An encoding microsphere and its application in simultaneous detection of multiple markers of extracellular vesicles

Abstract

The application belongs to the technical field of synthesis and detection, and particularly relates to a coded microsphere and application thereof in simultaneous detection of multiple markers of extracellular vesicles. The coded microsphere comprises the following raw materials: polystyrene microspheres, liposomes, nucleic acid aptamer, hairpin probe I, and hairpin probe II. The nucleic acid aptamer can specifically recognize the protein on the surface of EVs. The hairpin probe I is used for detecting miRNA, and the hairpin probe II is used for binding different t-EVs subgroups. In combination with the flow cytometry technology, the fluorescent and size combination coded signals and report signals can be simultaneously displayed, miRNA decoding and quantification are respectively carried out, and multiple detection of tumor-derived extracellular vesicles is realized, thereby providing a new way for cancer diagnosis and screening.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of synthesis and detection, and particularly relates to a coding microsphere and application thereof in simultaneous detection of multiple markers of extracellular vesicles. BACKGROUND

[0002] Tumor cell-derived extracellular vesicles (t-EVs) are nanoscale vesicles secreted by tumor cells, which can mediate communication between tumor cells. The expression levels of biological molecules such as membrane proteins and internal nucleic acids of t-EVs are closely related to the occurrence and development of tumors. Therefore, sensitive and accurate detection of EVs biomarkers is an effective method for non-invasive diagnosis and treatment monitoring of diseases.

[0003] MicroRNAs (miRNAs) are a class of short, non-coding single-stranded RNAs (about 22 nucleotides), which play an important role in gene expression regulation by inhibiting the translation of target genes or degrading the transcripts of target genes. Abnormal expression of miRNAs is closely related to the pathogenesis of various cancers. Given that multiple miRNAs are involved in the occurrence and development of ovarian cancer, multiplex analysis of t-EVs-miRNA is expected to improve the accuracy and reliability of cancer diagnosis.

[0004] Membrane proteins, as an important component of t-EVs, mediate the recognition process of t-EVs and target cells, and are involved in the regulation of the occurrence and development of various diseases such as tumors, immunity, and neurodegeneration, and can be used as diagnostic markers of diseases. However, due to the expression difference of membrane proteins, different EVs subgroups play different biological functions. By detecting the miRNA expression of multiple t-EVs subgroups and drawing the miRNA map, the accuracy of cancer diagnosis can be effectively improved.

[0005] Existing t-EVs-miRNA analysis usually requires lysing EVs to extract internal miRNA, which has complicated operation steps and requires a long analysis time, which is easy to cause miRNA degradation, thereby affecting the accuracy and reliability of the detection results. In addition, due to the limitation of fluorescence spectrum overlap, fluorescence analysis can at most simultaneously perform 3-4 color imaging, which limits the types of miRNA detection and is not conducive to the multiplex detection of miRNA.

[0006] Therefore, it is urgent to develop a multiplex, in-situ and accurate detection method for detecting t-EVs-miRNA derived from tumor cells, and to become a clinically feasible tool for disease screening, classification and progression monitoring, and to promote the development of liquid biopsy field. SUMMARY

[0007] In view of the problems existing in the detection of biomarkers of extracellular vesicles at the present stage, the application provides a coding microsphere and application thereof in simultaneous detection of multiple markers of extracellular vesicles, wherein the coding microsphere comprises polystyrene microspheres, a lipid layer formed by wrapping a liposome on the surface of the polystyrene microspheres, and a hairpin probe II modified on the surface of the microspheres by cholesterol membrane insertion, wherein the hairpin probe II has a complementary sequence of an aptamer, and the microspheres are wrapped with a hairpin probe I in the mesoporous interior.

[0008] The technical scheme of the application is as follows:

[0009] In a first aspect, a coding microsphere is provided, and the raw materials comprise: polystyrene microspheres, a liposome, an aptamer, a hairpin probe I, and a hairpin probe II.

[0010] The hairpin probe I is used for detecting miRNA.

[0011] The aptamer is used for specifically recognizing the protein on the surface of EVs.

[0012] The hairpin probe II is used for specifically binding to a t-EVs subgroup.

