Pancreatic cancer exosome detection method based on DNA chain capture

Abstract

The invention belongs to the technical field of biochemical analysis, discloses a pancreatic cancer exosome detection method based on DNA chain capture, and particularly discloses a reagent which comprises an aptamer DNA long chain, the aptamer DNA long chain comprises nucleic acid aptamer sequences of target protein which are alternately repeated, and the target protein comprises tumor marker protein GPC1. The invention provides the reagent containing the aptamer DNA long chain, the separation, enrichment and detection of the exosome can be realized by using the reagent, the separation, enrichment and detection steps are simple, expensive instruments are not needed, and in the process of capturing the exosome, the structure stability of the exosome is ensured, the protein of the exosome is not damaged, and the safety of the exosome is ensured. The biological activity can be well maintained.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemical analysis, and particularly relates to a method for detecting pancreatic cancer exosomes based on DNA chain capture. Background Art

[0002] Exosomes are present in all biological fluids and are a type of extracellular vesicle. They originate from endosomes and are microscopic vesicles ranging in size from approximately 40 to 160 nm. Exosomes mediate interactions between the tumor microenvironment and the tumor, delivering bioactive substances such as proteins, lipids, and non-coding RNA to target cells. By altering the tumor microenvironment, exosomes can control the progression of certain cancers, angiogenesis, and immune evasion. Given these characteristics, exosomes are expected to become a key tool in the early diagnosis of pancreatic cancer.

[0003] Currently, the gold standard for exosome isolation is ultracentrifugation. Ultracentrifugation can be divided into differential centrifugation and density gradient centrifugation. Ultracentrifugation isolates exosomes by gradually increasing the centrifugation speed. Density gradient centrifugation is an optimization of the former. During the ultracentrifugation step, sucrose solutions with varying density gradients are prepared, dispersing exosomes of varying sizes and densities into different density layers. This method yields exosomes with higher purity than differential centrifugation. However, the multiple centrifugation steps and expensive centrifugation equipment hinder efficient and convenient separation, and yields are relatively low. Polymer precipitation methods, which form a polymer network to entangle and adsorb all particles in the solution, can isolate exosomes relatively quickly, but they can also lead to issues such as low purity, contamination from impurities, and high biotoxicity from polymer chains. Membrane filtration methods can separate exosomes based on pore size, but due to the specific adsorption of exosomes to membranes and the clogging of pores, recovery and purity are low. The immunoaffinity-based method separates exosomes through the specific binding of antibodies and exosome surface membrane proteins. However, due to the low probability of spatial collision between antigens and antibodies, this method has low separation efficiency and long separation time, and may cause damage to exosomes during the elution process. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a reagent.

[0005] The purpose of the second aspect of the present invention is to provide the use of the reagent of the first aspect of the present invention in isolating, enriching, detecting exosomes or preparing a product for isolating, enriching, and detecting exosomes.

[0006] The third aspect of the present invention aims to provide a method for isolating and enriching exosomes.

[0007] The fourth aspect of the present invention aims to provide a method for visual detection of exosomes.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a reagent comprising a long aptamer DNA chain, wherein the long aptamer DNA chain comprises intermittently repeated nucleic acid aptamer sequences targeting a target protein, wherein the target protein comprises the tumor marker protein GPC1.

[0010] In some embodiments of the present invention, the nucleic acid aptamer sequence is shown in SEQ ID NO: 3.

[0011] In some embodiments of the present invention, the long aptamer DNA chain is a long single-stranded DNA molecule generated based on rolling circle amplification technology.

[0012] In some embodiments of the present invention, the aptamer DNA long chain is prepared by the following steps:

[0013] Circularizing the linear template DNA to obtain a circularized DNA template;

[0014] The circularized DNA template is subjected to rolling circle amplification reaction under the action of DNA polymerase to obtain a long chain of aptamer DNA.

[0015] In some embodiments of the present invention, the linear template DNA includes the nucleic acid fragment shown in SEQ ID NO: 3.

[0016] In some embodiments of the present invention, the nucleotide sequence of the linear template DNA is as shown in SEQ ID NO: 1.

[0017] In some embodiments of the present invention, the circularization comprises the following steps: mixing the linear template DNA, the connecting strand and the buffer, annealing at 94-96° C. for 8-15 minutes to obtain an annealing solution; mixing the annealing solution with T4 buffer and T4 DNA ligase, circularizing at 14-20° C. for 4-6 hours, and inactivating at 64-67° C. for 8-12 minutes to obtain a circularized DNA template.

