-CD9 antibody used when fixing and capturing EVs), fluorescently labeled anti-CD81 antibody (used when fixing and capturing EVs) anti-CD81 antibody), fluorescently labeled anti-HER2 antibody (anti-CD9/CD81 antibody used when fixing and capturing EVs).
  • fluorescently labeled anti-EpCAM antibodies the same anti-EpCAM antibodies used when fixing captured EVs
  • fluorescently labeled Anti-CD63 antibody anti-CD63 antibody used when fixing EVs
  • the cells were washed three times with 200 ⁇ L of 0.1% F-TPBS, and the fluorescence intensity spectrum in the wavelength range from 520 nm to 660 nm was recorded using a CLARIOstar Plus (BMG Labtech) multifunctional disk analyzer.
  • EVs isolated from human plasma using anti-EpCAM antibodies clearly expressed the expression of cancer marker proteins, and the EV marker protein CD63 was also clearly detected in EVs isolated from human plasma.
  • the levels of EpCAM and/or CD63 protein in EVs isolated from human plasma may affect the performance of the CD63-BP aptamer-based fluPADE assay in detecting EVs isolated from cancer cell lines in the following aspects.
  • the presence of EpCAM in human plasma EVs may cause the reduction of HT29-derived EVs, which occurs due to the competition between the capture anti-EpCAM antibodies in human plasma and the EpCAM protein in plasma EVs and the same protein in HT29-derived EVs.
  • CD63 expression level of EVs in human plasma is much lower than that in HT29-source EVs (fluorescence intensity 6206.5 corresponds to human plasma EVs of 9.2 ⁇ 10 8 and fluorescence intensity 84345.1 corresponds to HT29-source EVs of 2.0 ⁇ 10 8 )
  • human plasma The interaction between EV's CD63-BP aptamer and CD63 protein may facilitate the detection of the total FP signal. The synergy of these effects will result in an increase in the apparent LOD from 5 ⁇ 10 6 to 5 ⁇ 10 7 EVs/mL and a decrease in the LDR range from 5.0 ⁇ 10 8 -2.0 ⁇ 10 10 to HT29-derived EVs bound to human plasma.
  • Fluorescence intensity is the most commonly used detection method in fluorescence detection, and fluorescence polarization is rarely used as a detection mode, especially in the field of medical diagnosis.
  • the present invention uses the same EV fixation method and fluorescently labeled aptamers to compare EV performance based on fluorescence intensity and fluorescence polarization mode, specifically presented through LOD and LDR.
  • EVs in human plasma were then detected using the same detection method as EVs in PBS.
  • Figure 11 shows the relationship between fluorescence intensity and EV concentration, LOD and LDR using CD63-BP aptamer, HER2-HApt aptamer or HER2-2A aptamer pair from F-PBS (Fig. 11A,C,E ) or HT29-derived EVs or SKBR3-derived EVs in human plasma (Fig. 11B, D, F) were measured for fluorescence intensity.
  • the LOD of fluorescence intensity detection of EVs secreted by cancer cells in F-PBS using fluorescently labeled CD63-BP aptamer was 2.0 ⁇ 10 8 HT29 EVs/mL ( Figure 11A), and using fluorescently labeled HER2-HApt aptamer to detect SKBR3
  • the LOD of fluorescence intensity detection of source EVs was 5.0 ⁇ 10 8 SKBR3 EVs/mL ( Figure 11C)
  • the LOD of fluorescently labeled HER2-2A aptamer was 2.0 ⁇ 10 8 SKBR3 EVs/mL ( Figure 11E).
  • the present invention also determines the LODs of the fluorescence intensity of EVs in human plasma.
  • the LOD of the fluorescence intensity measured by the CD63-BP aptamer in human plasma increased by about 2.5 times, specifically 5.0 ⁇ 10 8 HT29 EVs/mL.
  • the LOD of the HER2-Hapt aptamer increased by 2 times, specifically 1.0 ⁇ 10 9 , HER2
  • the LOD of the -2A aptamer increased 4-fold, specifically 8.0 ⁇ 10 8 SKBR3 EVs/mL ( Figure 11D, Figure 11F).
  • the significant correlation difference in LOD between EVs in PBS and EVs in human plasma may be related to biomarkers in EVs in human plasma.
  • the LOD of the FluPADE assay was much lower compared to the fluorescence intensity assay using the same assay aptamer.
  • the LOD detected by FluPADE using the CD63-BP aptamer was 4 times lower than the LOD detected by fluorescence intensity in PBS and human plasma (5.0 ⁇ 10 6 EVs/mL and 2.0 ⁇ 10 8 HT29 EVs/mL, respectively). and 10 times (5.0 ⁇ 10 7 EVs/mL and 5.0 ⁇ 10 8 HT29 EVs/mL respectively).
  • FP detection using the HER2-HApt aptamer in PBS and human plasma was approximately 17-26 times more sensitive than fluorescence intensity detection.
  • the LOD of the HER2-HApt aptamer in PBS is 3.0 ⁇ 10 7 SKBR3 EVs/mL
  • the LOD of the HER2-2A aptamer is 1.0 ⁇ 10 7 SKBR3EVs/mL
  • the LOD of the HER2-HApt aptamer in human plasma is 5.0.
  • the LOD of HER2-2A aptamer is 3.0 ⁇ 10 7 SKBR3 EVs/mL.
  • the fluorescence intensity LDR of the fluorescently labeled CD63-BP aptamer is 3.0 ⁇ 10 8 -2.0 ⁇ 10 9 EVs/mL
  • the fluorescence intensity LDR of the HER2-2A aptamer is 1.0 ⁇ 10 9 -1.0 ⁇ 10 10 EVs/mL
  • the aptamer-based FluPADE assay especially for the detection of HER2-positive EVs, is able to detect cancer EVs in PBS and human plasma at lower concentrations and perform EV detection over a wider range of EV concentrations. Quantification, while detection based on the fluorescence intensity of aptamers cannot achieve such an effect.
  • Figure 12 shows the relationship between fluorescence intensity and EV concentration, LOD and LDR based on antibody testing.
  • anti-CD63 antibody detected FI with an LOD of 1.0 ⁇ 10 7 HT29 EVs/mL and an LDR of 5.0 ⁇ 10 7 -1.0 ⁇ 10 9 HT29 EVs/mL ( Figure 12A); using anti-HER2 antibody
  • the LOD for detecting FI was 1.0 ⁇ 10 9 SKBR3 EVs/mL, and the LDR was 2.0 ⁇ 10 9 -2.0 ⁇ 10 10 SKBR3 EVs/mL ( Figure 12C).
  • anti-CD63 antibody detected FI with an LOD of 1.0 ⁇ 10 7 HT29 EVs/mL and an LDR of 5.0 ⁇ 10 7 -1.0 ⁇ 10 9 HT29 EVs/mL ( Figure 12A); using anti-HER2
  • the LOD of antibody detection of FI is 5.0 ⁇ 10 7 HT29 EVs/mL, and the LDR is 1.0 ⁇ 10 8 -2.0 ⁇ 10 9 HT29 EVs/mL ( Figure 12B);
  • the LOD of detection of FI using anti-HER2 antibody is 3.0 ⁇ 10 9 SKBR3 EVs/mL, LDR is 5.0 ⁇ 10 9 -1.0 ⁇ 10 10 SKBR3 EVs/mL ( Figure 12D).
  • FluPADE provided by the present invention is superior to traditional fluorescence intensity-based detection methods in detecting cancer-derived EVs in buffers and human plasma, with higher sensitivity and wider dynamic range (Table 1) .
  • the LOD of the detection method is ⁇ 5 ⁇ 10 7 EVs/mL, and the LDR is 5 ⁇ 10 8 -2 ⁇ 10 10 EVs/mL;
  • the nucleic acid aptamer is When the aptamer is HER2-HApt, the LOD of the detection method is ⁇ 5 ⁇ 10 7 EVs/mL, and the LDR is 8 ⁇ 10 7 -2 ⁇ 10 10 EVs/mL;
  • the detection method The LOD of the detection method is ⁇ 3 ⁇ 10 7 EVs/mL, and the LDR is 2 ⁇ 10 8 -2 ⁇ 10 10 EVs/mL.
  • One of the key challenges in EV-based cancer diagnosis is the low abundance of cancer-derived EVs in samples, as cancer-derived EVs are released into an EV reservoir that contains approximately 200 types derived from biological fluids. EVs released by healthy human cells. This situation makes the identification of cancer cell-derived EVs equivalent to finding a needle in a haystack.
  • the present invention continuously divided EpCAM-positive EVs derived from HT29 cells into five ratios: 1:2000, 1:1000, 1:500, 1:100 and 1:10. Titrate into HEK293 cell-derived EpCAM-negative EVs to control the total concentration of EVs at 1.0005 ⁇ 10 10 EVs/mL.
  • EVs from HER2-positive SKBR3 cells were titrated sequentially at 1:1000, 1:750, 1:500, 1:100, and 1:10 ratios.
  • the total EV concentration was fixed at 3.0030 ⁇ 10 10 EVs/mL (HER2-HApt aptamer) and 1.0010 ⁇ 10 10 EVs/mL (HER2-2A aptamer).
  • FP-based EV detection in PBS For FP-based EV detection in PBS, add 8 ⁇ g/mL of anti-EpCAM antibody for HT29-derived EVs or anti-CD9/CD81 antibody for SKBR3-derived EVs to streptavidin-coated microwells. Incubate at room temperature for 30 minutes and wash, then fix the EVs at 4°C for more than 16 hours. Add 100 ⁇ L of PBS containing one of the following aptamers (5 nM) to each microwell: fluorescently labeled CD63-BP, Fluorescently labeled HER2-HApt and fluorescently labeled HER2A were then cultured on a shaker (Thermoline Scientific, model: TL400) at room temperature in the dark. The culture time for the first two aptamers was 1 hour, and the culture time for the third one was 1 hour. The incubation time for aptamers is 1.5 hours.
  • the FP signal was measured on a multifunctional disk analyzer CARIOstar Plus (BMG Labtech) with an excitation filter of 485 nm and an emission filter of 535 nm.
  • the FP control wells of free ligand were prepared in the same way as above except that EV was not added. It is worth noting that the total concentration of EVs needs to be maintained at a level of 10 10 EVs/mL to simulate the physiological concentration of EVs in the systemic circulation. In this way, the results of the present invention are clinically relevant and can be applied to pathology experiments.
  • the sensitivity of the present assay is defined as the lowest ratio at which the detected FP signal is equal to or higher than the FP signal at the LOD.
  • the sensitivity of the HER2-HApt aptamer-based FluPADE assay is that it can detect at least 1 HER2-positive EV in a system with a background of 750 HER-2-negative EVs ( Figure 13) (HER2-HApt aptamer).
  • EV while the sensitivity of the assay using the HER2-2A aptamer is that at least 1 HER2-positive EV can be detected in the background of 1000 HER2-negative EVs, and the detection sensitivity based on the HER2-HApt aptamer is higher.
  • a mature liquid biopsy method for detecting cancer-derived EVs not only needs to be highly sensitive, but also should be able to distinguish EVs from different sources using the same biomarkers.
  • EVs released by tumors at the primary site and EVs released from metastatic sites belong to genetically and phenotypically different entities. Even EVs released by cancer cells from the same patient may qualitatively express preferred biomarkers. But it is conceivable that in different primary sites, different growth stages or different clinical courses There were quantitative differences in the abundance of surface biomarker proteins of tumor-released EVs. From a diagnostic perspective, the FluPADE provided by the present invention is very conducive to detecting different EV populations from different cancer cells based on quantitative differences in surface biomarker abundance.
  • the present invention established a model system in which EVs were derived from 3 cell lines of 3 common solid cancers.
  • the present invention first determined the differences in CD63 and HER2 abundance in EVs prepared from colorectal cancer (HT29), breast cancer (SKBR3) and hepatocellular carcinoma (HepG2).
  • the method for measuring CD63 and HER2 abundance in these EVs was similar to the method described above, except that 50 nM of Alexa -conjugated anti-HER2 antibody (BioLegend, Cat No.: 324412) to quantify HER-positive EVs.