[0013] EVs are first incubated with specific aptamers (Extended 1, Extended 2, and specific sequences are shown in Table 1, SEQ ID No. 15-SEQ ID No. 16), and the EVs are labeled with aptamer recognition sequences, so that the surface hairpin probe II (PS1, PS2, PS 3, and specific sequences are shown in Table 1, SEQ ID No. 12-SEQ ID No. 14) of the coding microsphere of different sizes recognizes the aptamer label for specific capture and detection. In order to detect EVs expressing two t-EV biomarkers, proximity ligation (PLA) is used to create a sequence that can be recognized by a specific hairpin probe (PS 3). In order to effectively realize the recognition and binding of the hairpin probe, it is necessary to expose the connection sequence, therefore, a DNA single strand (Linker 2, and specific sequence is shown in Table 1, SEQ ID No. 18) is introduced, which is complementary to Linker 1 (and specific sequence is shown in Table 1, SEQ ID No. 17) to initiate the ligation reaction, so as to realize the recognition and detection of double-expressed EVs.

[0014] When the hairpin probe on the surface of the microsphere specifically recognizes the aptamer on the surface of the EV, the distance between the microsphere and the EV is shortened, leading to membrane fusion between the microsphere and the corresponding EV, and the miRNA in the EV enters the mesoporous inside the microsphere. When the hairpin probe in the mesoporous detects the corresponding miRNA, the fluorescence is restored, and the miRNA in the EV is quantified according to the fluorescence intensity. Different sizes of microspheres are modified with different hairpin probes II to combine different EV subgroups, and different fluorescence encoding microspheres are packaged with different hairpin probes I to detect different miRNAs. Combined with flow cytometry technology, the fluorescence and size combination encoding signals and the report signals can be displayed at the same time, and the decoding and quantification of miRNA are carried out respectively, realizing the one-time multiplex detection of tumor-derived extracellular vesicles and providing a new way for the diagnosis and screening of cancer.

[0015] According to the specific embodiment of the present application, the encoding microsphere is a polystyrene microsphere surface wrapped with a liposome, and the surface is modified with a hairpin probe II, and the inside is wrapped with a hairpin probe I.

[0016] According to the specific embodiment of the present application, the hairpin probe II is a hairpin probe modified with cholesterol.

[0017] According to the specific embodiment of the present application, the hairpin probe II comprises a sequence segment complementary to the nucleic acid aptamer.

[0018] According to the specific embodiment of the present application, the hairpin probe I comprises an MB; the nucleotide sequence of the MB comprises: FAM-CCGCG-complementary sequence of the target miRNA-CGCGG-BHQ1.

[0019] In a second aspect, a preparation method of the above-mentioned encoding microsphere is protected, comprising the following steps:

[0020] (1) preparation of a liposome;

[0021] (2) liposome wrapping of the encoding microsphere;

[0022] (3) internal loading of the encoding microsphere;

[0023] (4) external modification of the encoding microsphere.

[0024] According to the specific embodiment of the present application, the process of step (1) adopts a thin film hydration method to prepare the liposome; specifically comprising: dissolving dioleoyl lecithin (DOPC), dioleoyl phosphatidyl ethanolamine (DOPE), cholesterol, and dioleoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DOPE-PEG2000) in chloroform, drying or evaporating into a lipid film, and dissolving with DPBS to form a liposome solution.

[0025] According to the specific embodiment of the present application, the process of step (2) is completed by self-assembly, specifically including that the 5' end FAM and 3' end quencher modified hairpin probe is loaded into the mesoporous of the coding ball through the self-assembly process.

[0026] According to the specific embodiment of the present application, step (3) adopts the method of shaking bed incubation, specifically including that the microspheres of step (2) are resuspended in DPBS, 500 nM of the modified cholesterol hairpin probe II is taken, 37 degrees shaking bed for 1 h, 8000 rpm, 5 min, washed for three times, resuspended in DPBS, and stored at 4 degrees for subsequent use.

[0027] According to the specific embodiment of the present application, step (2) further includes staining; preferably, Cy3-NHS, Cy5-NHS dyes are adopted.

[0028] The polystyrene microspheres have amino groups on the surface, and the Cy3 and Cy5 are modified to the surface of the microspheres through the click chemistry crosslinking of the amino group and NHS. Different proportions of Cy3 and Cy5 can distinguish the microspheres into different microsphere groups in the flow cytometry characterization, different microsphere groups load different MB probes, and the fluorescence coding microspheres detect different types of miRNA.

[0029] In a third aspect, the application protects the use of the above-mentioned coding microspheres or the coding microspheres prepared by the above-mentioned method in the simultaneous detection of multiple markers of extracellular vesicles.