[0018] In some embodiments of the present invention, the rolling circle amplification reaction comprises the following steps: mixing the circularized DNA template with a buffer (such as Phi29 buffer), a DNA polymerase (such as Phi29 DNA polymerase), primers, and dNTPs, reacting at 28-35° C. for 30-60 min, and inactivating at 64-67° C. for 8-12 min to obtain a long aptamer DNA chain.

[0019] The second aspect of the present invention provides the use of the reagent of the first aspect of the present invention in isolating, enriching, detecting exosomes or preparing a product for isolating, enriching, and detecting exosomes.

[0020] In some embodiments of the present invention, the product includes a kit and a chip.

[0021] The third aspect of the present invention provides a method for isolating and enriching exosomes, comprising the following steps: mixing the reagent of the first aspect of the present invention with a biological sample, and incubating.

[0022] In some embodiments of the present invention, the incubation condition is 35-40° C. for 1-3 hours.

[0023] In some embodiments of the present invention, the incubation condition is 35-37° C. for 1-2 hours.

[0024] In some embodiments of the present invention, the method further comprises a separation step.

[0025] In some embodiments of the present invention, the separation step includes performing solid-liquid separation on the mixed solution after incubation.

[0026] In some embodiments of the present invention, the separation includes performing solid-liquid separation on the mixed liquid by centrifugation.

[0027] In some embodiments of the present invention, the separation step further comprises a step of releasing exosomes using an endonuclease (such as DNase I).

[0028] In some embodiments of the present invention, the biological sample is cell culture fluid, strain culture fluid and tissue culture fluid.

[0029] In some embodiments of the present invention, the biological sample is at least one of blood, urine, saliva, semen, bile, cerebrospinal fluid, and respiratory tract washing fluid.

[0030] In some embodiments of the present invention, the cells include tumor cells, such as pancreatic cancer cells.

[0031] A fourth aspect of the present invention provides a method for visualizing detection of exosomes, comprising the following steps:

[0032] Mixing the reagent of the first aspect of the present invention with a biological sample and incubating the mixture to obtain exosomes;

[0033] Exosomes are loaded onto a paper chip, and after adding an antibody against a marker protein on the exosomes to bind to the exosomes, an enzyme-linked secondary antibody is used to bind to the antibody against the marker protein on the exosomes;

[0034] Add enzyme substrate for color development reaction.

[0035] In some embodiments of the present invention, the material of the paper chip includes polyvinylidene fluoride and polyethersulfone membrane.

[0036] In some embodiments of the present invention, the incubation condition is 35-40° C. for 1-3 hours.

[0037] In some embodiments of the present invention, the incubation condition is 35-37° C. for 1-2 hours.

[0038] In some embodiments of the present invention, the anti-exosome marker protein antibody includes an anti-GPC1 antibody.

[0039] In some embodiments of the present invention, a paper chip is used to filter a solution containing exosomes, thereby loading the exosomes on the paper chip.

[0040] In some embodiments of the present invention, the visualization detection method further comprises a step of collecting images of the paper chip after color development.

[0041] In some embodiments of the present invention, the image acquisition includes using an image acquisition device to acquire an image of the color-developed paper chip, and using image processing software to analyze and obtain color intensity data on the paper chip.

[0042] In some embodiments of the present invention, the visualization detection method includes: adding an anti-exosome marker protein antibody dropwise to a paper chip containing exosomes, reacting at 35-40°C for 20-30 minutes, and washing; adding an enzyme-linked secondary antibody solution, reacting at 5-40°C for 20-30 minutes, washing, adding TMB and H2O2, reacting at room temperature for 3-7 minutes, collecting images, and analyzing the color intensity data on the paper chip to achieve detection of exosomes.

[0043] In some embodiments of the present invention, the biological sample is cell culture fluid, strain culture fluid and tissue culture fluid.

[0044] In some embodiments of the present invention, the biological sample is at least one of blood, urine, saliva, semen, bile, cerebrospinal fluid, and respiratory tract washing fluid.

[0045] In some embodiments of the present invention, the cells include tumor cells, such as pancreatic cancer cells.

[0046] The beneficial effects of the present invention are:

[0047] The present invention provides a reagent containing long chains of aptamer DNA. Using this reagent, the separation, enrichment, and detection of exosomes can be achieved. The separation, enrichment, and detection steps are simple and do not require the use of expensive instruments. In addition, during the capture of exosomes, the structural stability of the exosomes is ensured while the proteins in the exosomes are not damaged, thereby better maintaining their biological activity.