  • EVs derived from different cancers displayed different amounts of surface marker proteins on the surface of EV biomarkers (CD63) and cancer biomarkers (HER2). Differences in the abundance of EV surface marker proteins will lead to differences in the degree of fluorescence polarization produced when the detected fluorescently labeled aptamers immobilize EVs on the surface.
  • the ratio of EVs secreted by cancer cell lines to plasma EVs was 1:10 (v/v), and the total EV concentration in all 18 samples was the same, 1.0 ⁇ 10 10 EVs/mL.
  • EVs from three different types of solid cancers were detected using either a fluorescently labeled EV marker (CD63) or a fluorescently labeled cancer cell marker (HER2), each spiked into plasma from six blood donors, as shown in Figure 14B and shown in Figure 14D.
  • CD63 fluorescently labeled EV marker
  • HER2 fluorescently labeled cancer cell marker
  • fluorescence polarization changes based on CD63 aptamer detection can be used to differentiate anti-EpCAM antibody-immobilized EVs from three different cancer cells (Fig. 14C).
  • the present invention designed a set of interactive experiments, in which the corresponding antibodies of two EV markers (CD9 and CD81) were used to immobilize EVs from three different types of cancer, and HER2's Corresponding aptamers for cancer biomarkers.
  • Figure 14E in human plasma, the ⁇ FP of HER2-HApt aptamer in detecting HT29-derived EVs was higher than that in detecting SKBR3-derived EVs.
  • Figure 14F the changes in the 3 cancer-derived EVs from 3 different cell sources were very obvious.
  • a second aspect of the present invention provides an application of the nucleic acid aptamer-based extracellular vesicle fluorescence polarization detection method, which is applied to the qualitative and quantitative analysis of extracellular vesicles secreted by cancer cells.
  • the cancer cells are derived from colorectal cancer, breast cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, esophageal cancer, nasopharyngeal cancer, throat cancer, endometrial cancer, lung cancer, head and neck cancer, Any of kidney cancer, bladder cancer, thyroid cancer, skin cancer, ovarian cancer, cervical cancer, prostate cancer, and penile cancer.
  • the detection method can distinguish EVs secreted by cancer cells in different primary sites; the primary sites include intestine, breast, liver, stomach, pancreas, esophagus, epidermis, skin and soft tissue, ovary, Any of the cervix, prostate, penis.
  • the detection method can distinguish EVs secreted by cancer cells at different growth stages; the growth stages include any one of the in situ cancer stage, regional lymph node metastasis stage, and distant metastasis stage; excellent
  • the detection method can distinguish extracellular vesicles secreted by cancer cells at different growth stages during anti-cancer treatment.
  • the detection method can be directly performed on an automatic biochemical analyzer in a clinical laboratory without the need for special equipment or specially-made instruments.
  • the detection method can be directly performed on an automatic immunoassay analyzer in a clinical laboratory without the need for special equipment or specially-made instruments.
  • This application uses nucleic acid aptamers as detection substances and uses fluorescence polarization to detect extracellular vesicles secreted by cancer cells. It can effectively distinguish extracellular vesicles that contain the same biomarker protein but originate from different cell populations and can be used for cancer in vitro. Qualitative and quantitative analysis of liquid biopsy. At the same time, the present invention can specifically detect extracellular vesicles secreted by cancer cells in the blood. The detection process is not interfered by free tumor marker proteins, tumor cell membrane fragments or tumor extracellular vesicle membrane fragments in the blood, and the detection results are accurate and effective.
  • Figure 1 Schematic diagram of the steps for fluorescence polarization detection of cancer cell-derived EVs
  • Figure 2 Schematic diagram of the working principle of the extracellular vesicle fluorescence polarization detection method based on nucleic acid aptamers
  • FIG. 3 EV characteristics of HT29, SKBR3, HepG2, HEK293 and MDA-MB-231 cells with HER2 gene knocked out; specifically, A is the EV particle size distribution measured by nanoparticle tracking analysis technology; B is scanning electron microscopy observation The EV morphology obtained; C is the test results of immunoblotting assay by Alix, CD63, calnexin, EpCAM and HER2; D is the EV surface biomarker proteins or cancer biomarkers analyzed by flow cytometry (including CD9, CD63, CD81, EpCAM and HER2) expression; E is a histogram of EV surface marker protein expression detected and analyzed by flow cytometry, in which red is the background control (IgG isotype control-coated magnetic beads used to immobilize EVs), blue The color shows the specific antibody-coated magnetic beads used for EV immobilization.
  • A is the EV particle size distribution measured by nanoparticle tracking analysis technology
  • B scanning electron microscopy observation The EV morph
  • FIG. 4 A is a schematic diagram of detecting fixed EVs with fluorescently labeled anti-CD63 antibodies (BioLegend, Cat No.: 353006); B is a schematic diagram of using biotinylated anti-EpCAM antibodies under the conditions of 16 hours of action in a cold room.
  • Immobilized HT29-derived EV (BioLegend, Cat No.: 324216) and SKRB3-derived EV immobilized using 1:1 anti-CD9/CD81 antibody (BioLegend, anti-CD9 antibody: Cat No.: 312112; anti-CD81 antibody :CatNo.:349514); in which biotin-labeled antibodies are coated on streptavidin-coated microwells (Thermo Fisher Scientific, CatNo.:15503); fixed EVs are treated with 1% Triton X-100 or physiological saline Treatment (p ⁇ 0.0001); C is the fluorescence intensity of biotinylated anti-EpCAM antibodies for HT29-derived EVs and biotinylated anti-CD9 for EVs from SKBR3 sources compared to fluorescence intensity of EVs immobilized with 5 ⁇ g/mL antibody.
  • FIG. 5 Binding affinity of antibodies and nucleic acid aptamers to immobilized EVs and fluorescence polarization properties of EVs using antibodies or nucleic acid aptamers;
  • A is the binding curve and apparent dissociation constant (K) of CD63 antibody-immobilized HT29 source EVs D );
  • B is the binding curve and apparent dissociation constant (K D ) of CD63-BP aptamer-immobilized HT29 source EV;
  • C is the use of anti-CD63 antibody or CD63-BP aptamer at 5 nM, K D and 2 K D concentrations.
  • D is the binding curve and K D of anti-HER2 antibody-immobilized SKBR3 source EV
  • E is the binding curve and K D of SKBR3 source EV immobilized by HER2-HApt aptamer
  • F is 5nM, KD and 2KD concentrations Differences in fluorescence polarization of SKBR3 source EVs immobilized using anti-HER2 antibody and HER2-HApt aptamer
  • Figure 7 Determine the optimal incubation time between aptamers and immobilized EVs to obtain the best fluorescence polarization signal;
  • A is the CD63-BP aptamer acting on HT29 source EVs;
  • B is the HER2-HApt aptamer acting on SKRB3 source EV;
  • C is the effect of HER2-2A aptamer on SKRB3 source EV;
  • FIG. 8 Differences in fluorescence polarization of CD63 and HEER2 aptamers in different samples; A is the difference in fluorescence polarization of 5nM fluorescently labeled CD63-BP aptamer in 6 samples acting on anti-EpCAM antibody-immobilized HT29 source EVs; Fluorescent labeling Aptamers do not bind to CD63 and anti-EpCAM-immobilized HT29 source EVs; the anti-EpCAM-immobilized HT29 source EVs are derived from Triton X-100 lysis (anti-EpCAM antibody isotype matching negative control), HEK293 sources that do not express EpCAM EV or free EpCAM protein (50nM); B is the fluorescence polarization difference of 5nM fluorescently labeled HER2-HApt-BP aptamer in 5 different samples: anti-CD9/CD81 antibody will immobilize SKRB3 source EV and does not interact with HER2 and anti- Fluorescently labeled
  • Figure 9 Fluorescence polarization signal as a function of EV concentration.
  • the LOD and LDR of the FluPADE detection method were determined based on a linear curve of the internal fluorescence signal as a function of log10(lg) EV concentration measured in PBS ( Figures A, C, E ) or human plasma (1:10, v/v; panels B, D, F) based on CD63-BP aptamer (A-B), HER2-HApt aptamer (C-D) or HER2-2A (E-F) aptamer detection .
  • FP and ⁇ FP values are expressed in millipolarization (mP) units; all FP values are expressed in millipolarization (mP) units, and the FP value for each sample was calculated from the average of 3 different wells. The FP value is the average of three measurements of the hole.
  • Figure 10 Background signal measurement of EV marker proteins and cancer biomarkers in EV assay in human plasma; the red spectrum in A shows the detection of EpCAM and CD63 using anti-EpCAM antibody, and the detection of CD81 and HER2 using anti-CD9/CD81 antibody.
  • the fluorescence intensity spectrum (emission wavelength of 575nm) of the PE-anti-CD9 antibody at an excitation wavelength of 570nm to 690nm (red) was also recorded, as well as the use of IgG isotypes Fluorescence intensity spectrum (blue) of EV isolated from human plasma using a control antibody.
  • B is the measurement of anti-EpCAM antibody (ex: 485nm, em: 535nm), anti-CD63 antibody (ex: 485nm, em: 535nm), anti-CD9 antibody (ex: 560nm, em: 575nm), and anti-CD81 antibody respectively.
  • FIG. 11 LOD and LDR of fluorescence intensity detection based on aptamer; specifically, the detection of HT29 source EV using CD63-BP aptamer (A-B) and the detection using HER2-HApt aptamer (C-D) or HER2-2A aptamer (E-F)
  • A-B CD63-BP aptamer
  • C-D HER2-HApt aptamer
  • E-F HER2-2A aptamer
  • FIG. 12 LOD and LDR for fluorescence intensity detection based on antibodies; specifically using anti-CD63 antibody (AB) The relationship between EV concentration and internal fluorescence intensity as well as LOD and LDR results when measuring HT29-derived EVs and using anti-HER2 antibody (CD) to determine SKRB3-derived EVs; the cell line-derived EVs were diluted with PBS ( Figure A, C) and Human plasma was used for dilution (Panels B, D).
  • Figure 13 The sensitivity of the aptamer-based fluorescence polarization method for detecting cancer-derived EVs; specifically, the changes in the aptamer fluorescence polarization signal under the action of a linear concentration of cancer-derived EVs.
  • A is the ratio of EpCAM-positive HT29 source EVs and EpCAM-negative HEK293 source EVs detected using 5.0nM CD63-BP aptamer;
  • B and C are respectively the HER2-positive results detected using 5.0nM HER2-HApt aptamer or HER2-2A aptamer.
  • FIG. 14 FluPADE is able to differentiate EVs from different sources based on dual analysis of fluorescence polarization of biomarkers.
  • the first row of pictures in A shows the expression levels of CD63 in HT29-source EVs, SKBR3-source EVs and HepG2-source EVs isolated using anti-EpCAM antibody-coated magnetic beads;
  • the second row of pictures shows the expression levels of CD63 in HT29-source EVs, SKBR3-source EVs and HepG2-source EVs separated using anti-EpCAM antibody-coated magnetic beads;
  • Red is the background fluorescence of EVs cultured on magnetic beads coated with IgG isotype matching control antibodies; blue is the fluorescence intensity of EVs isolated from magnetic beads coated with anti-EpCAM antibodies (first row) and anti-CD9/CD81 antibody coated Fluorescence intensity of EVs separated by magnetic beads (second row).
  • B is a schematic diagram of the FluPADE assay using the CD63-BP aptamer to detect EVs in human plasma isolated from 3 different cancer cell lines using anti-EpCAM antibodies from 6 blood donors.
  • C is the fluorescence polarization signal difference of EVs in human plasma separated by anti-EpCAM antibodies of three different cancer cell lines acting on the CD63-BP aptamer; the human plasma was derived from 6 blood donors.
  • D is a schematic diagram of the FluPADE assay using the HER2-HApt aptamer to detect EVs in human plasma isolated from anti-CD9/CD81 antibodies from 3 different cancer cell lines from 6 blood donors.
  • E is the fluorescence polarization signal difference of EVs in human plasma separated by anti-CD9/CD81 antibodies of 3 different cancer cell lines acting on HER2-HApt aptamer; the human plasma was derived from 6 blood donors.
  • Extracellular vesicle fluorescence polarization detection method based on CD63-BP aptamer in PBS.
