A method for detecting extracellular vesicles and subtypes thereof in a separation-free system and applications thereof

Abstract

The present application relates to a kind of detection method and application of extracellular vesicle and its subtype in no separation system, the present application provides a detection probe, it includes probe I, probe II and probe III;One end of the probe I is labeled with biotin, and the other end is labeled with first recognition unit;One end of the probe II is labeled with fluorescent dye;One end of the probe III is labeled with second recognition unit;The probe I is partially paired with the probe II, so that the biotin and the fluorescent dye are close to each other;The probe I, the probe II is partially paired with the probe III respectively, so that the probe I, probe II and probe III are hybridized to form T type structure under the condition that target exists.The present application also provides corresponding detection method, can realize the detection of extracellular vesicle in no separation system, with the advantages such as simple operation, rapid detection, high sensitivity, not susceptible to fluorescence intensity fluctuation, fluorescence bleaching and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of extracellular vesicle analysis detection, and particularly relates to a detection method of extracellular vesicles and subtypes thereof in a separation-free system and application. BACKGROUND

[0002] Extracellular vesicles (EVs) are a class of extracellular vesicles with lipid bilayer membrane structure secreted by most living cells, containing cell-specific proteins, lipids and nucleic acids, which can be transmitted to other cells as signal molecules to affect the functions of other cells. Among extracellular vesicles, exosomes with smaller size are studied more. Studies have found that extracellular vesicles can reflect tumor progression in real time. The specific biomarkers (such as proteins, lipids and nucleic acids) of tumor EVs all have the characteristics of tumor cells, and EVs produced by different organs can be used as molecular markers for tumor diagnosis. Due to the heterogeneity of tumors, patients may have the same pathological changes, but may be caused by completely different genetic changes, so precise diagnosis and individualized treatment of tumors are more important. As a real-time dynamic reflection of tumor state, tumor extracellular vesicles also have a wide application prospect in precision medicine.

[0003] Extracellular vesicles contained in body fluids are a collection of EVs produced by different organs and cells, most of which are derived from blood or normal cells, and the number of tumor-related EVs is very small. How to distinguish and detect tumor-related EVs is particularly important. Studies have shown that to meet the accuracy of tumor-related EV subtype detection, a single target is often not enough, and an EV needs to have two or more targets expressed simultaneously to make a more accurate diagnosis of tumors (Nature Communications, 2019, 10, 3854). This poses a challenge to the development of methods for detecting EV subtypes that can detect multiple targets expressed simultaneously.

[0004] Currently, the detection of EVs subtypes in body fluids is mainly through the separation and enrichment of the capture, and then the interaction with multiple fluorescently labeled target antibodies / aptamers, and the co-localization detection under fluorescence microscope. These methods need to separate EVs, which is time-consuming, inconvenient, easy to cause loss and pollution of EVs, and is not conducive to the routine development in clinic. Based on this, in recent years, the field has begun to develop EVs subtype detection methods without separation extraction, such as thermal swimming enrichment, logic gate operation, joint amplification, etc. These methods hope to realize the detection of EVs subtypes without separation extraction. However, due to the complexity of body fluids, there are few non-separation methods that can be used for clinical body fluid samples, or the sensitivity is not high, or it needs to rely on expensive instruments such as digital PCR to obtain satisfactory sensitivity. Therefore, it is of great significance to develop a simple, rapid, high-sensitivity, inexpensive, non-analytical extraction, and easy-to-produce clinical detection kit for EVs subtype detection method in clinical diagnosis. SUMMARY

[0005] The purpose of the present application is to provide a detection method and application of extracellular vesicles and its subtypes in a non-separation system. The detection method provided by the present application can realize the detection of extracellular vesicles based on fluorescence polarization technology in a non-separation system, and has the advantages of simple operation, rapid detection, high sensitivity, and being not easily affected by fluorescence intensity fluctuation and fluorescence bleaching.

[0006] To this end, in a first aspect, the present application provides a detection probe, which comprises probe I, probe II and probe III; one end of the probe I is labeled with biotin, and the other end is labeled with a first recognition unit; one end of the probe II is labeled with a fluorescent dye; one end of the probe III is labeled with a second recognition unit;

[0007] The probe I and the probe II are partially paired, so that the biotin and the fluorescent dye are close to each other; under the condition that the target exists, the probe I and the probe II are partially paired with the probe III through joint amplification, so that the probe I, the probe II and the probe III hybridize to form a T-shaped structure;

[0008] The first recognition unit specifically recognizes cell membrane, first target protein, first target polysaccharide or first target lipid; and the second recognition unit specifically recognizes cell membrane, second target protein, second target polysaccharide or second target lipid.

