Coding microsphere and application thereof in simultaneous detection of various markers of extracellular vesicles

Abstract

The invention belongs to the technical field of synthesis and detection, and particularly relates to a coding microsphere and application thereof in simultaneous detection of various markers of extracellular vesicles. The coding microsphere is prepared from the following raw materials: polystyrene microspheres, lipidosome, a nucleic acid aptamer, a hairpin probe I and a hairpin probe II, the nucleic acid aptamer can specifically recognize protein on the surface of EVs; the hairpin probe I is used for detecting miRNA; and the hairpin probe II is used for combining different t-EVs subgroups. By combining the flow cytometry technology, a fluorescence and size combined coding signal and a report signal can be displayed at the same time, decoding and quantification of miRNA are performed respectively, multiple detection of tumor-derived extracellular vesicles is realized, and a new way is provided for diagnosis and screening of cancers.

Description

Technical Field

[0001] The present invention belongs to the field of synthesis and detection technology, and specifically relates to a coding microsphere and an application thereof in the simultaneous detection of multiple types of extracellular vesicle markers. Background Art

[0002] Tumor cell-derived extracellular vesicles (t-EVs) are nanoscale vesicles secreted by tumor cells that can mediate communication between tumor cells. The expression levels of biological molecules such as membrane proteins and internal nucleic acids 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) that play an important role in gene expression regulation by inhibiting the translation of target genes or degrading the transcripts of target genes. Abnormally expressed miRNAs are closely related to the pathogenesis of various cancers. Given that multiple miRNAs are involved in the occurrence and progression of ovarian cancer, multiplex analysis of t-EVs-miRNAs is expected to improve the accuracy and reliability of cancer diagnosis.

[0004] Membrane proteins are important components of t-EVs. They mediate the recognition process between t-EVs and target cells, participate in regulating the occurrence and development of various diseases such as tumors, immunity, and neurodegeneration, and can be used as disease diagnostic markers. However, due to differences in membrane protein expression, different EVs subpopulations play different biological functions. By detecting the miRNA expression of multiple t-EVs subpopulations and drawing miRNA maps, the accuracy of cancer diagnosis can be effectively improved.

[0005] Existing t-EVs-miRNA analysis usually requires lysing EVs to extract the internal miRNA, which is cumbersome and takes a long time to analyze, which can easily lead to miRNA degradation, thus affecting the accuracy and reliability of the test results. In addition, due to the limitation of fluorescence spectrum overlap, fluorescence analysis can only perform 3-4 colors of imaging at the same time, which limits the types of miRNA detection and is not conducive to multiple detection of miRNA.

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

[0007] In view of the problems existing in the current detection of biomarkers of extracellular vesicles, the present invention provides a coding microsphere and its application in the simultaneous detection of multiple types of extracellular vesicle markers. The coding microsphere comprises a polystyrene microsphere surface coated with liposomes to form a lipid layer, and then a hairpin probe II is modified to the surface of the microsphere by cholesterol insertion, the hairpin probe II has a complementary sequence of a nucleic acid aptamer, and the hairpin probe I is wrapped inside the microsphere mesopore.

[0008] The technical solution of the present invention is as follows:

[0009] In the first aspect, a coding microsphere is protected, wherein the raw materials include: polystyrene microspheres, liposomes, nucleic acid aptamers, hairpin probes I and II;

[0010] The hairpin probe I is used to detect miRNA;

[0011] The nucleic acid aptamer is used to specifically recognize proteins on the surface of EVs;

[0012] The hairpin probe II is used to specifically bind to a subpopulation of t-EVs.