  • Extracellular vesicle fluorescence polarization detection method based on HER2-HApt aptamer in PBS.
  • HT29 source EV and SKBR3 source EV were immobilized in microwells. Specifically, biotin-labeled antibodies were placed in microwells coated with streptavidin and incubated at room temperature for 30 minutes and washed, and then EVs were cultured at 4°C for more than 16 hours.
  • HER2A aptamer-based extracellular vesicle fluorescence polarization detection method in PBS HER2A aptamer-based extracellular vesicle fluorescence polarization detection method in PBS.
  • the method for detecting fluorescence polarization of extracellular vesicles based on CD63-BP aptamer in human plasma is the same as Example 1; the difference is that in step S1, before fixing EVs, a target amount of EVs is added to human plasma. (ratio is 1:10, v/v), and then use 8.0 ⁇ g/mL anti-EpCAM antibody and anti-CD9/CD81 antibody (mass ratio 1:1) respectively to remove EVs derived from cell lines in human plasma. Fixed in microwells.
  • the method for detecting fluorescence polarization of extracellular vesicles based on HER2-HApt aptamer in human plasma is the same as that in Example 4; the difference is that the CD63-BP aptamer in step S2 is replaced with HER2-HApt aptamer. .
  • the method for detecting fluorescence polarization of extracellular vesicles based on HER2A aptamer in human plasma the specific implementation method is the same as Example 4; the difference is that the CD63-BP aptamer in step S2 is replaced with HER2A aptamer; in S2 The incubation time is 1.5 hours.
  • the method for detecting fluorescence intensity of extracellular vesicles based on CD63-BP aptamer in PBS is the same as Example 1; the difference is:
  • the S2 step is to add 100 ⁇ L of PBS buffer containing 800 nM fluorescently labeled CD63-BP aptamer, and then add 100 ⁇ L of PBS containing 50 nM fluorescently labeled CD63 antibody or 50nM fluorescently labeled HER2 antibody was then cultured on a shaker (Thermoline Scientific, model: TL400) at room temperature in the dark for 1 hour.
  • the S3 step is, after washing three times with 200 ⁇ L of washing buffer, use a multifunctional disk analyzer CLARIOstar Plus (BMG Labtech) to measure the fluorescence intensity under a 485nm excitation filter and a 535nm emission filter.
  • CLARIOstar Plus BMG Labtech
  • the method for detecting the fluorescence intensity of extracellular vesicles based on the HER2-HApt aptamer in PBS is implemented in the same manner as in Example 7; the difference is that the CD63-BP aptamer in the S2 step is replaced with the HER2-HApt aptamer.
  • the specific implementation method for detecting the fluorescence intensity of extracellular vesicles based on the HER2-HApt aptamer in PBS is the same as in Example 7; the difference is that the CD63-BP aptamer in the S2 step is replaced with the HER2-2A aptamer.
  • the method for detecting the fluorescence intensity of extracellular vesicles based on anti-CD63 antibody in PBS is implemented in the same manner as in Example 7; the difference is that the CD63-BP aptamer in step S2 is replaced with an anti-CD63 antibody.
  • the method for detecting the fluorescence intensity of extracellular vesicles in PBS based on anti-HER2 antibodies is implemented in the same manner as in Example 7; the difference is that the CD63-BP aptamer in the S2 step is replaced with an anti-HER2 antibody.
  • Example 7 The difference is that in step S1, before fixing EVs, the target amount of EVs is added to human plasma (ratio is 1:10, v/v), and then 8 ⁇ g/mL anti-EpCAM antibody is used respectively. and anti-CD9/CD81 antibody (mass ratio 1:1), to immobilize EVs derived from cell lines in human plasma in microwells.
  • the specific implementation method for detecting the fluorescence intensity of extracellular vesicles based on the HER2-HApt aptamer in human plasma is the same as in Example 12; the difference is that the CD63-BP aptamer in the S2 step is replaced with the HER2-HApt aptamer.
  • Example 12 The difference is that the CD63-BP aptamer in the S2 step is replaced by the HER2-2A aptamer.
  • the specific implementation method for detecting the fluorescence intensity of extracellular vesicles in human plasma based on anti-CD63 antibodies is the same as in Example 12; the difference is that the CD63-BP aptamer in step S2 is replaced with an anti-CD63 antibody.
  • the specific implementation method for detecting the fluorescence intensity of extracellular vesicles in human plasma based on anti-CD63 antibodies is the same as in Example 12; the difference is that the CD63-BP aptamer in the S2 step is replaced with an anti-HER2 antibody.
  • the LOD of the above examples is measured as the EV concentration determined by the test signal, which is equal to the signal of the control sample plus 3 times the standard deviation of the control sample result.
  • the linear dynamic range is defined by linear regression of the EV concentration signal; the measurement results are shown in Table 1.

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Abstract

一种基于核酸适体的细胞外囊泡荧光极化检测方法及其应用。检测方法包括以下步骤：S1.通过抗体与单细胞外囊泡（EV）表面生物标记蛋白或EV表面癌症标志物作用，对EV进行固定；然后快速清洗，去除游离EV，蛋白质，膜碎片和脂质；S2.分别添加与EV标志物或癌细胞标志物适配的核酸适体进行培养，核酸适体经过荧光标记；S3.对S2步骤的产物进行荧光极化检测，实现对癌细胞分泌的EV的定性、定量分析。检测方法能够特异性地检测血液中癌细胞分泌的细胞外囊泡，检测过程不受血液中游离肿瘤标志蛋白、肿瘤细胞膜碎片或肿瘤细胞外囊泡膜碎片的干扰，检测结果精准有效。

Description

一种基于核酸适体的细胞外囊泡荧光极化检测方法及应用 技术领域
本发明涉及C12N15/115(与靶分子特异性、高亲和力结合的适体)技术领域，尤其涉及一种基于核酸适体的细胞外囊泡荧光极化检测方法及其应用。
背景技术