[0009] Further, the first recognition unit is cholesterol, a first nucleic acid aptamer or a first antibody.

[0010] Further, the second recognition unit is cholesterol, a second nucleic acid aptamer or a second antibody.

[0011] Further, the probe I, the probe II, and the probe III are single-stranded nucleotides.

[0012] Further, the probe I, the probe II, and the probe III are each independently selected from single-stranded deoxyribonucleotides (ssDNA), single-stranded ribonucleotides (ssRNA), hybrid single-stranded nucleotides of deoxyribonucleotides and ribonucleotides (hybrid single-stranded nucleotides of ssDNA and ssRNA).

[0013] In some embodiments, the probe I, the probe II, and the probe III are single-stranded deoxyribonucleotides.

[0014] Further, in the probe I, one end marked with the first recognition unit has a first non-paired sequence, which cannot be complementarily paired with other sequences in the detection probe.

[0015] Further, in the probe III, one end marked with the second recognition unit has a second non-paired sequence, which cannot be complementarily paired with other sequences in the detection probe.

[0016] Further, the first non-paired sequence and the second non-paired sequence are each independently selected from 0-80 bp, such as 1 bp, 5 bp, 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, etc.

[0017] In some embodiments, the first non-paired sequence and the second non-paired sequence are each independently selected from oligo-thymine nucleotides (oligo-T), oligo-adenine nucleotides (oligo-A), oligo-cytosine nucleotides (oligo-C), oligo-guanine nucleotides (oligo-G), and oligo-uracil nucleotides (oligo-U).

[0018] According to the technical solution of the present application, the T-shaped structure formed by hybridization of the probe I, the probe II, and the probe III is unstable under the temperature condition of the detection system in the absence of the extracellular vesicle target. For example, the Tm value of the partial pairing of the probe I and the probe II, the Tm value of the partial pairing of the probe I and the probe III, and the Tm value of the partial pairing of the probe II and the probe III are all less than the temperature of the detection system. Only in the presence of the extracellular vesicle target, the T-shaped structure formed by hybridization of the probe I, the probe II, and the probe III can stably exist under the temperature condition of the detection system through the combined action. In the case of meeting this condition, there is no special requirement for the sequence length of the partial pairing of the probe I and the probe II, the sequence length of the partial pairing of the probe I and the probe III, and the sequence length of the partial pairing of the probe II and the probe III.

[0019] In some embodiments, the length of the partially paired sequence of the probe I and the probe II, the length of the partially paired sequence of the probe I and the probe III, and the length of the partially paired sequence of the probe II and the probe III are each independently selected from 5-30 bp, such as 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 12 bp, 15 bp, 18 bp, 20 bp, 22 bp, 25 bp, 28 bp, 30 bp, etc.

[0020] In some embodiments, the probe I comprises, in order from 5' end to 3' end: biotin, a sequence that pairs with the probe II, a sequence that pairs with the probe III, an optional first non-paired sequence, a first recognition unit;

[0021] The probe II comprises, in order from 5' end to 3' end: a sequence that pairs with the probe III, a sequence that pairs with the probe I, a fluorescent dye;

[0022] The probe III comprises, in order from 5' end to 3' end: a sequence that pairs with the probe I, a sequence that pairs with the probe II, an optional second non-paired sequence, a second recognition unit.

[0023] In another embodiment, the probe I comprises, in order from 3' end to 5' end: biotin, a sequence that pairs with the probe II, a sequence that pairs with the probe III, an optional first non-paired sequence, a first recognition unit;

[0024] The probe II comprises, in order from 3' end to 5' end: a sequence that pairs with the probe III, a sequence that pairs with the probe I, a fluorescent dye;

[0025] The probe III comprises, in order from 3' end to 5' end: a sequence that pairs with the probe I, a sequence that pairs with the probe II, an optional second non-paired sequence, a second recognition unit.