[0013] EVs were first incubated with specific nucleic acid aptamers (Extended 1, Extended 2, see SEQ ID No. 15-SEQ ID No. 16 in Table 1 for specific sequences), and these EVs were labeled with aptamer recognition sequences so that the surface hairpin probes II (PS1, PS2, PS 3, see SEQ ID No. 12-SEQ ID No. 14 in Table 1 for specific sequences) on different-sized encoded microspheres could recognize the aptamer tags for specific capture and detection. In order to detect EVs expressing two t-EV biomarkers, proximity ligation (PLA) was used to create a sequence that could be recognized by a specific hairpin probe (PS 3). In order to effectively achieve recognition and binding of the hairpin probes, the linker sequence needs to be exposed, so a single DNA strand (Linker 2, see SEQ ID No. 18 in Table 1 for specific sequences) was introduced, which was complementary to Linker 1 (see SEQ ID No. 17 in Table 1 for specific sequences) to initiate the ligation reaction, thereby achieving recognition and detection of dual-expressing EVs.

[0014] When the hairpin probe modified on the surface of the microsphere specifically recognizes the aptamer on the surface of EVs, the distance between the microsphere and EVs is shortened, guiding the microsphere to fuse with the corresponding EVs, and the miRNA inside the EVs enters the mesopores inside the microspheres. When the hairpin probe in the mesopore detects the corresponding miRNA, the fluorescence is restored, and the miRNA in the EVs is quantified according to the fluorescence intensity. Different hairpin probes II are modified on the surface of microspheres of different sizes to bind to different EVs subpopulations, and different hairpin probes I are encapsulated inside different fluorescence-encoded microspheres to detect different miRNAs. Combined with flow cytometry technology, fluorescence and size combination encoding signals and reporter signals can be displayed simultaneously, and miRNA decoding and quantification can be performed respectively, realizing a multiplex detection of tumor-derived extracellular vesicles, providing a new way for the diagnosis and screening of cancer.

[0015] According to a specific embodiment of the present application, the coding microsphere is a polystyrene microsphere with liposomes coated on the surface, the surface is modified with hairpin probe II, and the interior is coated with hairpin probe I.

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

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

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

[0019] In a second aspect, a method for preparing the above-mentioned encoded microspheres is provided, comprising the following steps:

[0020] (1) Preparation of liposomes;

[0021] (2) liposomal encapsulation of encoded microspheres;

[0022] (3) Internal loading of coded microspheres;

[0023] (4) External modification of encoded microspheres.

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

[0025] According to a specific embodiment of the present application, the process of step (2) is completed by self-assembly, specifically comprising loading the hairpin probe modified with FAM at the 5' end and quencher at the 3' end into the mesopore of the coding ball through a self-assembly process.

[0026] According to the specific implementation of the present application, the step (3) adopts a shaking incubation method, which specifically includes dissolving the microspheres of step (2) in DPBS, taking 500nM of the modified cholesterol hairpin probe II, shaking at 37 degrees for 1 hour, 8000rpm, 5min, washing three times, dissolving in DPBS, and storing at 4 degrees for subsequent use.

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

[0028] The surface of polystyrene microspheres has amino groups. Cy3 and Cy5 are modified to the surface of microspheres through click chemical cross-linking of amino groups and NHS. Different proportions of Cy3 and Cy5 can distinguish the microspheres into different microsphere groups in flow cytometry characterization. Different microsphere groups are loaded with different MB probes, so that fluorescently encoded microspheres can detect different types of miRNA.

[0029] The third aspect is to protect the use of the above-mentioned encoded microspheres or the encoded microspheres prepared by the above-mentioned method in the simultaneous detection of multiple types of extracellular vesicle markers.

[0030] In a fourth aspect, a method for simultaneously detecting multiple types of extracellular vesicle markers is provided, using the above-mentioned encoded microspheres or the encoded microspheres prepared by the above-mentioned method;

[0031] According to the specific implementation of the present application, it specifically includes: isolating EVs from cell lines, adding encoded microspheres and incubating. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0033] Figure 1 It is the size-coded and fluorescence-coded microsphere grouping situation of the present invention;

[0034] Figure 2 It is a transmission electron microscope (TEM) image of the liposome of the present invention and a particle size distribution and concentration image;

[0035] Figure 3 is a scanning electron microscope (SEM) image of the surface mesopores and sizes of the polystyrene microspheres of the present invention;