芯片实验室设备，纳米颗粒追踪分析技术(NTA)，高分辨率单细胞外囊泡(EV)分析技术，流式细胞术和酶联免疫吸附试验(ELISA)是目前以高通量方式检测和量化细胞外囊泡的主要方法。然而，微流体和微机械芯片等芯片实验室技术需要高级微加工技术和精密仪器来读取信号并分析数据，局限了这些技术在实验室和临床领域中的EV研究中的广泛应用。高灵敏度流式细胞仪不仅用于检测和量化EV，还能够表征具有高通量和多重分析潜力的单个EV，在这项技术广泛应用于EV研究和临床实践之前，时间成本、群体效应和温和的再现性势必会推动这一技术的广阔发展。虽然ELISA是一种常用的生化检测分析方法，但ELISA检测EV的检测限仅在109-1010EVs/mL范围内较高。因此，探究一种简便、特异、灵敏的检测和量化EV的方法，是实验室和临床领域亟待解决的问题。
在Weber研究之后的过去几十年里，荧光极化已被用于临床环境和高通量分析(即药物发现)中。这一技术的飞速发展背后可归因于其良好的重现性、高度的自主性、对小体积(大约10μL)样本的适应性以及在均相分析中不分离游离和结合配体。分离未结合和结合物种的异质技术不仅更加费力，而且存在着对实际生物分子相互作用的不准确量化的问题。同时，其它基于荧光的均相测定，例如荧光共振能量转移(FRET)、时间分辨荧光(TRF)或时间分辨FRET(TR-FRET)需要多个标记，而不是荧光偏振中使用的单个标记。此外，荧光极化作为一种比率测量方法，可消除由内部过滤效应触发的伪影，减少分析环境的影响，并且不受其它化合物的吸光度或颜色猝灭的影响。
荧光极化(FP)检测方法的开发过程中，选择分子量应小于15kDa的探测物质是至关重要的要素。基于这样的情况，如PKH26和PKH67,以及碳花青染料(DiI,DiO)等亲脂性染料作为配体时，虽然其分子量很小(～1kDa)，但其本身具有固有的局限性，例如与非EV颗粒的非特异性结合、低标记效率以及染料聚集时的额外影响。虽然抗体特异性结合EV标记物，但其大尺寸(约150kDa)有可能不适用于FP。相比之下，核酸适体(化学抗体)的尺寸要小得多(<15kDa)，化学合成更为稳定，因此是基于FP检测EV的理想探测物质。
现有技术中只有两篇关于使用FP检测EV的报道。Kalimuthu等人使用5-十二烷基氨基荧光素(C12-FAM，一种亲脂性染料)作为探测物质来检测源自HT29细胞系和TCMK1细胞系的EV。然而，染料分子的聚集以及探测物质(C12-FAM)与非EV目标(如EV溶液中的膜碎片或脂蛋白)之间的非特异性相互作用会干扰FP信号的观测。在另一报道(Z.Zhang et al.,Aptamer-based fluorescence polarization assay for separation-free exosome quantification,Nanoscale,2019,11,10106-10113.)中，CD63适体被用于直接检测A549细胞系或人血浆中的EV，无需事先分离EV。尽管本申请人证实了该实验方法对CD63阳性EV的敏感性远高于其它可溶性CD63蛋白，暂不论它在癌症诊断中的用途，在提取 EV或人血浆中，所用的探针会与可溶性蛋白和/或EV中的蛋白质聚集体的交互作用，导致检测阳性结果的被检测到的靶点为可溶性蛋白或膜碎片而不是来源于癌细胞分泌的细胞外囊泡，因为适体的FP检测方法的LOD可达86aM或28aM。
本申请提供了一种基于核酸适体的细胞外囊泡荧光极化检测方法(fluorescence polarization using aptamers for the detection of extracellular nanovesicles，FluPADE)。得益于抗体和适体的高特异性以及适体的小尺寸，该方法能够高效准确地检测细胞培养基或人血浆中的癌源性EV。
发明内容
本发明第一方面提供了一种基于核酸适体的细胞外囊泡荧光极化检测方法，如图1所示，包括以下步骤：
S1.通过抗体与EV表面生物标记蛋白或EV表面癌症标志物作用，对EV进行固定；然后快速清洗，去除游离EV，蛋白质，膜碎片和脂质；
S2.分别添加与EV标志物或癌细胞标志物适配的核酸适体进行孵育，所述核酸适体经过荧光标记；
S3.对S2步骤的产物进行荧光极化检测，实现对癌细胞分泌的EV的定性、定量分析，操作过程中无需清洗。
这一检测方法中的生物化学过程和生物物理学测定原理如图2所示。
癌细胞分泌的EV其实是利用各种生物标志物对EV进行捕获和检测时，使荧光极化信号得以呈现的诱因。本发明在FluPADE检测中采用3种不同的核酸适体，以证明可以通过靶向结合EV表面生物标志物的多种适体以及适体的尺寸规格的探究，实现检测方法的优化。同时，本发明采用3种来源于不同类型实体肿瘤细胞系的癌细胞分泌的EV，具体为结直肠癌(HT29)，乳腺癌(SKBR3)和肝细胞癌(HepG2)，论证本方法的普适性。因此，FluPADE检测的灵敏度、操作简便性和功能化决定了这一方面能够很好地应用于临床上诸多肿瘤学科的研究。
本实验室使用标准超滤方法(Centricon Plus-70离心过滤装置，MWCO 10 kDa，Merck，Cat UFC701008)制备5种源自不同的人类细胞系的细胞外囊泡，包括HT29、SKBR3、HepG2、HEK293和MDA-MB-231细胞，以及HER2基因被敲除的MDA-MB-231细胞。使用NTA技术研究EV的粒径分布及其在总颗粒浓度中所占比例。图3A显示了5种来源的EV的平均尺寸。这其中，敲除了HER2基因的MDA-MB-231源EV的尺寸最大(142.3±10.3nm)，而HT29细胞系的EV尺寸最小(98.7±8.5nm)。五种EV的主体尺寸均符合小型EV的尺寸范围(尺寸＜200nm)。
进一步地，使用扫描电子显微镜对5种不同来源的EV的表面形貌和形状进行了表征。5种来源的EV形状均为球形(图3B)。虽然5种EV的尺寸都呈多分散状，但其中大部分EV的直径都小于200nm。有趣的是，敲除HER2基因的MDA-MB-231源EV的平均尺寸最大，与NTA技术确定的尺寸分布结果保持一致(图3A)。
在一些优选的实施方式中，所述EV标志物包括CD9，CD63，CD81中的至少一种。
在一些优选的实施方式中，所述癌细胞标志物包括EpCAM和/或HER2。
参照国际细胞外囊泡协会的规定，EV应当至少包含一种跨膜蛋白、一种胞质蛋白，且不存在负调节蛋白。因此，本申请通过免疫印迹试验证明了EV中CD63(一种跨膜蛋白)和 Alix(一种胞质蛋白)的存在，并通过其它癌症标志物(EpCAM和HER2)证明无负调节蛋白(calnexin，内质网标志物)的存在。如图3C所示，CD63和Alix在所有的EV中均出现，而calnexin在5种EV中均未出现。就癌症标志物而言，在源自HT29，SKBR3和HepG2的EV中均发现EpCAM，但HEK293源EV中未出现EpCAM的表达。重要的是，只在源自HT29，SKBR3和HepG2的EV中发现了HER2的基因表达，但是在敲掉HER2基因的MDA-MB-231源EV中未发现(图3C)。
EV表面蛋白是本发明中的直接结合靶标，因此，除了通过印迹法表征蛋白质外，本发明还采用磁珠流式细胞仪对5种来源的EV的几种生物标志物进行了表征，包括三种EV标记蛋白(CD9、CD63和CD81)和两种癌细胞标志物(EpCAM和HER2)。首先，以10倍体积的结合缓冲液(经过0.22μm过滤，成分为含5％牛血清白蛋白的磷酸缓冲液)对链霉亲和素包被的10μm磁珠(Merck,Cat No.:LSKMAGT02)进行洗涤，并用10倍体积的结合缓冲液在室温下封闭1小时。弃掉上清液，封闭后的磁珠在结合缓冲液中重悬浮以分离EV。将0.5μg生物素化抗体与2.5μg封闭的链霉亲和素包被磁珠混合，在室温下温和混合培养30min。使用足量的洗涤缓冲液(经过0.22μm过滤，成分为含0.1％吐温20的磷酸缓冲液)对抗体包被的磁珠洗涤3次，然后加入100μL的5×108EV和100μL的结合缓冲液，在4℃条件下过夜培养。最后，用足量的洗涤缓冲液对EV-磁珠复合物洗涤三次。
在流式细胞仪中，将2μL5μg的EV-磁珠复合物与100μL如下抗体浓度均为50nM的结合缓冲液混合进行培养。APC-anti-human CD9(Thermo Fisher Scientific,Cat No.:A15698)或PE-conjugated anti-human CD9抗体(BioLegend,Cat No.:312106)被用来检测捕获EVanti-CD63抗体(BioLegend,CatNo.:353017)和anti-CD81抗体(BioLegend,CatNo.:349514)，APC-anti-human CD63(Thermo Fisher Scientific,Cat No.:A15712)或荧光标记的anti-human CD63抗体(BioLegend,Cat No.:353006)被用来检测捕获EV的anti-CD9抗体(BioLegend,Cat No.:312112)和anti-CD81抗体(BioLegend,Cat No.:349514)的混合物。相同浓度(50nM)的如下抗体：APC-anti-human CD81(Thermo Fisher Scientific,Cat No.:17-0819-42)或荧光标记的anti-human CD81抗体(BioLegend,Cat No.:349504)被用来检测捕获EV的anti-CD9抗体(BioLegend,Cat No.:312112)和anti-CD63抗体(BioLegend,Cat No.:353017)的混合物。Alexa -anti-human EpCAM抗体(50nM,R&D Systems,CatNo.:FAB9601R100UG)被用来检测捕获EV的anti-CD9抗体(BioLegend,CatNo.:312112)，anti-CD63抗体(BioLegend,CatNo.:353017)和anti-CD81抗体(BioLegend,CatNo.:349514)。PE-anti-human HER2抗体(50nM,BioLegend,Cat No.:324405)被用来检测捕获EV的anti-CD9抗体(BioLegend,Cat No.:312112)，anti-CD63抗体(BioLegend,Cat No.:353017)和anti-CD81抗体(BioLegend,Cat No.:349514)。将含有抗体和固定有EV的磁珠的试管置于HulaMixer样品混合器(Thermo Fisher Scientific,Cat No:15920D)中室温下放置30分钟。在经过流式细胞仪分析(每次试验分析10000个样品)之前，在磁铁支架上用足量的洗涤缓冲液洗涤EV包被磁珠3次。用BDFACS-CantoTM II流式细胞仪记录对应的中位荧光强度(MFI)和荧光柱状图，然后用FlowJoTM(v10.6.2)进行分析。
图3D总结了5种细胞来源的EV表面生物标志物的概况。图3E展示了流式细胞仪检测分析的EV表面标记蛋白表达的直方图。如图所示，除了HepG2源EV中无CD81的表达之外，所有来源的EV都有标记蛋白(CD9，CD63和CD81)的表达。就癌细胞标志物而言，来源于HT29、 HepG2和SKBR3EV的EV存在EpCAM的表达，但是来源于HEK293的EV则未出现EpCAM的表达。这些通过流式细胞仪检测分析的结果(图3D，图3E)和免疫印迹分析的结果完全一致(图3C)。同样的，在来源于HT29，SKBR3和HepG2的EV中存在着HER2蛋白的表达，但是在敲掉HER2基因的MDA-MB-231源EV中未显现。本申请基于这些特征良好的EV的形态、生物化学和细胞生物学特性，开创性地开展基于核酸适体的荧光极化分析方法的研究。
本发明的目的是开发一种能够选择性地灵敏检测癌症来源的EV的试验方法。因此，我们在一个试验中同时使用两种不同抗体，通过抗体与EV标志物或癌细胞标志物的作用来捕获EV。例如，生物素化anti-human EpCAM抗体用于捕获HT29源EV，生物素化anti-human CD9/CD81抗体用于涂有黑色链霉亲和素的96孔板中固定SKBR3源的EV。首先，本发明证明了生物素化抗体和涂有链霉亲和素的微孔板的确可以确保EV的分离。
在一些优选的实施方式中，所述S1步骤在微孔板中进行，具体采用涂有链霉亲和素的微孔捕获抗体，然后对EV进行固定。
荧光标记的anti-human CD63抗体用于检测捕获的癌症EV(图4A)，图4B显示了经过/未经过Triton X-100处理过的HT29源EV、SKBR3源EV的anti-human CD63抗体的荧光强度(FI)。在室温下用1％Triton X-100处理固定的EV30分钟，以裂解所有囊泡。在未使用Triton X-100预处理的样品中，anti-EpCAM抗体捕获的EV的CD63信号显著高于(p<0.0001)用Triton X-100处理过的样品，且显著高于同型匹配阴性对照anti-IgG抗体(BioLegend，Cat No.：400104)包被孔板，该样品中无EV被捕获。这些数据证明了捕获癌细胞释放的EV的可行性和特异性。
在一些优选的实施方式中，所述S1步骤中，当EV来源于HT29时，所述抗体为生物素化anti-human EpCAM抗体，所述抗体的浓度为2.0-15.0μg/mL；进一步优选为8.0μg/mL。
在一些优选的实施方式中，所述S1步骤中，当EV来源于SKBR3时，所述抗体为生物素化anti-human CD9/CD81抗体(质量比1：1)，抗体的浓度为2.0-15.0μg/mL；进一步优选为8.0μg/mL。