[0048] The present invention develops a method for real-time detection of exosomes (such as pancreatic cancer exosomes) based on long aptamer DNA and ELISA method with simple operation and rapid process ( Figure 1 ), designed a template strand containing a complementary sequence to the GPC1 aptamer, and used rolling circle amplification to prepare a long DNA strand containing the GPC1 aptamer. The aptamer DNA strand can specifically capture pancreatic cancer exosomes while reducing nonspecific adsorption of impurities due to the porous structure of the DNA hydrogel. After the exosomes are captured, they are filtered through a PVDF membrane. Exosome aggregates captured by the DNA strand remain on the membrane, while impurities such as lipoproteins that were not specifically captured are filtered out. After the exosomes are separated, the exosome concentration is visually detected using a paper-based enzyme-linked immunosorbent assay (P-ELISA). This method, combining the aptamer DNA strand with the enzyme-linked immunosorbent assay, allows for real-time detection of exosome concentration, reducing the time and cost of exosome isolation and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a schematic diagram of the principle of the instant detection of pancreatic cancer exosomes based on long aptamer DNA chains of the present invention.

[0050] Figure 2 The feasibility study verification results of the aptamer DNA long chain capturing GPC1 protein; wherein, A is the PAGE image of the binding between the GPC1 aptamer and GPC1 protein; B is the specific verification of the binding between the GPC1 aptamer and GPC1; C is the RCA reaction agarose gel verification image.

[0051] Figure 3 TEM identification results of exosomes; A is the TEM image of PANC-1 exosomes; B is the TEM image of HELA exosomes; C is the TEM image of HPNE exosomes.

[0052] Figure 4 These are the NTA identification results of exosomes; A is the NTA image of PANC exosomes; B is the NTA image of HELA exosomes; and C is the NTA image of HPNE exosomes.

[0053] Figure 5 Western blotting was used to detect the protein expression of exosomal protein markers (CD63 and CD81) and tumor marker protein (GPC1) in PANC-1 exosomes, HPNE exosomes, and HELA exosomes.

[0054] Figure 6 Figure 1 is the TEM identification result of exosomes captured by aptamer DNA long chains; A is the TEM image of aptamer DNA long chains bound to PANC-1 exosomes after centrifugation; B is the TEM image of aptamer DNA long chains bound to PANC-1 exosomes without centrifugation; C is the TEM image of PANC-1 exosomes released by DNase I enzyme cleavage of aptamer DNA long chains.

[0055] Figure 7 NTA image of PANC-1 exosomes captured by long aptamer DNA chains.

[0056] Figure 8 Western blotting was used to detect the protein expression of exosome protein markers (CD81) and tumor marker proteins (GPC1) in PANC-1 exosomes captured by aptamer DNA long chains.

[0057] Figure 9 Confocal microscopy images of exosomes stained with CM-DiI. Scale bar: 10 μm.

[0058] Figure 10 Confocal microscopy images of PANC-1 exosomes incubated with aptamer DNA long chains and random DNA long chains. Confocal microscopy images of HELA exosomes and HPNE exosomes incubated with aptamer DNA long chains. Scale bar: 10 μm.

[0059] Figure 11 Verification of the specific capture of PANC exosomes by long aptamer DNA chains.

[0060] Figure 12 Comparison of the yield of exosomes isolated from cell culture medium by DNA body chain capture method and ultracentrifugation method.

[0061] Figure 13 Comparison of total protein concentration and purity of exosomes obtained by aptamer DNA chain capture method and ultracentrifugation method; where A is the total protein concentration of exosomes separated by aptamer DNA chain capture method and ultracentrifugation method, and B is the purity comparison of exosomes separated from cell culture medium by chain capture method and ultracentrifugation method.

[0062] Figure 14 Figure 3 shows the detection results of pancreatic cancer exosomes captured by visualization of aptamer DNA long chains; A shows the detection of exosomes at different concentrations by paper-based enzyme-linked immunosorbent assay (P-ELISA); B shows the relationship between BM and exosome concentration (n=3). DETAILED DESCRIPTION

[0063] The present invention is further described in detail below through specific examples.

[0064] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0065] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0066] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0067] Example 1 Preparation and Characterization of Aptamer DNA Long Chains

[0068] The preparation of aptamer DNA long chain includes the following steps:

[0069] (1) Annealing: 0.6 μL template strand (5'-P-TCGTTTGATGTTCCTAATC CAAAAGGGGGATG GATTCGCTG, SEQ ID NO: 1, P is a modified phosphate group, the underlined portion is the complementary sequence of the aptamer DNA), 0.6 μL of connecting strand (5'-TAGGAACATCAAACGACAGCGA-3', SEQ ID NO: 2), 0.6 μL of NE buffer 2 (10×) and 4.2 μL of DEPC water were prepared into a 6 μL mixture and annealed at 95°C for 10 min to obtain an annealing solution.

[0070] The nucleotide sequence of the aptamer DNA is 5'-CATCCCCCTTTTG-3' (SEQ ID NO: 3).