[0036] Figure 4This is DLS verification of liposome-encapsulated microspheres and successful surface modification of hairpin probes II;

[0037] Figure 5 Figure Ⅱ is the confocal verification of the internal loading of MB and surface modified liposomes and hairpin probes in microspheres;

[0038] Figure 6 This is the confocal verification FRAP fluorescence recovery image of the lipid layer on the surface of the microsphere;

[0039] Figure 7 It is a transmission electron microscopy (TEM) image of the extracellular vesicles of the present invention and a particle size distribution and concentration image;

[0040] Figure 8 is a fluorescence recovery image of the fusion of the microspheres of the present invention with the extracellular vesicle membrane;

[0041] Fig. 9 It is a confocal image of membrane fusion between the microspheres of the present invention and EVs;

[0042] Fig.10 The cell flow cytometry of the present invention verifies that the membrane fusion of microspheres and EVs is mediated by nucleic acid aptamers and hairpin probe II;

[0043] Fig.11 It is the specificity of membrane fusion between the microspheres of the present invention and specific EVs subpopulations;

[0044] Fig.12 The double positive recognition microspheres of the present invention and specific EVs subpopulations (EPCAM + PD-L1 + )Specificity of membrane fusion;

[0045] Fig.13 The EPCAM recognition microspheres of the present invention and specific EVs subpopulations (EPCAM + )Specificity of membrane fusion;

[0046] Fig.14 The successful connection of the double positive nucleic acid aptamer was verified by gel electrophoresis of the present invention;

[0047] Fig.15 The cell flow cytometry verification microspheres of the present invention successfully detect miRNA in EVs;

[0048] Fig.16 The present invention verifies the difference in miRNA expression between normal ovarian cell lines and ovarian cancer cell lines.

[0049] Beneficial Effects of the Invention

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

[0051] (2) High-throughput detection of t-EVs: Flow cytometry is used as a high-throughput detection platform. After t-EVs fuse with the microsphere membrane, they reach the cell-level size. Through cell flow cytometry detection, the effect of high-throughput detection of t-EVs can be achieved.

[0052] (3) Accurate multiplex detection of biomarkers of different t-EVs subpopulations; flow cytometry is used as a platform for co-detection of fluorescence and size. Different size-coded microspheres can represent different t-EVs subpopulations, and different fluorescence-coded microspheres can represent accurate multiplex 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 descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

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

[0056] The present invention is further described below with reference to the embodiments.

[0057] Example 1

[0058] A method for preparing coded microspheres comprises the following steps:

[0059] (1) Synthesis of liposome solution LNP: Liposomes were prepared by thin film hydration method. DOPC, DOPE, cholesterol, and DOPE-PEG2000 (molar ratio 50:24.5:25:0.5) were dissolved in chloroform and rotary evaporated at the bottom of a round-bottom flask to form a lipid film. The film was hydrated with DPBS (37°C, 1 h) and extruded through a 200 nm membrane 50 times to form a liposome solution. The solution was stored at 4°C and used within one week.

[0060] (2) Preparation of coded sphere 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 the volume ratios being 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 coded 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 the coding sphere: MB modified with FAM at the 5' end and quencher at the 3' end was loaded into the mesopores of the coding sphere through a self-assembly process. 50 mL MB (20 mM) + 300 mL LSNPS (20 mg / ml) was vortexed for 5 min, and then 300 mL LNP (2.5 mg / ml) was added. The mixture was gently shaken at 37 degrees overnight, and then centrifuged to remove excess MB and LNP. The mixture was centrifuged three times (8000 rpm, 5 min), and then redissolved in DPBS. 500 nM of the cholesterol-modified hairpin probe II was taken, shaken at 37 degrees for 1 hour, 8000 rpm, 5 min, washed three times, redissolved in DPBS, and stored at 4 degrees for subsequent use.