如图4C所示，在这两种情况下，CD63信号在抗体使用量2.0μg/mL～8.0μg/mL范围内呈递增趋势，当抗体浓度超过8.0μg/mL时，CD63信号无明显变化。因此，使用生物素化anti-EpCAM抗体或生物素化CD9/CD81抗体捕获EV的最优浓度为8.0μg/mL。
进一步优选，所述捕获抗体的时间为0.1-1.5小时；所述固定的时间为4-20小时，固定温度为4℃。
如图4D所示确定涂有链霉亲和素的微孔捕获抗体所需的最佳时间。当捕获抗体在链霉亲和素包被的微孔中培养时间为0.5小时，1小时和1.5小时的时候，检测到2种来源EV的抗体的信号均无明显差异，表明培养时间为0.5小时即可。综上，固定后的捕获抗体培养EV的最优时间为16小时(图4E)。
FP是一种能够真实量化配体和靶标相互作用的同源基因技术；需要注意的是，FP表征结果依赖于交互过程中的结合亲和力。为了比较抗体和核酸适体与EV作用时的FP的响应差异，本案首先确定了这些配体与本发明中使用的EV的结合亲和力。所有核酸适体最初都是通过线性寡核苷酸合成，使用前将它们折叠成合适的3D结构，且所采用的折叠方案经过严谨调控得以确定。
在一些优选的实施方式中，所述核酸适体包括CD63-BP，HER2-HApt，HER2-2A中的 至少一种。
在一些优选的实施方式中，所述核酸适体在使用前经过折叠处理，具体步骤包括：用添加有0.5-2.0mM MgCl2的磷酸缓冲液，将核酸适体稀释至目标浓度，然后在90-98℃条件下变性处理2-10min，冰上或室温培养5-20分钟，然后在35-38℃再折叠10-30分钟。
进一步优选的，对于CD63-BP适体和阴性对照DNA适体，用添加有1.0mMMgCl2的磷酸缓冲液，将核酸适体稀释至目标浓度，然后在95℃变性处理5min，冰上培养10分钟，然后在37℃再折叠15分钟。对于HER2-HApt适体，用添加有1.0mMMgCl2的磷酸缓冲液，将核酸适体稀释至目标浓度，然后在95℃变性处理5min，冰上培养15分钟，然后在37℃再折叠15分钟。对于HER2-2A适体，核酸适体由磷酸缓冲液稀释，并加入2.5mMMgCl2，然后在95℃变性处理5min，室温培养10分钟，然后在37℃再折叠至少15分钟。
有趣的是，实验结果显示抗体的结合亲和力显着高于相应的核酸适体(图5)。具体而言，anti-CD63抗体和CD63-BP适体(5'-荧光标记的CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TA-idT-3’，Angew Chem Int Edit,2017,56,11916-11920.)对HT29源EV的表观解离常数(KD)分别是13.84nM和41.74nM。anti-HER2抗体和HER2-HApt适体(5'-荧光标记的GCA GCG GTG TGG GGG CAG CGG TGT GGG GGC AGC GGT GTG GGG-idT-3’，Proc.Natl.Acad.Sci.U.S.A.,2013,110,8170-8175.)对SKBR3源EV的KD分别是2.92nM和77.28nM。理论上，FP试验中探测物质的浓度不应超过该探测物质与其靶标的结合亲和力的两倍，以避免探测物质的化学计量滴定。因此，本发明比较了3种不同浓度的探测物质下的FP响应，包括浓度等于KD、两倍KD和5nM。如图5C，图5F所示，当使用anti-CD63抗体和anti-HER2抗体作为探测物质对固定的EV进行检测时，三个浓度下均呈现出无EV(仅游离配体)和有EV(部分结合配体)样品的ΔFP值趋于负向变化，说明这两种抗体均不能被FP检测到。与此形成鲜明对比的是，荧光标记的CD63-BP适体和荧光标记的HER2-HApt适体在三个浓度下呈现的ΔFP均为正值(图5C，5F)。结果显示使用荧光标记的CD63-BP适体的FP(ΔFP)随适体浓度从5nM(ΔFP为10.8±0.3mP)到2KD(1.8±0.2mP)降低。同样，HER2-HApt适体的ΔFP从5nM时的8.7±0.4mP降低到2KD时的0.3±0.3mP。ΔFP随适体配体浓度的增加而降低很可能是由于未结合配体的比例增加，这有助于观察到的荧光去极化。因此，本申请证明在三种不同浓度的CD63和HER2适配体的检测中，固定的HT29和SKBR3源EV都可以由FP清晰检测，但与EV表面相同靶标蛋白结合的抗体却不能被检测到。
进一步优选，所述核酸适体的目标浓度为1-8nM；更进一步优选为5nM。
在进行正式的FluPADE检测之前，为了确定所用的核酸适体的最佳浓度，本申请探究了各种浓度的荧光标记的CD63-BP适体/HER2-HApt适体/HER2-2A适体(5’-荧光标记的TTT CCT CCA TTG G-inverted thymidine-3’,#202111267773.2)的平行和垂直荧光强度的信噪比(S/N)。如图6所示，平行和垂直荧光强度的信噪比随着3种核酸适体浓度的增加均出现上升(图6A)，CD63-BP适体的平行/垂直荧光强度比从1nM时的6.9±0.7或7.6±3.7增加到50nM时的341.2±2.1或419.0±5.9，HER2-HApt适体的平行/垂直荧光强度比从1nM时的8.6±0.8或10.6±2.1增加到50nM时的303.5±2.3或402.3±6.4。HER2-2A适体的平行/垂直荧光强度比的范围为1nM时的8.8±0.6或11.4±1.9至50nM时的313.3±5.7或467.4±6.4。本领域技术人员知晓，FluPADE检测中配体的最佳浓度应尽可能选择最低浓度，在该浓度下背景的平行和垂直荧光强度对最终FP的贡献极小，这样FP信号主要来自于 核酸适体，而不是来自于背景噪声。在本发明中，3种核酸适体在浓度为5nM时，平行和垂直荧光强度的信噪比均高于30(图6B)。基于这些比值，最终的FP信号中检测适体的平行和垂直荧光强度占主导地位。进一步地，5nM的浓度远低于核酸适体的KD，确保了FP信号检测的可靠性。此外，当适体浓度达到5nM及以上时，FP信号保持不变(图6B)。因此，3种荧光标记的核酸适体的最佳浓度优选为5nM。
在一些优选的实施方式中，所述S2步骤具体为，将60-140μL含有荧光标记的核酸适体的缓冲液添加至S1步骤所得产物中，将微孔板置于摇床上室温避光培养0.5-2小时。
在一些优选的实施方式中，所述S3步骤中荧光极化的信号通过多功能盘式分析仪进行读取；所述多功能盘式分析仪配置有475-490nm的激发滤光片和520-565nm的发射滤光片。
本发明对3种核酸适体与固定的EV的培养时间进行了探究，并对FluPADE检测方案进行了优化。如图7A所示，CD63-BP适体的ΔFP从0.5小时的7.5±0.4mP显著增加至1.0小时的10.8±0.3mP(p<0.05)，在1.5小时后ΔFP不再增加。同样，HER2-HApt适体的最佳培养时间为1.0小时，ΔFP峰值为8.7±0.4mP(图7B)。另一方面，HER2-2A适体在1.5小时时达到ΔFP峰值5.8±0.5mP(图7C)。
4℃条件下，在96孔板中使用8μg/mL的捕获抗体将EV固定16小时以上，经过洗涤，在结合缓冲液中加入100μL5.0nM的荧光标记适体之一(CD63-BP适体：PBS加1.0mM的MgCl2；HER2-HApt适体：PBS加5.0mM的MgCl2；HER2-2A适体：PBS加2.5mM的MgCl2)。将微孔板置于摇床(Thermoline Scientific，型号：TL400)中室温避光培养1.5小时。最后，FP信号由多功能盘式分析仪CLARIOstar Plus(BMG Labtech)读出采用485nm的激发滤光片和535nm的发射滤光片进行FP值的测量。所有FP值均以毫极化(mP)单位表示，每个样品的FP值由3个不同孔的平均值计算得到，每个孔的FP值为该孔3次测量值的平均值。对照孔(仅含游离适体)采用同样的方法进行制备，不同之处在于孔中仅加入PBS。
为了证明本发明检测的是EV，并非可溶性膜碎片或游离蛋白质，试验中设置了一个对照组，其中固定的EV用1％Triton X-100(聚乙二醇辛基苯基醚)室温处理15分钟，以确保无完整的EV残留。对于游离EV和蛋白质的单因素对照样品，在孔中加入100μL的50nM的EpCAM蛋白，对不表达EpCAM或HER2的细胞的EV进行固定，作为对照组进行进一步的特异性控制。室温处理15分钟，以确保无完整的EV残留。对于游离EV和蛋白质的单因素对照样品，在孔中加入100μL的50nM的EpCAM蛋白，对不表达EpCAM或HER2的细胞的EV进行固定，作为对照组进行进一步的特异性控制。
在一些优选的实施方式中，所述缓冲液包括合成型缓冲液或人血浆。
进一步优选的，所述合成型缓冲液为PBS缓冲液。
进一步优选的，所述人血浆来源于血型为A型，B型，AB型，O型，Rh+，Rh-的任一种的献血者；
进一步优选的，所述人血浆来源于年龄为0-120岁的献血者；
进一步优选的，所述人血浆来源于健康人或非健康人；进一步优选的，所述人血浆来源于非健康人，所述非健康人优选为肿瘤患者。
在液体活检中，要检测的EV悬浮在人血浆而非PBS中；基于此，为了模拟液体活检条件，细胞系来源的EV被添加至人血浆中。为此，在分离EV之前，将细胞系衍生的EV以1:10 (v/v)的比例加至人血浆中，用8μg/mL所需抗体捕获人血浆中细胞系来源的EV。对照孔的处理方法与之类似，不同之处在于，将细胞系来源的EV替换为相同体积的PBS。
如图8A所示，除使用anti-IgG抗体的同型匹配阴性对照外，用适配EpCAM的抗体固定HT29源EV。本试验中使用荧光标记的CD63-BP适体检测捕获的EV的存在，发现阴性对照适体的ΔFP(荧光极化变化)明显低于HT29源EV(ΔFP为10.1±0.7mP，p<0.0001)，所述阴性对照适体包括不与CD63结合的乱序核苷酸序列，溶解EV的Triton X-100，以及不表达EpCAM的HEK293源EV。事实上，这些阴性对照的ΔFP水平低于本试验方法的检测限。此外，使用同型匹配对照抗体捕获EV的样品的ΔFP为-0.6±0.3mP，与上述结果完全一致，进一步证明该方法对HT29源EV的选择性。为了排除ΔFP是由CD63适体和被anti-EpCAM抗体捕获的游离EpCAM蛋白结合所导致的可能性，本发明进一步设置一个阴性对照，在涂有anti-EpCAM的孔中只添加游离的EpCAM蛋白，而未添加EpCAM阳性EV。如图8A所示，该阴性对照的ΔFP为-0.5±0.4mP，表明阳性ΔFP并非由荧光标记的CD63-BP适体与固定在微孔中的游离EpCAM蛋白的交互作用所引起。综上所述，图8A清楚地证明本试验对EpCAM阳性的HT29源EV具有选择性和特异性，且荧光、HT29源EV和抗体涂层之间的交互作用对FP信号的影响微乎其微。
进一步地，本发明检测了荧光标记的HER2DNA适体的FP信号对微孔板中anti-CD9和CD81抗体捕获的EV的特异性。以类似的模式，试验中来源于HER2阳性EV的HER2-HApt适体和HER2-2A适体的ΔFP(分别为8.7±0.4mP和5.8±0.5mP)显著高于阴性对照样品的ΔFP，所述阴性对照样品包括乱序核苷酸序列适体，溶解EV的洗涤剂，同型匹配对照抗体涂层，来源于被敲除HER2基因的MDA-MB-231细胞的EV(p<0.0001，图8B，图8C)。事实上，这些阴性对照样品的ΔFP水平也低于本试验方法的检测限(图9)。
在本发明中，使用癌细胞系中的EV，用缓冲液(F-PBS，经0.2微米滤膜过滤后的磷酸缓冲液)或人血浆配制成宽浓度范围的混合液，用来确定试验方法的检测限(LOD)和线性动态范围(LDR)。
FluPADE中每个检测适体检测的F-PBS或人血浆中的EV的LOD和LDR见图9。有趣的是，与在PBS中稀释的EV的检测值和范围线性动态不同(分别比较图9A，C，E和图9B，D，F)，使用人血浆EV进行的FluPADE试验呈现为LOD增加，LDR下降。对于基于CD63-BP适体的FluPADE试验，EV的LOD从PBS中的5.0×106HT29 EVs/mL增加到人血浆稀释的EV的5.0×107HT29 EVs/mL。PBS总EV的LDR为5.0×108-2.0×1010HT29 EVs/mL，比人血浆中EV的LDR(5.0×108-1.0×1010)更宽(图9A和图9B)。同样的，HER2-HApt适体的FluPADE结果显示，SKBR3源EV在PBS中的LOD较低，为1.0×107EVs/mL；而在人血浆中的LOD为3.0×107SKBR EVs/mL。同时试验结果显示PBS中EV的LDR下移，具体为2.0×109-2.0×1010SKBR3 EVs/mL；而人血浆中EV的LDR更宽，具体为2.0×108-1.0×1010SKBR3 EVs/mL(图9D和图9F)。本发明FluPADE检测作用于PBS和人血浆中的SKBR3源EV中的HER2，使用HER2-2A适体测定的LOD相比使用HER2-HApt适体的LOD更低，但前者的LDR相比于HER2-HApt适体的LDR更窄(图9C，E与图9D，F)。
试验中使用相同的检测适体对PBS或人血浆中的同种EV进行分析，发现LODs和LDRs的差异是由PBS中细胞系来源的EV以及人血浆中的EV中的可用生物标记蛋白的总量之间的差异决定。微孔中捕获抗体固定的EV量和PBS或血浆中阳性生物标志物EV是导致荧光极化信号的最终输出差异的原因。因此，本发明探究了人血浆中EV标记蛋白量的贡献。