[0071] (2) Circularization reaction: 0.75 μL annealing solution, 0.5 μL T4 buffer (10×), 1 μL T4 DNA ligase (100 U / μL), and 2.75 μL DEPC water were prepared into a 5 μL mixture, cyclized at 16°C for 5 h, and inactivated at 65°C for 10 min to obtain a circularized solution.

[0072] (3) RCA reaction: 5 μL of cyclization solution, 1 μL of Phi29 buffer (10×), 1 μL of Phi29 DNA polymerase (2.5 U / μL), 0.8 μL of recombinant albumin (RA, purchased from New England, catalog number: M0269L) (1.25 μg / μL), 1.25 μL of dNTPs (10 mM), and 0.95 μL of DPEC water were mixed and reacted at 30°C for 1 h, and inactivated at 65°C for 10 min to obtain the aptamer DNA long chain (i.e., GPC1 aptamer segment).

[0073] The prepared aptamer DNA strands were subjected to agarose gel electrophoresis as follows: 10 μL of the aptamer DNA strands were mixed with 6× DNA loading buffer and loaded onto a 15% polyacrylamide gel. Electrophoresis was performed at 80 V, followed by staining with 4S-GelRed and photographing using a gel imaging system.

[0074] Agarose gel electrophoresis Figure 2 As shown in C.

[0075] Example 2 Feasibility Study of Capturing GPC1 Protein with Aptamer DNA

[0076] Principle Electrophoresis Verification: The final concentration of the nucleic acid used for verification was set at 2.0 mg / μL. GPC1 aptamer segments and GPC1 protein (purchased from Shenzhen Tianqishun Biotechnology Co., Ltd., Catalog No. TQ-FY-KY5176, final protein concentration: 100 ng / μL) were incubated for 2 hours, bringing the final solution volume to 10 μL. After adding 2 μL of 6× Loading Buffer and vortexing to mix, the sample was loaded onto a 12% agarose gel for electrophoresis analysis using 0.5× TBE buffer at 90 V for 60 minutes.

[0077] Flow cytometry verification: PANC-1 (human pancreatic cancer cells), HPNE (human pancreatic ductal cells), HELA (cervical cancer cells), and HcerEpic (human cervical epithelial cells) cells in the logarithmic growth phase were grown in 6-well plates, with the number of cells in each well being approximately 1.0 × 10 6 The serum-free medium was replaced, and the four cells were incubated with 2 μM aptamer-FAM (CATCCCCCTTTTG, SEQ ID NO: 3) at 37°C for 2 h. As a negative control, the four cells were incubated with serum-free medium at 37°C for 2 h. After incubation, unbound aptamers were washed with PBS, and the cells were resuspended in 400 μL PBS and fluorescence detection was performed by flow cytometry.

[0078] Verification of DNA long chains generated by RCA reaction: The final concentration of the nucleic acid of the GPC1 aptamer segment (Example 1) and the random DNA chain (the nucleotide sequence of the template chain of the random DNA chain is considered to be 5'-P-TCGTTTGATGTTCCTAATGATTCGCTG-3', SEQ ID NO: 4) generated by the RCA reaction was set to 2.0 mg / μL, and the final solution volume was 10 μL. Finally, 2 μL of 6× Loading buffer was added and shaken to mix thoroughly. The sample was then loaded onto a 12% agarose gel for electrophoresis analysis using 0.5× TBE buffer at 90 V for 60 min.

[0079] The binding of the generated GPC1 aptamer segments to the GPC1 protein was verified by 12% PAGE gel and flow cytometry, and the RCA reaction was characterized by 12% agarose gel. Figure 2 As shown in the PAGE diagram, the electrophoresis band after the GPC1 aptamer segment and the GPC1 protein bind is above 15000 bp, indicating that the GPC1 aptamer segment and the GPC1 protein bind ( Figure 2 Subsequently, the same concentration of FAM fluorescently labeled aptamers were incubated with four cell lines (PANC-1, HPNE, HELA, and HcerEpic cells) and analyzed by flow cytometry to verify their specific binding to GPC1 protein. Figure 2 As shown in Figure B, the GPC1 aptamer binds to the GPC1 protein of PANC cells. The end sequences of the aptamer template strand and the random template strand are designed to complement the base sequence of the connecting strand, and the phosphate group is modified at the 5' end of the template strand to ensure the successful generation of the circular template strand. Then, through the RCA reaction, a long DNA single strand with repeated circular template fragments is generated under the action of Phi-29 polymerase. In the agarose gel image, lanes 3, 4 and lanes 5, 6 show the successful generation of the RCA reaction, and the concentration of the long DNA strand generated by the 60-minute RCA reaction is higher than that of the 30-minute reaction ( Figure 2 Middle C).