[0030] In a fourth aspect, the application protects a method for simultaneously detecting multiple markers of extracellular vesicles, which adopts the above-mentioned coding microspheres or the coding microspheres prepared by the above-mentioned method.

[0031] According to the specific embodiment of the present application, specifically including: separating the EVs of cell line origin, and adding the coding microspheres for incubation. BRIEF DESCRIPTION OF DRAWINGS

[0032] The drawings accompanying the specification of the present application are used to provide further understanding of the present application, the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application.

[0033] Figure 1 is the size coding and fluorescence coding microsphere grouping condition of the present application;

[0034] Figure 2 is the liposome transmission electron microscopy (TEM) and particle size distribution and concentration chart of the present application;

[0035] Figure 3 is the scanning electron microscopy (SEM) of the mesoporous and size of the polystyrene microsphere surface of the present application;

[0036] Figure 4Figure 1 is a confocal image showing that the microspheres are wrapped by liposomes and the surface is successfully modified with hairpin probe II;

[0037] Figure 5 Figure 2 is a confocal image showing that the microspheres are wrapped by liposomes and the surface is successfully modified with hairpin probe II;

[0038] Figure 6 Figure 3 is a FRAP fluorescence recovery image showing that the lipid layer on the surface of the microspheres is successfully modified;

[0039] Figure 7 Figure 4 is a transmission electron microscope (TEM) image of the extracellular vesicles of the application, together with a particle size distribution and concentration chart;

[0040] Figure 8 Figure 5 is a fluorescence recovery image showing that the microspheres of the application fuse with the membrane of the extracellular vesicles;

[0041] Figure 9 Figure 6 is a confocal image showing that the microspheres of the application fuse with the membrane of the EVs;

[0042] Figure 10 Figure 7 is a cell flow cytometry image showing that the membrane fusion between the microspheres and the EVs is mediated by the aptamer and hairpin probe II;

[0043] Figure 11 Figure 8 is the specificity of the membrane fusion between the microspheres and the specific EVs subpopulation;

[0044] Figure 12 Figure 9 is the specificity of the membrane fusion between the double-positive recognition microspheres and the specific EVs subpopulation (EPCAM + PD-L1 + );

[0045] Figure 13 Figure 10 is the specificity of the membrane fusion between the EPCAM recognition microspheres and the specific EVs subpopulation (EPCAM + );

[0046] Figure 14 Figure 11 is a gel electrophoresis image showing the successful ligation of the double-positive aptamer;

[0047] Figure 15 Figure 12 is a cell flow cytometry image showing that the microspheres successfully detect the miRNA in the EVs;

[0048] Figure 16 Figure 13 is an image showing the difference in miRNA expression between normal ovarian cell lines and ovarian cancer cell lines.

[0049] Advantages of the application

[0050] (1) The microspheres are simple to modify, and do not require long incubation during the detection process;

[0051] (2) t-EVs high-throughput detection; flow cytometry as a high-throughput detection platform, t-EVs and microspheres membrane fusion to reach the cell level size, through the cell flow detection, to achieve the effect of t-EVs high-throughput detection.

[0052] (3) Biomarker accurate multiple detection of different t-EVs subgroups; flow cytometry as a platform for fluorescence and size co-detection, different size coding microspheres can represent different t-EVs subgroups, and different fluorescence coding microspheres can represent accurate multiple detection of different miRNAs.

[0053] (4) In situ detection of EVs-miRNA, miRNA is not interfered by other substances in the system. DETAILED DESCRIPTION

[0054] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0055] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and furthermore, it should be understood that when the terms "comprise" and / or "include" are used in the specification, there is a presence of the features, steps, operations, devices, components and / or combinations thereof.

[0056] The present application is further illustrated below in conjunction with examples.

[0057] Example 1

[0058] A method for preparing coding microspheres, comprising the following steps:

[0059] (1) Synthesis of liposome solution LNP: liposomes were prepared by thin film hydration method, DOPC, DOPE, cholesterol, DOPE-PEG2000 (molar ratio 50:24.5:25:0.5) were dissolved in chloroform, rotary evaporation to lipid film at the bottom of round bottom flask, hydration with DPBS (37℃, 1h), 200nm membrane extrusion 50 times, to form liposome solution, 4℃ storage, use within one week.