[0062] (4) Isolation of cell line-derived EVs: 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 under 5% CO. 2 , 37°C. When the cell growth density reached 70%-80%, the cells were starved, and the cell supernatant was collected after 48 hours of culture in serum-free medium. The cell debris in the lower layer was discarded by centrifugation at 3000g for 20min, and the supernatant was filtered through a 0.22mm filter membrane, centrifuged at 100000g for 2h to separate EVs, resuspended in DPBS, and stored at -80°C for later use.

[0063] (4) Detection of miRNA in EVs: Take 10 mL of cell line EVs (1*10 9 After 1 hour of reaction at 16℃ with T4 ligase, 1uM Linker2 was added and reacted at 37℃ for 1 hour. Then, 10mL microspheres (1*10 7 The mixture was mixed with 100 μl of PBS and incubated at 37°C for 2 h. After dilution, the samples were analyzed by flow cytometry, and the FAM intensity was analyzed using FlowJo software.

[0064] Effect test:

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

[0066] Liposome-encapsulated microspheres were incubated with Dil (membrane dye) and then dropped onto a confocal dish and covered with a coverslip. The fluorescence intensity before photobleaching was measured using a confocal microscope, a 100x objective, and 1% laser power. The edge area of ​​the microspheres was then photobleached using full laser power, and the fluorescence intensity of the lipid layer was recorded for 80 seconds by continuous imaging. The photobleached area was defined as the region of interest (ROI). The fluorescence recovery curve was analyzed using Lecia software.

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

[0068] 100 μL, 1*10 9 After staining at 37°C for 30 min, free dye was removed by filtration using a 100 kDa centrifugal filter (3000 g, 20 min). 9 particle / ml) and microspheres (100μL, 1*10 7 After mixing, the mixture was incubated at 37°C for 2 h and the fluorescence recovery of DiO was monitored using a fluorescence spectrometer.

[0069] (3) Fluorescence diffusion confocal image after membrane fusion prepared in Example 1 ( Fig. 9 )

[0070] 100 μL, 1*10 9 After staining EVs with 100 μL and 100 μL of EVs at 37°C for 30 min, free dye was removed by filtration using a 100 kDa centrifugal filter (3000 g, 20 min). 9 particle / ml) and microspheres (100μL, 1*10 7 After mixing, incubate at 37°C for 2 hours. Take 10ul of the mixed solution and drop it on a glass slide, cover it with a cover glass. Under a fluorescence microscope with a 100x objective lens, take images of the microspheres before and after 2 hours of incubation.

[0071] (4) Flow cytometry and fluorescence images of membrane fusion-binding specific subpopulations prepared in Example 2 ( Fig.13 )

[0072] DiO (10 μM) was used to treat 100 μL, 1*109 particles / ml of MCF-7EVs (EPCAM + ) and U251 EVs (EPCAM-) were stained at 37°C for 30 min, and then filtered using a 100 kDa centrifugal filter (3000 g, 20 min) to remove free dye. 9 particle / ml) and EPCAM nucleic acid aptamer modified microspheres (100μL, 1*10 7 After mixing with 50 μg / ml, the membranes were fused at 37°C for 2 h, and then cell flow cytometry and fluorescence detection were performed to show that the fusion was specific.

[0073] (5) Flow cytometry of the encoded microspheres prepared in Example 2 to detect miRNA in extracellular vesicles of different concentrations ( Fig.16 )

[0074] Take 10 μL A375 EVs (EPCAM + PD-L1 + )(1*10 9 particle / ml or 1*10 8 particle / ml) and 10 μL of double positive aptamer modified microspheres (with miR-21 hairpin probe I encapsulated inside) (1*10 7 The mixture was mixed with 50 μl of 4% paraformaldehyde (particles / ml) and membrane fusion was carried out at 37°C for 2 h. After dilution, the samples were analyzed by flow cytometry, and the FAM intensity was analyzed using FlowJo software.