[0080] Example 3 Identification and characterization of exosomes isolated by ultracentrifugation

[0081] 1. Cell Culture

[0082] Human cervical cancer cells HELA, human cervical epithelial cells HcerEpic, and human pancreatic cancer cells PANC-1 were purchased from the Punosai Cell Bank, and human normal pancreatic ductal cells hTERT-HPNE were purchased from the Warner Cell Bank. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator.

[0083] 2. Ultracentrifugation to separate exosomes

[0084] After cells have grown to fill four T225 flasks, the complete medium is replaced with DMEM supplemented with 1% penicillin-streptomycin. After incubation at 37°C and 5% CO₂ for 48 hours, the cell suspension is harvested and centrifuged at 3000×g for 10 minutes. The supernatant is then collected and removed of cells and cell debris. Subsequently, the supernatant is centrifuged at 10,000×g for 10–30 minutes, and large vesicles and protein aggregates are removed. The resulting 72 mL supernatant is centrifuged at 130,000×g for 75 minutes at 4°C. The supernatant is carefully aspirated and the exosome pellet is resuspended in 72 mL of PBS. The pellet is then centrifuged again at 130,000×g for 75 minutes at 4°C. The pellet is carefully collected and resuspended in 200 μL of PBS buffer. The exosomes are aliquoted and stored at -80°C. Exosomes are isolated by differential centrifugation. The isolated exosomes were characterized by western blotting (WB), transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA).

[0085] Identification and characterization of exosomes

[0086] TEM: Take 1 μL of exosome resuspension and dilute it 10 times with ultrapure water, then immediately drop it onto a copper grid supported by an ultrathin carbon film. After drying at room temperature, add 1% phosphotungstic acid solution and let it stand for 10 seconds. Then, immediately wash away the residual phosphotungstic acid with ultrapure water. After placing the copper grid in a hollow place to dry for 12 hours, use TEM to characterize the morphology of exosomes.

[0087] NTA: NTA analyzes the concentration and size of exosomes in the resuspension. In this analysis, a 520 nm monochromatic laser beam is applied to the diluted exosome resuspension. A 60-second video is captured at 25 frames per second, and the Brownian motion of the exosomes in solution is analyzed using NanoSight NS300 software. The data are maintained constant between samples, and analysis and calculations are performed for each video to provide estimates of concentration and size.

[0088] Western blot: Add RIPA strong lysis buffer to the exosome resuspension at a 1:1 ratio. Lyse on ice for 30 minutes. Collect the lysate and determine protein concentration using a BCA protein assay kit. Add 6× loading buffer to the lysate and cook the protein in a metal bath at 75°C for 10 minutes. Prepare a 12% SDS-PAGE separating gel and a 5% SDS-PAGE stacking gel. Set the voltage for the separating gel at 80V for 30 minutes and the stacking gel at 120V for 45 minutes. Transfer the protein to a 0.22μm PVDF membrane using wet transfer at 250A for 45 minutes. Block with 5% nonfat dry milk at room temperature for 1.5 hours, then wash with TBST. Incubate with primary antibody overnight at 4°C, wash three times with TBST, and incubate with secondary antibody at room temperature for 1 hour. Apply ECL luminescent solution to the PVDF membrane and photograph using a gel imaging scanner.

[0089] The results of TEM characterization of exosome morphology are as follows Figure 3 As shown in the figure, the morphology of the exosomes is cup-shaped with a depression in the middle. After negative staining with phosphotungstic acid, the phospholipid bilayer membrane structure is clear and the particle size is about 100 nm.

[0090] The particle size of exosomes was further analyzed using NTA, and the test results were as follows Figure 4 As shown in Figure 2, the particle sizes of the three exosomes are distributed between 100 and 250 nm, the peak particle size of PANC exosomes is 124 nm, and the concentration of the peak particle size is 2.5×10 6 Particles / mL, the peak particle size of HPNE exosomes was 133 nm, and the concentration of the peak particle size was 5.0×10 5 Particles / mL, the peak particle size of HELA exosomes was 110 nm, and the concentration of peak particle size was 5.5×10 6 Particles / mL.

[0091] To further verify the expression of surface protein markers, the expression of surface membrane proteins CD63, CD81, and GPC1 of the three exosomes was verified by Western blot. The results are as follows Figure 5 As shown in the results, PANC-1 exosomes highly expressed the surface membrane proteins CD63, CD81, and GPC1, while HPNE and HELA exosomes highly expressed the surface membrane proteins CD63 and CD81, but did not express the surface membrane protein GPC1. These results demonstrate that the exosomes extracted by ultracentrifugation have a morphology, particle size distribution, and membrane protein expression consistent with the literature.