[0060] (2) Preparation of code-spheres PSNPS: 20 mL of aminated mesoporous polystyrene spheres (25 mg / mL) were incubated with different volume ratios of Cy3-NHS (10 mg / mL) and Cy5-NHS (10 mg / mL) dyes at 37°C for 2 h, with volume ratios of 15 mL:0 mL, 10 mL:5 mL, 7.5 mL:7.5 mL, 5 mL:10 mL and 0 mL:15 mL, respectively. The code-spheres were centrifuged three times (8000 rpm, 5 min) and washed three times with DPBS to remove excess dye.

[0061] (3) Internal loading and external modification of code-spheres: 5' end FAM and 3' end quencher modified MBs were loaded into the mesopores of code-spheres through a self-assembly process. After 50 mL of MBs (20 mM) were vortexed with 300 mL of PSNPS (20 mg / mL) for 5 min, 300 mL of LNP (2.5 mg / mL) was added, and the mixture was shaken gently at 37°C overnight. Excess MBs and LNP were removed by centrifugation three times (8000 rpm, 5 min), and the code-spheres were resuspended in DPBS. The modified cholesterol hairpin probe II 500 nM was added and shaken at 37°C for 1 h. The mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was discarded. The code-spheres were washed three times with DPBS and stored at 4°C for subsequent use.

[0062] (4) Isolation of EVs from cell lines: IOSE80, OVCAR-3, A2780, A375, U251 and MCF-7 cells were cultured in DMEM containing 10% FBS (EV-Free), 100 units / mL penicillin and 100 units / mL streptomycin, 5% CO2, 37°C. When the cell density reached 70%-80%, the cells were starved for 48 h in serum-free medium, and the cell supernatant was collected. The lower cell debris was removed by centrifugation at 3000 g for 20 min, and the supernatant was filtered through a 0.22 mm filter. EVs were isolated by centrifugation at 100,000 g for 2 h, resuspended in DPBS, and stored at -80°C for later use.

[0063] (4) miRNA detection in EVs: 10 mL of cell line EVs (1*10 9 particles / mL) + Extended 1 (500 nM) + Extended 2 (500 nM) + Linker 1 (750 nM) were reacted with T4 ligase at 16°C for 1 h, and then 1 uL of Linker2 was added and reacted at 237°C for 1 h. Subsequently, 10 mL of microspheres (1*10 7 particles / mL) were mixed and incubated at 37°C for 2 h. After dilution, flow cytometry analysis was performed, and the FAM intensity was analyzed using FlowJo software.

[0064] Effect test:

[0065] (1) Fluorescence recovery graph of LNP@PS prepared in Example 1 after photobleaching Figure 6 )

[0066] After incubating the liposome-encapsulated microspheres with Dil (membrane dye), drop them on a confocal dish and cover them with a cover glass. Using a confocal microscope, a 100x objective, and 1% laser power, measure the fluorescence intensity before photobleaching. Then, using full laser power, photobleach the edge area of the microspheres, and record the fluorescence intensity of the lipid layer for 80 s by continuous imaging. Define the photobleached area as the region of interest (ROI). Analyze the fluorescence recovery curve using Lecia software.

[0067] (2) Fluorescence recovery graph of EVs after membrane fusion prepared in Example 1 Figure 8 )

[0068] Stain 100 μL, 1*10 9 particle / ml of EVs with DiO (10 μM) and Dil (10 μM) at 37°C for 30 min, and then filter out the free dye using a 100 kDa centrifugal filter device (3000 g, 20 min). After mixing the double-stained EVs (100 μL, 1*10 9 particle / ml) with microspheres (100 μL, 1*10 7 particle / ml), incubate them at 37°C for 2 h, and then monitor the fluorescence recovery of DiO using a fluorescence spectrometer.

[0069] (3) Fluorescence diffusion confocal graph after membrane fusion prepared in Example 1 Figure 9 )

[0070] Stain 100 μL, 1*10 9 particle / ml of EVs with Dil (10 μM) at 37°C for 30 min, and then filter out the free dye using a 100 kDa centrifugal filter device (3000 g, 20 min). After mixing the single-stained EVs (100 μL, 1*10 9 particle / ml) with microspheres (100 μL, 1*10 7 particle / ml), incubate them at 37°C for 2 h. Take 10 μL of the mixture and drop it on a glass slide, and cover it with a cover glass. Under a 100x objective fluorescence microscope, take images of the microspheres before and after 2 h of incubation, respectively.