[0075] (6) Detection of multiple miRNAs in different subpopulations of extracellular vesicles from different cell lines using the encoded microspheres prepared in Example 2

[0076] A variety of fluorescent coded microspheres were synthesized by modifying the surface of the microspheres with different ratios of Cy3 and Cy5, 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, the specific sequences are shown in SEQ ID No.1-SEQ ID No.11 in Table 1) were wrapped inside to detect different miRNAs. 10 μL IOSE80 (human normal ovarian cells), A2780 (human ovarian cancer cells), OVCAR-3 (human ovarian cancer cells) EVs (1*10 9 particle / ml) and 10μL coded microspheres (1*10 7 The mixture was mixed with 50 μl of 4% paraformaldehyde (particles / ml) and membrane fusion was carried out at 37°C for 2 h. After dilution, the samples were analyzed by flow cytometry, and the FAM intensity was analyzed using FlowJo software.

[0077] Table 1

[0078]

[0079]

[0080] The present invention is only an embodiment and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A coded microsphere, characterized in that: The raw materials include: polystyrene microspheres, liposomes, nucleic acid aptamers, hairpin probe I, and hairpin probe II; The hairpin probe I is used to detect miRNA; The nucleic acid aptamer is used to specifically recognize proteins on the surface of EVs; The hairpin probe II is used to specifically bind to a subpopulation of t-EVs.

2. The coded microsphere according to claim 1, characterized in that: The coding microsphere is a polystyrene microsphere with liposomes wrapped on the surface, the surface is modified with hairpin probe II, and the inside is wrapped with hairpin probe I; And / or, the hairpin probe I includes MB; the nucleotide sequence of the MB includes: FAM-CCGCG-complementary sequence of the target miRNA-CGCGG-BHQ1.

3. The coded microsphere according to claim 1 or 2, characterized in that: The hairpin probe II is a cholesterol-modified hairpin probe; And / or, the hairpin probe II includes a sequence segment complementary to the nucleic acid aptamer.

4. A method for preparing the coded microspheres according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of liposomes; (2) liposomal encapsulation of encoded microspheres; (3) Internal loading of coded microspheres; (4) External modification of encoded microspheres.

5. The preparation method according to claim 4, characterized in that: The process of step (1) adopts a thin film hydration method to prepare liposomes; preferably, it specifically includes: dissolving dioleoylphosphatidylcholine DOPC, dioleoylphosphatidylethanolamine DOPE, cholesterol, and dioleoylphosphatidylethanolamine-polyethylene glycol 2000 in chloroform, drying or evaporating to form a lipid film, and dissolving with DPBS to form a liposome solution.

6. The preparation method according to claim 4, characterized in that: The process of step (2) is completed by self-assembly, specifically comprising loading the hairpin probe modified with FAM at the 5' end and quencher at the 3' end into the mesopore of the coding ball through the self-assembly process.

7. The preparation method according to claim 4, characterized in that: The step (3) adopts a shaking incubation method, specifically comprising re-dissolving the microspheres of step (2) in DPBS, taking 500 nM of the cholesterol-modified hairpin probe II, shaking at 37 degrees for 1 hour, 8000 rpm, 5 minutes, washing three times, re-dissolving in DPBS, and storing at 4 degrees for subsequent use; Preferably, a cholesterol-modified hairpin probe II is also included.

8. The preparation method according to claim 4, characterized in that: The step (2) also includes dyeing; preferably, Cy3-NHS or Cy5-NHS dyes are used.

9. Use of the encoded microspheres described in any one of claims 1 to 3 or the encoded microspheres prepared by the method described in any one of claims 4 to 8 for the simultaneous detection of multiple types of extracellular vesicle markers.

10. A method for simultaneously detecting multiple types of extracellular vesicle markers, characterized in that: An encoded microsphere prepared by the encoded microsphere described in any one of claims 1 to 3 or the method described in any one of claims 4 to 8; Preferably, the method specifically comprises: isolating EVs from cell lines, adding the encoded microspheres described in any one of claims 1 to 3 or the encoded microspheres prepared by the method described in any one of claims 4 to 8, and incubating.

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