简而言之，采用如上所述的用抗体分离人细胞系来源的EV的方法对来源于人血浆 的EV进行分离，然后用100μL的50nM抗体对来自人血浆的固定的EV进行培养，所述抗体包括荧光标记的anti-EpCAM抗体(与固定捕获EV时使用的anti-EpCAM抗体相同)，荧光标记的anti-CD63抗体(固定EV时使用的anti-CD63抗体)，PE-conjugated anti-CD9抗体(固定捕获EV时使用的anti-CD9抗体)，荧光标记的anti-CD81抗体(固定捕获EV时使用的anti-CD81抗体)，荧光标记的anti-HER2抗体(固定捕获EV时使用的anti-CD9/CD81抗体)。室温培养30分钟后，用200μL的0.1％F-TPBS洗涤3次，使用CLARIOstar Plus(BMG Labtech)多功能盘式分析仪记录520nm至660nm波长范围内的荧光强度光谱。
如图10所示，使用anti-EpCAM抗体从人血浆中分离出的EV明显存在着癌症标记蛋白的表达，而且从人血浆中分离的EV中也明显检测出EV标记蛋白CD63。从人血浆中分离的EV中EpCAM和/或CD63蛋白的水平可能会在如下几个方面，对基于CD63-BP适体的fluPADE检测方法检测癌细胞系分离的EV的性能产生影响。首先，人血浆EV中存在的EpCAM可能引起HT29源EV的减少，这一情况出现在人血浆中捕获anti-EpCAM抗体与血浆EV中的EpCAM蛋白质以及HT29源EV中同种蛋白质之间的竞争性结合过程中，这从F-PBS中HT29源EV具有更强的EpCAM信号也能得到佐证。研究中输出的EV信号差异表明，微孔上固定的anti-EpCAM抗体的数量有限。微孔中的结合位点数量与人血浆EV以及HT29源EV的竞争性结合，使得anti-EpCAM抗体的可用结合位点出现饱和，导致相比于PBS中分离出的大量HT29源EV，从人血浆中分离出的HT29数的量明显少得多。其次，虽然人血浆中EV的CD63表达水平比在HT29源EV中低得多(荧光强度6206.5对应人血浆EV为9.2×108，荧光强度84345.1对应HT29源EV为2.0×108)，人血浆EV的CD63-BP适体和CD63蛋白之间的相互作用有可能促进总FP信号被检测出来。这些效应的协同将导致涉及到与人血浆结合的HT29源EV的表观LOD从5×106上升到5×107EVs/mL，LDR的范围从5.0×108-2.0×1010降低到5.0×108-1.0×1010EVs/mL。同样的，试验发现CD9、CD81和HER2存在于通过anti-CD9/CD81抗体分离的人血浆EV中(图10)。人血浆EV中这一类EV生物标记蛋白的存在可以为观察到的悬浮在PBS或人血浆中的SKBR3源EV之间的FP性能变化提供解释。
荧光强度是荧光检测中最常用的检测方式，荧光极化很少被用作检测模式，尤其是在医疗诊断领域。如图11所示，本发明采用相同的EV固定方法和荧光标记适体，来对比基于荧光强度和荧光极化模式的EV表现，具体通过LOD和LDR进行呈现。
对于基于荧光强度的PBS中EV的检测，通过anti-EpCAM抗体或anti-CD9/CD81抗体的混合物将不同浓度的HT-29源EV和SKBR3源EV在孔中4℃固定16小时，分别用CD63或HER2进行检测。洗涤后，加入100μL含如下适体之一的缓冲液：800nM荧光标记CD63-BP适体、800nM荧光标记的HER2-HApt适体或800nM荧光标记的CD63-BP适体。然后加入100μL含50nM的荧光标记的CD63抗体或50nM荧光标记的HER2抗体的PBS。将固定的EV与探测物质在摇床(Thermoline Scientific,Model No.:TL400)上室温避光培养1小时。用200μL的洗涤缓冲液洗涤3次后，用多功能盘式分析仪CLARIOstar Plus(BMG Labtech)在485nm激发滤光片和535nm发射滤光片下测量荧光强度。用如上所述的方法制备背景荧光的对照孔，并使用生物素标记的IgG同型匹配的对照抗体替代生物素标记的EV标记物特异性抗体。
对于基于荧光强度的人血浆中来自细胞系的EV的检测，在固定EV之前将所需数量的EV以1:10(v/v)的比例添加到人血浆中。然后对人血浆中EV进行检测，检测方法与PBS中EV的检测方法相同。
图11显示了荧光强度与EV浓度、LOD和LDR之间的关系，具体为使用CD63-BP适体、HER2-HApt适体或HER2-2A适体对来自F-PBS(图11A，C，E)或人血浆(图11B，D，F)中的HT29源EV或SKBR3源EV进行荧光强度的测定。使用荧光标记的CD63-BP适体对F-PBS中癌细胞分泌的EV进行荧光强度检测的LOD为2.0×108HT29 EVs/mL(图11A)，使用荧光标记的HER2-HApt适体对SKBR3源EV进行荧光强度检测的LOD为5.0×108SKBR3 EVs/mL(图11C)，使用荧光标记的HER2-2A适体的LOD为2.0×108SKBR3 EVs/mL(图11E)。
本发明还测定了人血浆中EV的荧光强度的LODs。人血浆中CD63-BP适体测定荧光强度的LOD增加了约2.5倍，具体为5.0×108HT29 EVs/mL，HER2-Hapt适体的LOD增加了2倍，具体为1.0×109，HER2-2A适体的LOD增加了4倍，具体为8.0×108SKBR3 EVs/mL(图11D，图11F)。PBS中的EV与人血浆中的EV之间LOD的显著相关性差异可能与人血浆EV中的生物标记物有关。有趣的是，与使用相同检测适体的荧光强度检测相比，FluPADE检测的LOD要低得多。具体的，使用CD63-BP适体的FluPADE检测的LOD在PBS和人血浆中分别比荧光强度检测的LOD低4倍(分别为5.0×106EVs/mL和2.0×108HT29 EVs/mL)和10倍(分别为5.0×107EVs/mL和5.0×108HT29 EVs/mL)。类似的，在PBS和人血浆中使用HER2-HApt适体的FP检测比荧光强度检测灵敏约17-26倍。具体的，PBS中HER2-HApt适体的LOD为3.0×107SKBR3 EVs/mL，HER2-2A适体的LOD为1.0×107SKBR3EVs/mL，人血浆中HER2-HApt适体的LOD为5.0×107SKBR3 EVs/mL，HER2-2A适体的LOD为3.0×107SKBR3 EVs/mL。
除了基于HER2-HApt适体进行荧光强度检测时其LDR的下限值升高、上限值降低(图11)，人血浆中EV荧光强度的LDR上下限值均有上升趋势。具体来看，在PBS和人血浆中，荧光标记CD63-BP适体的荧光强度LDR为3.0×108-2.0×109EVs/mL，HER2-2A适体的荧光强度LDR为1.0×109-1.0×1010EVs/mL从这些结果中可以明显看出，人血浆中三种适体的荧光强度LDR的下限值均高于PBS中的LDR下限值。LDR的这种上移趋势可能是由于在EV固定和检测过程中，人血浆中EV上的生物标记物的附加竞争性结合所致。相比之下，基于适体的FluPADE试验，尤其是对HER2阳性EV进行检测时，能够在较低浓度下检测PBS和人血浆中的癌症EV，并在更宽的EV浓度范围下对EV进行定量，而基于适体的荧光强度进行检测则无法达到这样的效果。
在使用适体探究荧光强度测试方法性能的基础上，虽然使用抗体不能有效实现EV的FP检测，本申请还使用抗体探究了荧光强度的检测性能。图12显示了基于抗体测试的荧光强度与EV浓度、LOD和LDR之间的关系。对于PBS中的EV检测，anti-CD63抗体检测FI的LOD为1.0×107HT29 EVs/mL，LDR为5.0×107-1.0×109HT29 EVs/mL(图12A)；使用anti-HER2抗体检测FI的LOD为1.0×109SKBR3 EVs/mL，LDR为2.0×109-2.0×1010SKBR3 EVs/mL(图12C)。对于人血浆中的EV检测，anti-CD63抗体检测FI的LOD为1.0×107HT29 EVs/mL，LDR为5.0×107-1.0×109HT29 EVs/mL(图12A)；使用anti-HER2抗体检测FI的LOD为5.0×107HT29 EVs/mL，LDR为1.0×108-2.0×109HT29 EVs/mL(图12B)；使用anti-HER2抗体检测FI的LOD为3.0×109SKBR3 EVs/mL，LDR为5.0×109-1.0×1010SKBR3 EVs/mL(图12D)。
这些结果有力地证明，本发明提供的FluPADE在检测缓冲液和人血浆中癌症来源的EV方面优于传统的基于荧光强度的检测方法，具有更高的灵敏度和更宽的动态范围(表1)。
在一些优选的实施方式中，当核酸适体为CD63-BP时，所述检测方法的LOD≤5×107EVs/mL，LDR为5×108-2×1010EVs/mL；当核酸适体为HER2-HApt时，所述检测方法的LOD≤5×107EVs/mL，LDR为8×107-2×1010EVs/mL；当核酸适体为HER2-2A时，所述检测方法的LOD≤3×107EVs/mL，LDR为2×108-2×1010EVs/mL。
基于EV的癌症诊断的关键挑战之一是样本中癌症来源的EV的丰度较低，因为癌症来源的EV被释放到EV储库中，所述EV储库含有由生物体液中约200种类型的健康人类细胞释放的EV。这一情况使得癌细胞衍生的EV的确定相当于大海捞针。为了确定基于CD63-BP适体的FluPADE检测的灵敏度，本发明以1：2000，1：1000，1：500，1：100和1：10这五种比例，将HT29细胞源的EpCAM阳性EV连续滴定到HEK293细胞源的EpCAM阴性EV中，将EV的总浓度控制在1.0005×1010EVs/mL。
对于使用HER2-Hapt适体或HER2-2A适体的FluPADE检测，以1：1000，1：750，1：500，1：100和1：10的比例，将HER2阳性SKBR3细胞的EV连续滴定到敲除HER2基因的MDA-MB-231细胞的EV中，使得EV总浓度分别固定在3.0030×1010EVs/mL(HER2-HApt适体)和1.0010×1010EVs/mL(HER2-2A适体)
对于PBS中基于FP的EV检测，将8μg/mL用于HT29源EV的anti-EpCAM抗体或用于SKBR3源EV的anti-CD9/CD81抗体加入到链霉亲和素包被的微孔中，在室温下培养30分钟并洗涤，然后将EV在4℃条件下固定16小时以上，在每个微孔中加入100μL包含有以下适体之一(5nM)的PBS：荧光标记的CD63-BP、荧光标记的HER2-HApt、荧光标记的HER2A，然后在避光室温条件下在摇床(Thermoline Scientific，型号：TL400)上进行培养，其中前2个适体的培养时间为1小时，第3个适体的培养时间为1.5小时。
最后，在多功能盘式分析仪CARIOstar Plus(BMG Labtech)上测量FP信号，其中激发滤光片为485nm，发射滤光片为535nm。游离配体的FP对照孔除未加入EV外，制备方法与上述相同。值得注意的是，EV的总浓度需保持在1010EVs/mL的水平，以模拟体循环中EV的生理浓度。这样本发明的结果具有临床相关性，并且可以应用到病理学实验中。本发明试验的灵敏度定义为检测到的FP信号等于或高于LOD处的FP信号时的最低比值。
如图13所示，当总EV浓度保持不变时，ΔFP随着标记阳性EV与标记阴性EV比例的增加而增加。利用CD63-BP适体检测EV时，发现1：2000比值下的ΔFP低于3×SDblank的ΔFP(分别为0.2±0.1mP和0.6±0.1mP)。然而，该试验的ΔFP从比值为1：1000时的0.7±0.1显著增加到比值为1:1时的8.6±0.9mP(图13)。该结果表明，采用CD63-BP适体的FluPADE检测不仅能够在LOD(5×106EVs/mL)水平上检测，还能够在1000倍EpCAM阴性EV条件下检测EpCAM阳性的HT29源EVs。
如图13所示，基于HER2-HApt适体的FluPADE检测的灵敏度为在背景为750个HER-2阴性EV(图13)(HER2-HApt适体)的体系中能够检测到至少1个HER2阳性EV，而使用HER2-2A适体的测定的灵敏度为1000个HER2阴性EV的背景下能够检测到至少1个HER2阳性EV，基于HER2-HApt适体的检测灵敏度更高。
一种成熟的液体活检检测癌症来源的EV的方法，不仅需要有高灵敏度，而且还应当能够使用相同的生物标志物区分不同来源的EV。肿瘤在原发部位释放的EV和从转移部位释放的EV在遗传和表型上属于不同的实体。即使同一患者的癌细胞释放的EV都可能定性地表达偏好的生物标志物。但可以想象，在不同的原发部位、不同生长阶段或不同的临床过程 的肿瘤释放的EV的表面生物标记蛋白的丰度存在定量差异。从诊断的角度来看，本发明提供的FluPADE非常有利于根据表面生物标志物丰度的数量差异对来自不同癌细胞的不同EV群体进行检测。为了证明FluPADE在检测和/或区分不同来源的EV方面的能力，本发明设立了一个模型系统，其中EV来自于3种常见实体癌的3种细胞系。本发明首先确定了由结直肠癌(HT29)，乳腺癌(SKBR3)和肝细胞癌(HepG2)制备的EV中CD63和HER2丰度的差异。测定这些EV中CD63和HER2丰度的方法与上述方法相似，不同之处在于使用50nM的Alexa -conjugated anti-HER2 antibody(BioLegend,Cat No.:324412)来量化HER阳性EV。