[0092] The above results indicate that PANC-1 exosomes can serve as GPC1 + Exosome model for evaluating GPC1 capture by long aptamer DNA chains + The possibility of exosomes.

[0093] Example 4 Feasibility Study of Specific Capture of PANC-1 Exosomes by Long-chain Aptamer DNA

[0094] 1. Validation of DNA Strand Separation and Capture of Exosomes

[0095] The concentration of the aptamer DNA long chain (prepared in Example 1) used for verification was 2.0 mg / μL, and the concentration of the exosomes (prepared in Example 3) was 1×10 8 Particles / mL. 10 μL of exosomes was added to 10 μL of the aptamer DNA long-chain solution. After incubation at 37°C with slow rotation for 2 hours, the solution was centrifuged at 14,000 × g for 20 minutes. The precipitate was characterized using TEM. DNase I endonuclease was further added to release the exosomes. The exosomes separated from the ligand DNA long-chain were identified and characterized using Western blotting, NTA, and TEM (the specific procedures were the same as in Example 3).

[0096] The exosomes extracted in Example 3 were added to the aptamer DNA long chain solution, and the mixture was slowly rotated at 37°C for 2 h. After centrifugation, the supernatant was carefully aspirated, and the precipitate was resuspended in PBS. The morphology of the PANC-1 exosomes captured by the aptamer DNA long chain was characterized by TEM. Figure 6 As shown in A to B. TEM images show that the aptamer DNA long chain enriched dozens or hundreds of exosomes, forming 2-3 μm aggregates after centrifugation. To characterize the exosomes captured by the aptamer DNA long chain, the supernatant was removed after centrifugation, and DNase I endonuclease was added. The morphology of the exosomes was characterized by TEM. Figure 6 As shown in middle C, the morphology of the exosomes is cup-shaped with a depression in the middle. After negative staining with phosphotungstic acid, the phospholipid bilayer membrane structure is clear, and the particle size is about 100 nm.

[0097] The particle size of the captured exosomes was further analyzed using NTA, and the test results were as follows: Figure 7 As shown in Figure 2, the particle size is mainly distributed between 100 and 250 nm, with a peak particle size of 112 nm and a peak particle size concentration of 6.0×10 5 Particles / mL.

[0098] To further verify the expression of surface protein markers, the expression of surface membrane proteins CD81 and GPC1 of exosomes was verified by Western blot. Figure 8 As shown in the figure, the captured exosomes express CD81 and GPC1 proteins. These results demonstrate that the long-chain aptamer can successfully capture PANC-1 exosomes. Moreover, the capture process ensures the stability of the exosome structure without damaging the proteins in the exosomes, thus maintaining their biological activity.

[0099] 2. Validation of Specific Isolation of PANC Exosomes

[0100] The concentration of all aptamer DNA long chains used for validation was 2.0 mg / μL, and the concentration of exosomes was 1×10 7 Particles / mL were collected by differential centrifugation (the sample was first centrifuged at 300g to remove large particles and cells. The supernatant was then centrifuged at 2000g to remove cell debris. The supernatant was then centrifuged at 10,000g to remove impurities such as apoptotic bodies and protein aggregates. Finally, the exosomes were isolated by ultracentrifugation twice at 130,000g. Repeated centrifugation steps improved the purity of the exosomes.) The exosomes were stained with CM-DiI dye at a working concentration of 10 μM for 15 minutes, and the excess dye was filtered out using a 10 kDa ultrafiltration tube. The RCA-generated aptamer DNA long chains were stained with 1× Sybr Green II for 15 minutes. The stained aptamer DNA long chains were mixed with HELA exosomes, PANC-1 exosomes, and HPNE exosomes at a 1:1 ratio by volume and incubated for 2 hours. The random template DNA chains were also mixed with PANC-1 exosomes at a 1:1 ratio by volume and incubated for 2 hours. 5 μL of the mixture was pipetted onto a confocal slide, covered with a coverslip, and imaged under a confocal microscope. The mean red fluorescence intensity of the CM-DiI dye in the confocal image was then calculated using ImageJ image analysis software, with the mean fluorescence intensity value serving as the quantitative output signal.