[0071] (4) Flow cytometry and fluorescence graph of membrane fusion combined with specific subpopulation prepared in Example 2 Figure 13 )

[0072] Stain 100 μL, 1*10 9particle / ml of MCF-7 EVs (EPCAM + ) and U251 EVs (EPCAM-) were filtered by 100 kDa centrifugal filter unit (3000g, 20min) to remove free dye after staining at 37℃ for 30min. Single stained EVs (100μL, 1*10 9 particle / ml) were mixed with EPCAM aptamer modified microspheres (100μL, 1*10 7 particle / ml), after membrane fusion at 37℃ for 2h, cell flow cytometry and fluorescence detection were performed, and the fusion was specific.

[0073] (5) Flow cytometry of different concentrations of miRNA in extracellular vesicles detected by microspheres prepared in Example 2 Figure 16

[0074] Take 10μL A375 EVs (EPCAM + PD-L1 + ) (1*10 9 particle / ml or 1*10 8 particle / ml) and 10μL double positive aptamer modified microspheres (internal wrapping of miR-21 hairpin probe I) (1*10 7 particle / ml) were mixed, and membrane fusion was carried out at 37℃ for 2h. After dilution, flow cytometry analysis was performed, and FAM intensity was analyzed using FlowJo software.

[0075] (6) Detection of different subgroups of extracellular vesicles from different cell lines by microspheres prepared in Example 2

[0076] A variety of fluorescent encoding microspheres were synthesized by modifying different proportions of Cy3 and Cy5 on the surface of the microspheres, and different hairpin probes I (MB-21, MB-141, MB-200c, MB-200b, MB-1246, MB-30d-5p, MB-99a-5P, MB-145, MB-1290, MB-194, specific sequences see Table 1 SEQ ID No. 1-SEQ ID No. 11) were wrapped inside to detect different miRNAs. Take 10μL IOSE80 (human normal ovarian cells), A2780 (human ovarian cancer cells), OVCAR-3 (human ovarian cancer cells) EVs (1*10 9 particle / ml) and 10μL encoding microspheres (1*10 7 particle / ml) were mixed, and membrane fusion was carried out at 37℃ for 2h. After dilution, flow cytometry analysis was performed, and FAM intensity was analyzed using FlowJo software. ​

[0077] Table 1

[0078]

[0079]

[0080] It is also possible to make various changes in the application without departing from the spirit and scope of the application. Accordingly, the application is not limited to the embodiments described herein, but includes all changes and modifications that fall within the scope and spirit of the application.

Claims

1. An encoded microsphere, characterized in that, The raw material of the encoding microsphere comprises: polystyrene microspheres, liposomes, nucleic acid aptamer, hairpin probe I, hairpin probe II; The hairpin probe I is used for detecting miRNA; The nucleic acid aptamer is used for specifically recognizing the protein on the surface of EVs; The hairpin probe II is used for specifically binding t-EVs subpopulation; The encoding microsphere is polystyrene microsphere wrapped with liposomes, and the surface is modified with hairpin probe II, and the inside is wrapped with hairpin probe I; The hairpin probe I comprises MB; the nucleotide sequence of the MB comprises: FAM-CCGCG-complementary sequence of target miRNA-CGCGG-BHQ1; The hairpin probe II is a hairpin probe modified with cholesterol; The hairpin probe II comprises a sequence segment complementary to the nucleic acid aptamer.

2. A method of preparing the encoded microspheres of claim 1, wherein, The method comprises the following steps: (1) preparation of a liposome solution; (2) liposome wrapping of the encoding microsphere; (3) internal loading of the encoding microsphere; (4) external modification of the encoding microsphere.

3. The preparation method according to claim 2, characterized in that, The process of step (1) adopts a thin film hydration method to prepare the liposome solution.

4. The production method according to claim 3, characterized by, The process of step (1) specifically comprises: dissolving dioleoyl lecithin DOPC, dioleoyl phosphatidyl ethanolamine DOPE, cholesterol and dioleoyl phosphatidyl ethanolamine-polyethylene glycol 2000 in chloroform, drying or evaporating into a lipid film, dissolving with DPBS to form a liposome solution.

5. The preparation method according to claim 2, characterized in that, The process of step (2) is completed by self-assembly, and specifically comprises that the hairpin probe modified with FAM at the 5' end and quencher BHQ1 at the 3' end is loaded into the mesopore of the encoding sphere through the self-assembly process.

6. The production method according to any one of claims 2 to 5, characterized by, Before step (2), the process further comprises dyeing.

7. The production method according to claim 6, wherein The dyeing adopts Cy3-NHS and Cy5-NHS dyes.

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