如图14A所示，不同癌症来源的EV在EV生物标志物(CD63)和癌症生物标志物(HER2)的表面显示不同数量的表面标记蛋白。EV表面标记蛋白丰度的差异将导致所检测的荧光标记的适体固定表面EV时所产生的荧光极化程度的差异。
为确保使用从癌细胞系制备的EV获得的FluPADE结果不仅与液体活检的诊断设置相关，而且能够有效克服个体患者血浆中不同成分的可能干扰，本申请人仔细制备分析样品，将从癌症细胞系中制备的相同数量的EV注入6名不同献血者的血浆中。鉴于此，将人结直肠癌细胞(HT29)、乳腺癌细胞(SKRB3)和肝癌细胞(HepG2)制备的EV(1.0×109EV/mL)加入6名献血者的血浆中，血浆中EV浓度为9.0×109EV/mL。癌症细胞系分泌的EV与血浆EV的比率为1:10(v/v)，所有18个样品中总EV浓度相同，均为1.0×1010EVs/mL。用荧光标记的EV标记物(CD63)或荧光标记的癌细胞标志物(HER2)检测来自三种不同类型实体癌的EV，每一种都加入到来自6名献血者的血浆中，如图14B和图14D所示。有趣的是，尽管相同细胞来源的EV之间存在质间差异，但基于CD63适体检测的荧光极化变化可用于区分三种不同癌细胞的anti-EpCAM抗体固定的EV(图14C)。为了测试该试验方法的可靠性和稳定性，本发明设计了一组交互实验，其中使用两种EV标记物(CD9和CD81)的对应抗体固定来自3种不同类型癌症来源的EV，用HER2的对应适体用于癌症生物标志物。如图14E所示，在人血浆中，HER2-HApt适体检测HT29源EV的ΔFP比检测SKBR3源EV的ΔFP高。最重要的是，当这两组交互测试中单个分析物的ΔFP被绘制在1张图(图14F)中时，来自3个不同细胞来源的3种癌症源EV的变化非常明显。
这些结果进一步论证了FluPADE在医学诊断领域的应用前景广阔，因为它不仅可以高灵敏度检测癌症来源的EV，还可以辨别具有相同癌症生物标志物但在EV表面具有不同生物标志物丰度的不同EV群体。
本发明第二方面提供了一种所述的基于核酸适体的细胞外囊泡荧光极化检测方法的应用，所述检测方法应用于癌细胞分泌的细胞外囊泡的定性、定量分析。
在一些优选的实施方式中，所述癌细胞来源于结直肠癌，乳腺癌，肝细胞癌，胃癌，胰腺癌，食道癌，鼻咽癌，咽喉癌，子宫内膜癌，肺癌，头颈癌，肾癌，膀胱癌，甲状腺癌，皮肤癌，卵巢癌，宫颈癌，前列腺癌，阴茎癌中的任一种。
在一些优选的实施方式中，所述检测方法能够区分不同原发部位的癌细胞分泌的EV；所述原发部位包括肠，乳腺，肝，胃，胰腺，食道，表皮，皮肤软组织，卵巢，子宫颈，前列腺，阴茎中的任一种。
在一些优选的实施方式中，所述检测方法能够区分不同生长阶段的癌细胞所分泌的EV；所述生长阶段包括原位癌阶段，区域淋巴结转移阶段，远端转移阶段中的任一种；优 选的，所述检测方法能够区分抗癌治疗过程中在不同生长阶段的癌细胞所分泌的细胞外囊泡。
在一些优选的实施方式中，所述检测方法可直接在临床检验科的自动生化分析仪上施行，无需特殊设备或特制的仪器。
在一些优选的实施方式中，所述检测方法可直接在临床检验科的自动免疫分析仪上施行，无需特殊设备或特制的仪器。
有益效果：
本申请以核酸适体作为探测物质，用荧光极化来检测癌细胞分泌的细胞外囊泡，能够有效区分含有同种生物标记蛋白但来源于不同细胞群体的细胞外囊泡，可用于癌症体外液体活检的定性、定量分析。同时，本发明能够特异性地检测血液中癌细胞分泌的细胞外囊泡，检测过程不受血液中游离肿瘤标志蛋白、肿瘤细胞膜碎片或肿瘤细胞外囊泡膜碎片的干扰，检测结果精准有效。
附图说明
图1.癌细胞衍生的EV的荧光极化检测步骤示意图；
图2.基于核酸适体的细胞外囊泡荧光极化检测方法的工作原理示意图；
图3.HT29、SKBR3、HepG2、HEK293和敲除了HER2基因的MDA-MB-231细胞的EV特性；具体的，A为纳米颗粒跟踪分析技术测得的EV粒径分布；B为扫描电子显微镜观测到的EV形貌；C为通过Alix，CD63，calnexin，EpCAM和HER2进行的免疫印迹试验的测试结果；D为通过流式细胞术分析的EV表面生物标记蛋白或癌症生物标志物(包括CD9、CD63、CD81、EpCAM和HER2)的表达；E为流式细胞仪检测分析的EV表面标记蛋白表达的直方图，其中红色为背景对照(用于固定EV的IgG同型对照包被磁珠)，蓝色为用于EV固定的特异性抗体包被磁珠。
图4.A为用荧光标记的anti-CD63抗体检测固定的EV示意图(BioLegend,Cat No.:353006)；B为在冷室中作用16小时的条件下，用生物素化的anti-EpCAM抗体固定的HT29源EV(BioLegend,Cat No.:324216)以及使用1:1的anti-CD9/CD81抗体所固定的SKRB3源EV(BioLegend,anti-CD9 antibody:Cat No.:312112；anti-CD81 antibody:CatNo.:349514)；其中生物素标记抗体被包覆在涂有链霉亲和素包覆的微孔(Thermo Fisher Scientific,CatNo.:15503)；固定的EV经1％Triton X-100或生理盐水处理(p<0.0001)；C为与5μg/mL抗体固定的EV的荧光强度相比，用于HT29源EV的生物素化anti-EpCAM抗体和用于从SKBR3源EV的生物素化anti-CD9/CD81抗体的浓度优化，与5μg/mL抗体固定的EV的荧光强度相比，p<0.05；D为涂有链霉亲和素的微孔中固定HT29源EV的生物素化anti-EpCAM抗体以及固定SKBR3源EV的生物素化anti-CD9/CD81抗体的培养时间优化；E.HT29源EV/anti-EpCAM抗体或SKBR3源EV/anti-CD9/CD81的固定时间优化，与培养4小时的EV的荧光强度相比p<0.05；数据为平均值±标准差，n＝3。
图5.抗体和核酸适体与固定化EV的结合亲和力以及使用抗体或核酸适体作用于EV的荧光极化性能；A为CD63抗体固定HT29源EV的结合曲线和表观解离常数(KD)；B为CD63-BP适体固定HT29源EV的结合曲线和表观解离常数(KD)；C为在5nM，KD和2KD浓度下使用anti-CD63抗体或CD63-BP适体固定HT29源EV的荧光极化差异；D为anti-HER2抗体固定SKBR3源EV的结合曲线和KD；E为HER2-HApt适体固定SKBR3源EV的结合曲线和KD；F为5nM，KD和2KD浓度 下使用anti-HER2抗体和HER2-HApt适体固定SKBR3源EV的荧光极化差异；数据为平均值±标准差，n＝3。
图6.荧光极化试验中适体最佳浓度的测定；A为不同浓度下CD63-BP适体，HER2-HApt适体和HER2-2A适体的平行和垂直荧光强度的信噪比；B为不同浓度下适体荧光极化与浓度的关系；数据为平均值±标准差，n＝3。
图7.确定适体和固定的EV之间的最佳培养时间以获得最佳荧光极化信号；A为CD63-BP适体作用于HT29源EV；B为HER2-HApt适体作用于SKRB3源EV；C为HER2-2A适体作用于SKRB3源EV；数据为平均值±标准差，n＝3。
图8.不同样品对CD63和HEER2适体的荧光极化差异；A为6种样品中5nM荧光标记的CD63-BP适体作用于anti-EpCAM抗体固定HT29源EV的荧光极化差异；荧光标记适体不与CD63和anti-EpCAM固定的HT29源EV结合；所述anti-EpCAM固定的HT29源EV来源于Triton X-100裂解(anti-EpCAM抗体同型匹配阴性对照)，不表达EpCAM的HEK293源EV或游离EpCAM蛋白(50nM)；B为5种不同样品中5nM荧光标记HER2-HApt-BP适体的荧光极化差异：anti-CD9/CD81抗体会固定SKRB3源EV，不与HER2以及anti-CD9/CD81抗体固定的SKRB3源EV结合的荧光标记适体；经过anti-CD9/CD81抗体固定以及Triton X-100裂解的SKRB3源EV；涂有anti-IgG抗体的微孔中培养得到的SKRB3源细胞；敲除HER2基因的MDA-MB-231源EV(HER2阴性EV)；C为5nM荧光标记的HER2-2A适体在上述5种不同样品中的荧光计划差异，数据为平均值±标准差，n＝3。
图9.荧光极化信号随EV浓度的变化，基于内部荧光信号随log10(lg)EV浓度变化的线性曲线确定FluPADE检测方法的LOD和LDR，所述EV浓度由PBS(图A，C，E)或人血浆(1:10,v/v；图B，D，F)中基于CD63-BP适体(A-B)，HER2-HApt适体(C-D)或HER2-2A(E-F)适体检测确定。所有FP和ΔFP值均以毫极化(mP)单位表示；所有FP值均以毫极化(mP)单位表示，每个样品的FP值由3个不同孔的平均值计算得到，每个孔的FP值为该孔3次测量值的平均值。
图10.人血浆中EV测定中的EV标记蛋白和癌症生物标记物的背景信号测定；A中红色谱图为使用anti-EpCAM抗体检测EpCAM和CD63、使用anti-CD9/CD81抗体检测CD81和HER2分离人血浆中EV的过程中荧光标记的anti-EpCAM抗体、anti-CD63抗体、anti-CD81抗体、anti-HER2抗体在520～660nm激发波长(发射波长为535nm)的荧光强度；蓝色谱图为使用IgG同型对照抗体分离人血浆中EV的荧光强度。在使用anti-CD9/CD81抗体分离人血浆中EV时，还记录了在570nm～690nm激发波长下PE-anti-CD9抗体的荧光强度谱(发射波长为575nm)图(红色)，以及使用IgG同型对照抗体分离人血浆中EV的荧光强度谱图(蓝色)。B为分别测定anti-EpCAM抗体(ex:485nm,em:535nm)、anti-CD63抗体(ex:485nm,em:535nm)、anti-CD9抗体(ex:560nm,em:575nm)、anti-CD81抗体(ex:485nm,em:535nm)、anti-HER2抗体(ex:485nm,em:535nm)分离人血浆中EV的荧光强度。数据为平均值±标准差，n＝3。
图11.基于适体进行荧光强度检测的LOD和LDR；具体为使用CD63-BP适体(A-B)测定HT29源EV以及使用HER2-HApt适体(C-D)或HER2-2A适体(E-F)测定SKRB3源EV时EV浓度与内部荧光强度的关系图以及LOD和LDR结果；其中细胞系来源的EV分别用PBS稀释(图A，C，E)和人血浆进行稀释(图B，D，F)。
图12.基于抗体进行荧光强度检测的LOD和LDR；具体为使用anti-CD63抗体(A-B) 测定HT29源EV以及使用anti-HER2抗体(C-D)测定SKRB3源EV时EV浓度与内部荧光强度的关系图以及LOD和LDR结果；其中细胞系来源的EV分别用PBS稀释(图A，C)和人血浆进行稀释(图B，D)。
图13.基于适体的荧光极化方法检测癌源性EV的灵敏度；具体为线性浓度的癌源性EV的作用下适体荧光极化信号的变化。A为5.0nM的CD63-BP适体检测的EpCAM阳性HT29源EV与EpCAM阴性HEK293源EV的比值；B和C分别为使用5.0nM的HER2-HApt适体或HER2-2A适体检测的HER2阳性SKBR3源EV与HER2阴性MDA-MB-231源EV的比值。数据为平均值±标准差，n＝3。
图14.FluPADE能够基于生物标志物的荧光极化双重分析来区分不同来源的EV。A中第一行图为使用anti-EpCAM抗体包被的磁珠分离的HT29源EV、SKBR3源EV和HepG2源EV中CD63的表达水平；第二行图为使用anti-CD9/CD81抗体包被的磁珠分离的HT29源EV、SKBR3源EV和HepG2源EV中HER2的表达水平。红色为IgG同型匹配对照抗体包被磁珠培养的EV的背景荧光；蓝色为anti-EpCAM抗体包被的磁珠分离的EV的荧光强度(第一行)和anti-CD9/CD81抗体包被的磁珠分离的EV的荧光强度(第二行)。B为FluPADE检测中使用CD63-BP适体探测来自3种不同癌细胞系的anti-EpCAM抗体分离的人血浆中EV的示意图，所述人血浆来源于6位献血者。C为CD63-BP适体作用于3种不同癌细胞系的anti-EpCAM抗体分离的人血浆中EV的荧光极化信号差异；所述人血浆来源于6位献血者。D为FluPADE检测中使用HER2-HApt适体探测来自3种不同癌细胞系的anti-CD9/CD81抗体分离的人血浆中EV的示意图，所述人血浆来源于6位献血者。E为HER2-HApt适体作用于3种不同癌细胞系的anti-CD9/CD81抗体分离的人血浆中EV的荧光极化信号差异；所述人血浆来源于6位献血者。F为CD63适体和HER2-HApt适体检测HT29源EV、SKBR3源EV和HepG2源EV的ΔFP的聚类图(CD63适体：图14C；HER2-HApt适体：图14E)；数据为平均值±标准差，n＝3。
具体实施方式
实施例1.