[0101] CM-DiI is a commonly used membrane dye. The chloromethyl substitution group it carries can bind to the lipid molecules of the membrane structure, thereby achieving rapid staining of exosomes. SYBR-Green II is a nucleic acid dye that mainly binds to RNA and single-stranded DNA. In order to verify the specific capture of PANC-1 exosomes, the three exosomes were first stained with CM-DiI. After removing the free dye with a 10kDa ultrafiltration tube, the exosomes were captured with the dyed aptamer DNA long chain according to the above steps and characterized by fluorescence confocal microscopy. The results are shown in Figure 2. Figure 9 After the aptamer DNA long chain and exosomes were incubated for 2 hours, the results were as shown in Figure 10 As shown in the figure, the PANC-1 exosome test group captured by the aptamer DNA long chain was observed to have co-localized overlapping color blocks in the confocal image, while the other test groups were not observed to have overlapping color blocks in the confocal image. The mean fluorescence intensity of the red channel of the confocal image was then calculated using ImageJ image processing software. The fluorescence intensity of the PANC exosomes captured by the aptamer chain was 16.406MFI, which was 12.4 times that of the HELA exosomes captured by the aptamer ( Figure 11 ).

[0102] The above results prove that the GPC1 aptamer on the long DNA chain can specifically recognize the PANC-1 exosome surface membrane protein GPC1 + , thereby capturing PANC-1 exosomes.

[0103] Example 5 Performance comparison between aptamer DNA chain capture method and ultracentrifugation method

[0104] An exosome capture method (denoted as aptamer DNA chain capture method) comprises the following steps: taking 10 μL of a sample containing exosomes, adding 10 μL of a solution of aptamer DNA long chains (prepared in Example 1), incubating slowly with rotation at 37°C for 2 h, centrifuging at 14,000 × g for 20 min, collecting the precipitate, and further adding DNase I endonuclease to release the exosomes. The release takes 10 min, thereby obtaining PANC-1 exosomes.

[0105] The performance of the aptamer DNA chain capture method was further compared with the ultracentrifugation method used in the prior art.

[0106] Yield, purity and separation efficiency are the most important parameters for isolating exosomes and are the decisive factors for the good performance of exosomes in downstream applications. Exosome yield is defined as the number of exosomes isolated from a certain volume of sample (number of particles separated per microliter of solution, μL). -1 ), and its purity is defined as the number of exosomes per mg of protein (mg -1 The isolation efficiency of exosomes is defined as the yield of isolated exosomes per unit time (number of particles μL -1 h -1 )

[0107] First, the yield of exosomes isolated from cell culture medium by aptamer DNA chain capture method and ultracentrifugation method was calculated. Figure 12 As shown, the yield of the chain capture method was 5.5×10 6 mL -1 , is an ultracentrifugation method (1.95×10 5 mL -1 ) 45 times. Ultracentrifugation uses enormous centrifugal forces of up to 130,000 × g to separate exosomes. However, the high centrifugal forces and repeated centrifugation processes can easily damage the fragile membrane structure of exosomes, resulting in reduced isolation yield. However, the aptamer DNA chain capture method uses lower centrifugal forces to separate exosomes, avoiding the damage caused by high centrifugal forces and repeated centrifugation steps. This allows for non-destructive capture of exosomes and improves isolation yield.

[0108] The purity of the isolated exosomes was then calculated, and the protein concentration of the exosomes separated by the aptamer DNA chain capture method and the ultracentrifugation method was measured by BCA. For the convenience of calculation, the exosome concentration was set to 2.0×109 mL -1 , 20 μL of exosomes of the same concentration were taken for BCA protein concentration quantification. Figure 13 As shown, the total protein concentrations of exosomes isolated by chain capture and ultracentrifugation were 0.121 mg mL -1 and 0.134 mg mL -1 Therefore, the purity of exosomes separated by aptamer DNA chain capture and ultracentrifugation was calculated to be 1.66×10 10 mg -1 and 1.49×10 10 mg -1 The purity of exosomes isolated by aptamer DNA capture was 1.1 times higher than that obtained by ultracentrifugation. The high purity achieved by aptamer DNA capture is due to the high selectivity of aptamers for pancreatic cancer exosomes and their high resistance to adsorption of nonspecific proteins.

[0109] Next, the exosome separation efficiency was calculated. The time for exosome separation by aptamer DNA chain capture method and ultracentrifugation method was 1 h and 8 h, respectively. Therefore, the exosome separation efficiency of aptamer DNA chain capture method was calculated to be 5.5×10 6 mL -1 h -1 , is the ultracentrifugation method (2.4×10 4 mL -1 h -1 ). This is because the ultracentrifugation method requires repeated, prolonged centrifugation steps and a lengthy vacuum pretreatment step, which reduces the efficiency of exosome isolation. The aptamer DNA chain capture method optimizes the centrifugation step to a single round of high-speed centrifugation, which also eliminates the need for a vacuum pretreatment step, significantly improving exosome isolation efficiency.