在PBS中基于CD63-BP适体的细胞外囊泡荧光极化检测方法。
S1.分别使用8.0μg/mL的anti-EpCAM抗体和anti-CD9/CD81抗体(质量比1：1)，将HT29源EV和SKBR3源EV固定在微孔中。具体的，将生物素标记抗体置于涂有链霉亲和素的微孔中室温培养30分钟并洗涤，然后在4℃下培养EV16小时以上。
S2.向每个微孔中加入100μL5.0nM荧光标记的CD63-BP适体的PBS(经0.2微米滤膜过滤后的磷酸缓冲液含1.5mM MgCl2)，然后在避光室温条件下在摇床(Thermoline Scientific，型号：TL400)上进行培养，培养时间为1小时。
S3.使用配置有485nm的激发滤光片和535nm的发射滤光片的多功能盘式分析仪CLARIOstar Plus(BMG Labtech)读取FP信号。
用与上述相同的方法制备游离配体的FP对照样品，具体将相同体积、无EV的PBS添加至微孔中。
实施例2.
在PBS中基于HER2-HApt适体的细胞外囊泡荧光极化检测方法。
S1.分别使用8μg/mL的anti-EpCAM抗体和anti-CD9/CD81抗体(质量比1：1)，将 HT29源EV和SKBR3源EV固定在微孔中。具体的，将生物素标记抗体置于涂有链霉亲和素的微孔中室温培养30分钟并洗涤，然后在4℃下培养EV 16小时以上。
S2.向每个微孔中加入100μL5nM荧光标记的HER2-HApt适体的PBS(经0.2微米滤膜过滤后的磷酸缓冲液)，然后在避光室温条件下在摇床(Thermoline Scientific，型号：TL400)上进行培养，培养时间为1小时。
S3.使用配置有485nm的激发滤光片和535nm的发射滤光片的多功能盘式分析仪CLARIOstar Plus(BMG Labtech)读取FP信号。
用与上述相同的方法制备游离配体的FP对照样品，具体将相同体积、无EV的PBS添加至微孔中。
实施例3.
在PBS中基于HER2A适体的细胞外囊泡荧光极化检测方法。
S1.分别使用8μg/mL的anti-EpCAM抗体和anti-CD9/CD81抗体(质量比1：1)，将HT29源EV和SKBR3源EV固定在微孔中。具体的，将生物素标记抗体置于涂有链霉亲和素的微孔中室温培养30分钟并洗涤，然后在4℃下培养EV16小时以上。
S2.向每个微孔中加入100μL5nM荧光标记的HER2A适体的PBS(经0.2微米滤膜过滤后的磷酸缓冲液)，然后在避光室温条件下在摇床(Thermoline Scientific，型号：TL400)上进行培养，培养时间为1.5小时。
S3.使用配置有485nm的激发滤光片和535nm的发射滤光片的多功能盘式分析仪CLARIOstar Plus(BMG Labtech)读取FP信号。
用与上述相同的方法制备游离配体的FP对照样品，具体将相同体积、无EV的PBS添加至微孔中。
实施例4.
在人血浆中基于CD63-BP适体的细胞外囊泡荧光极化检测方法，具体实施方式同实施例1；不同点在于，S1步骤中，在固定EV之前，将目标量的EV加入人血浆中(比例为1:10，v/v)中，然后分别使用8.0μg/mL的anti-EpCAM抗体和anti-CD9/CD81抗体(质量比1：1)，将人血浆中细胞系来源的EV固定在微孔中。
用与上述相同的方法制备游离配体的FP对照样品，具体将100μL F-PBS添加到900μL人血浆中作为人血浆测定的FP对照样品。
实施例5.
在人血浆中基于HER2-HApt适体的细胞外囊泡荧光极化检测方法，具体实施方式同实施例4；不同点在于，将S2步骤中的CD63-BP适体替换为HER2-HApt适体。
实施例6.
在人血浆中基于HER2A适体的细胞外囊泡荧光极化检测方法，具体实施方式同实施例4；不同点在于，将将S2步骤中的CD63-BP适体替换为HER2A适体；S2中培养时间为1.5小时。
实施例7.
在PBS中基于CD63-BP适体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例1；不同点在于：
所述S2步骤为，加入100μL含800nM荧光标记CD63-BP适体的PBS缓冲液，然后加入 100μL含50nM的荧光标记的CD63抗体或50nM荧光标记的HER2抗体的PBS，然后在避光室温条件下在摇床(Thermoline Scientific，型号：TL400)上进行培养，培养时间为1小时。
所述S3步骤为，用200μL的洗涤缓冲液洗涤3次后，用多功能盘式分析仪CLARIOstar Plus(BMG Labtech)在485nm激发滤光片和535nm发射滤光片下测量荧光强度。
实施例8.
在PBS中基于HER2-HApt适体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例7；不同点在于，将S2步骤中的CD63-BP适体替换为HER2-HApt适体。
实施例9.
在PBS中基于HER2-HApt适体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例7；不同点在于，将S2步骤中的CD63-BP适体替换为HER2-2A适体。
实施例10.
在PBS中基于anti-CD63抗体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例7；不同点在于，将S2步骤中的CD63-BP适体替换为anti-CD63抗体。
实施例11.
在PBS中基于anti-HER2抗体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例7；不同点在于，将S2步骤中的CD63-BP适体替换为anti-HER2抗体。
实施例12.
在人血浆中基于CD63-BP适体的细胞外囊泡荧光强度检测方法，具体实施方式同
实施例7；不同点在于，S1步骤中，在固定EV之前，将目标量的EV加入人血浆中(比例为1:10，v/v)中，然后分别使用8μg/mL的anti-EpCAM抗体和anti-CD9/CD81抗体(质量比1：1)，将人血浆中细胞系来源的EV固定在微孔中。
用与上述相同的方法制备游离配体的FP对照样品，具体将100μLF-PBS添加到900μL人血浆中作为人血浆测定的对照样品。
实施例13.
在人血浆中基于HER2-HApt适体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例12；不同点在于，将S2步骤中的CD63-BP适体替换为HER2-HApt适体。
实施例14.
在人血浆中基于HER2-2A适体的细胞外囊泡荧光强度检测方法，具体实施方式同
实施例12；不同点在于，将S2步骤中的CD63-BP适体替换为HER2-2A适体。
实施例15.
在人血浆中基于anti-CD63抗体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例12；不同点在于，将S2步骤中的CD63-BP适体替换为anti-CD63抗体。
实施例16.
在人血浆中基于anti-CD63抗体的细胞外囊泡荧光强度检测方法，具体实施方式同实施例12；不同点在于，将S2步骤中的CD63-BP适体替换为anti-HER2抗体。
上述实施例的LOD以试验信号确定的EV浓度来衡量，所述EV浓度等于对照样品的信号加上3倍的对照样品结果的标准偏差。线性动态范围用EV浓度信号的线性回归来定义；测定结果见表1。
表1.实施例1～16检测方法的LOD和LDR结果汇总

Claims (16)

  1. 一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，包括以下步骤：
    S1.通过抗体与EV表面生物标记蛋白或EV表面癌症标志物作用，对EV进行固定；然后快速清洗，去除游离EV，蛋白质，膜碎片和脂质；
    S2.分别添加与EV标志物或癌细胞标志物适配的核酸适体进行培养，所述核酸适体经过荧光标记；
    S3.对S2步骤的产物进行荧光极化检测，实现对癌细胞分泌的EV的定性、定量分析，操作过程中无需清洗；
    所述EV标志物包括CD9，CD63，CD81中的至少一种；
    所述癌细胞标志物包括EpCAM和/或HER2；
    所述核酸适体包括CD63-BP，HER2-HApt，HER2-2A中的至少一种。
  2. 根据权利要求1所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述S1步骤中，当EV来源于HT29时，所述抗体为生物素化anti-human EpCAM抗体，所述抗体的浓度为2.0-15.0μg/mL。
  3. 根据权利要求1所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述S1步骤中，当EV来源于SKBR3时，所述抗体为生物素化anti-human CD9/CD81抗体，抗体的浓度为2.0-15.0μg/mL。
  4. 根据权利要求2或3所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述S1步骤在微孔板中进行，具体采用涂有链霉亲和素的微孔捕获抗体，然后对EV进行固定；
    所述捕获抗体的时间为0.1-1.5小时；所述固定的时间为4-20小时，固定温度为4℃。
  5. 根据权利要求1所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述核酸适体在使用前经过折叠处理，具体步骤包括：用添加有0.5-2.0mM MgCl2的磷酸缓冲液，将核酸适体稀释至目标浓度，然后在90-98℃条件下变性处理2-10min，冰上或室温培养5-20分钟，然后在35-38℃再折叠10-30分钟。
  6. 根据权利要求1或5所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述核酸适体的目标浓度为1-8nM。
  7. 根据权利要求6所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述S2步骤具体为，将60-140μL含有荧光标记的核酸适体的缓冲液添加至S1步骤所得产物中，将微孔板置于摇床上室温避光培养0.5-2小时。
  8. 根据权利要求7所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述缓冲液包括合成型缓冲液或人血浆；
    优选的，所述人血浆来源于血型为A型，B型，AB型，O型，Rh+，Rh-的任一种的献血者；
    优选的，所述人血浆来源于年龄为0-120岁的献血者；
    优选的，所述人血浆来源于健康人或非健康人；进一步优选的，所述人血浆来源于非健康人，所述非健康人优选为肿瘤患者。
  9. 根据权利要求1所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征在于，所述S3步骤中荧光极化的信号通过多功能盘式分析仪进行读取；所述多功能盘式分析仪配置有475-490nm的激发滤光片和520-565nm的发射滤光片。
  10. 根据权利要求9所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法，其特征 在于，当核酸适体为CD63-BP时，所述检测方法的LOD≤5×107EVs/mL，LDR为5×108-2×1010EVs/mL；当核酸适体为HER2-HApt时，所述检测方法的LOD≤5×107EVs/mL，LDR为8×107-2×1010EVs/mL；当核酸适体为HER2-2A时，所述检测方法的LOD≤3×107EVs/mL，LDR为2×108-2×1010EVs/mL。
  11. 一种根据权利要求1所述的基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述检测方法应用于癌细胞分泌的细胞外囊泡的定性、定量分析。
  12. 根据权利要求1所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述癌细胞来源于结直肠癌，乳腺癌，肝细胞癌，胃癌，胰腺癌，食道癌，鼻咽癌，咽喉癌，子宫内膜癌，肺癌，头颈癌，肾癌，膀胱癌，甲状腺癌，皮肤癌，卵巢癌，宫颈癌，前列腺癌，阴茎癌中的任一种。
  13. 根据权利要求11或12所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述检测方法能够区分不同原发部位的癌细胞分泌的EV；所述原发部位包括肠，乳腺，肝，胃，胰腺，食道，肺，胆囊膀胱，甲状腺，卵巢，子宫颈，前列腺，阴茎中的任一种。
  14. 根据权利要求11所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述检测方法能够区分不同生长阶段的癌细胞所分泌的EV；所述生长阶段包括原位癌阶段，区域淋巴结转移阶段，远端转移阶段中的任一种；优选的，所述检测方法能够区分抗癌治疗过程中在不同生长阶段的癌细胞所分泌的细胞外囊泡或者癌细胞在经过抗癌治疗后产生的具有抗药性的细胞外囊泡。
  15. 根据权利要求11所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述检测方法可直接在临床检验科的自动生化分析仪上施行，无需特殊设备或特制的仪器。
  16. 根据权利要求11所述的一种基于核酸适体的细胞外囊泡荧光极化检测方法的应用，其特征在于，所述检测方法可直接在临床检验科的自动免疫分析仪上施行，无需特殊设备或特制的仪器。
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