[0110] Example 6

[0111] In addition to simple and effective exosome capture, a simple, low-cost, and highly sensitive exosome detection method is essential for exosome-related research. Based on the specific capture and enrichment of pancreatic cancer exosomes by aptamer DNA long chains, a simple paper-based enzyme-linked immunosorbent assay (P-ELISA) method was developed for highly sensitive, visual, and quantitative detection of pancreatic cancer exosomes. This method utilizes the immunoaffinity between the aptamer and the target protein, PVDF membrane filtration, and an HRP-conjugated rabbit secondary antibody that catalyzes the colorimetric reaction of TMB and H2O2 to visually detect the concentration of captured pancreatic cancer exosomes. The specific detection process is as follows: After the exosomes are captured by the DNA chains (the process is the same as in Example 4), they are filtered through a 0.22μm polyvinylidene fluoride (PVDF) membrane. The PVDF membrane is blocked with 5% BSA solution for 10 minutes, and 10μL of 1μg / mL rabbit anti-GPC1 is added dropwise. After reacting on the PVDF membrane at 37°C for 30 minutes, the membrane is washed three times with PBST to remove excess anti-GPC1 antibody. Then, 10 μL of 0.2 μg / mL HRP-conjugated GPC1 secondary antibody was added dropwise and incubated at 37°C for 30 minutes. The excess secondary antibody was removed by washing three times. Finally, TMB and H₂O₂ were added dropwise and incubated at room temperature for 5 minutes. The color was recorded using a mobile phone camera, and the mean blue intensity (MB) on each filter paper was calculated using ImageJ software. The MB value was used as the quantitative output signal.

[0112] The results are as follows Figure 14 As shown in Figure A, the PVDF membrane in the blank control group (PBS without exosomes) is nearly colorless. However, as the exosome concentration increases, the blue color of oxTMB on the PVDF membrane gradually deepens. The color development of the PVDF membrane was recorded using a smartphone. The average blue intensity was analyzed using ImageJ image processing software. The BM value of the exosome sample group increased with increasing exosome concentration. A standard curve was drawn using the logarithm of the exosome concentration against BM, and the linear regression equation was BM = 189.2lgCsEV + 5.79, R 2 =0.977, limit of detection (LOD): 50 μL -1 ( Figure 14 Middle B).

[0113] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A reagent comprising a long aptamer DNA chain, wherein the long aptamer DNA chain comprises interspaced and repeated nucleic acid aptamer sequences targeting a target protein, wherein the target protein comprises the tumor marker protein GPC1.

2. The reagent according to claim 1, characterized in that The nucleic acid aptamer sequence is shown in SEQ ID NO:

3.

3. The reagent according to claim 1, characterized in that The aptamer DNA long chain is a long single-stranded DNA molecule generated based on rolling circle amplification technology.

4. The reagent according to claim 3, characterized in that The aptamer DNA long chain is prepared by the following steps: Circularizing the linear template DNA to obtain a circularized DNA template; The circularized DNA template is subjected to a rolling circle amplification reaction under the action of a DNA polymerase to obtain a long chain of aptamer DNA; preferably, the linear template DNA includes the nucleic acid fragment shown in SEQ ID NO: 3; Preferably, the nucleotide sequence of the linear template DNA is as shown in SEQ ID NO:

1.

5. Use of the reagent according to any one of claims 1 to 4 in isolating, enriching, detecting exosomes or preparing a product for isolating, enriching, and detecting exosomes.

6. A method for isolating and enriching exosomes, comprising the following steps: The reagent according to any one of claims 1 to 4 is mixed with a biological sample and incubated.

7. The method according to claim 6, characterized in that The incubation condition is 35-40°C for 1-3 hours; preferably, the method further comprises a separation step; Preferably, the separation step comprises performing solid-liquid separation on the mixed solution after incubation.

8. A method for visual detection of exosomes, comprising the following steps: Mixing the reagent according to any one of claims 1 to 4 with a biological sample and incubating the mixture to obtain exosomes; Exosomes are loaded onto a paper chip, and after adding an antibody against a marker protein on the exosomes to bind to the exosomes, an enzyme-linked secondary antibody is used to bind to the antibody against the marker protein on the exosomes; Add enzyme substrate for color development reaction.

9. The visual detection method according to claim 8, characterized in that: The material of the paper chip includes polyvinylidene fluoride and polyethersulfone membrane; and / or, the incubation condition is 35-40° C. for 1-3 hours; and or, the anti-exosome marker protein antibody includes anti-GPC1 antibody.

10. The visual detection method according to claim 8, characterized in that: The visual detection method further comprises the step of collecting images of the paper chip after color development; Preferably, the image acquisition includes using an image acquisition device to acquire an image of the color-developed paper chip, and using image processing software to analyze and obtain color intensity data on the paper chip.

Images