[0026] In some embodiments, the probe I is: biotin-GGAGTGATCTCAGTGAC-(T) n - AptX (5' to 3' direction); wherein, n = 0-80, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, etc.; AptX is a cholesterol, a first nucleic acid aptamer, or a first antibody;

[0027] The probe II is: TCTAACGTCGTCACTCC-fluorescent dye (5' to 3' direction);

[0028] The probe III is: GTCACTGAGACGACGTTAGA-(T) m-AptY (5' to 3' direction); wherein, m = 0-80, such as 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, etc.; AptY is cholesterol, a second aptamer or a second antibody.

[0029] Further, the fluorescent dye is tetramethylrhodamine (TMR), Texas Red or fluorescein (FAM).

[0030] In a second aspect of the present application, a detection system is provided, which comprises the detection probe of the first aspect of the present application and a buffer.

[0031] Further, the detection system further comprises streptavidin.

[0032] Further, the buffer comprises PBS buffer, 1-2 mM Mg 2+ .

[0033] In some embodiments, the buffer comprises: PBS buffer, 1-2 mM Mg 2+ , pH 7-7.5.

[0034] Further, in the detection system, the concentration of the detection probe is 0.5-5 nM, such as 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, etc.

[0035] Further, in the detection system, the molar ratio of the probe I, the probe II and the probe III is 1-1.2:1-1.2:1-1.2; such as 1-1.1:1-1.1:1-1.1, preferably 1:1:1.

[0036] Further, in the detection system, the concentration of streptavidin is 2-20 μg / mL; such as 2 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, etc.

[0037] In a third aspect of the present application, a kit is provided, which comprises the detection probe of the first aspect of the present application and / or the detection system of the second aspect of the present application.

[0038] In a fourth aspect of the present application, the use of the detection probe, the detection system or the kit in (a), (b), (c) is provided:

[0039] (a) detecting total extracellular vesicles;

[0040] (b) detecting extracellular vesicle subtypes by targeting a target protein;

[0041] (c) detecting a subtype of extracellular vesicles by simultaneously targeting two target proteins.

[0042] Further, the detected cells are from samples such as cell culture medium, urine, plasma, tissue fluid, etc.

[0043] According to the technical solution of the present application, the use is for non-disease diagnosis and treatment purposes.

[0044] In a fifth aspect, the present application provides a method for detecting total extracellular vesicles, comprising providing the detection probe as described in the present application, incubating the detection probe with a sample to be detected, and determining the fluorescence anisotropy change value and / or the fluorescence polarization change value.

[0045] In some embodiments, the probe I and the probe III are both labeled with cholesterol.

[0046] Further, between the incubation step and the determination step, the following step is further included: adding streptavidin for reaction.

[0047] Further, the incubation time is 20-90 min, such as 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc.

[0048] Further, the incubation temperature is 20-37℃, such as 20℃, 25℃, 30℃, 35℃, 37℃, etc. In some embodiments, the incubation is performed at room temperature.

[0049] Further, the time for adding streptavidin for reaction is 5-30 min; such as 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, etc.

[0050] In a sixth aspect, the present application provides a method for detecting a subtype of extracellular vesicles, comprising providing the detection probe as described in the present application, incubating the detection probe with a sample to be detected, and determining the fluorescence anisotropy change value and / or the fluorescence polarization change value.

[0051] In some embodiments, one of the probe I and the probe III is labeled with cholesterol, and the other is labeled with an aptamer or an antibody; the aptamer or the antibody specifically binds to one target protein of the subtype; or,

[0052] The probe I is labeled with a first aptamer or a first antibody, and the probe III is labeled with a second aptamer or a second antibody; the first aptamer / first antibody and the second aptamer / second antibody specifically bind to two target proteins of the subtype, respectively.

[0053] Further, the incubation step and the determination step further comprise the following step: adding streptavidin to react.

[0054] Further, the incubation time is 20-90min, such as 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, etc.

[0055] Further, the incubation temperature is 20-37℃, such as 20℃, 25℃, 30℃, 35℃, 37℃, etc. In some embodiments, the incubation is at room temperature.

[0056] Further, the time for adding streptavidin to react is 5-30min; such as 5min, 10min, 15min, 20min, 25min, 30min, etc.

[0057] According to the technical solution of the present application, the detection method is not for disease diagnosis and treatment purposes.

[0058] Compared with the prior art, the present application has the following significant progress:

[0059] (1) The detection probe provided by the present application can realize the detection of extracellular vesicles based on fluorescence polarization technology in a non-separation system, including the detection of total extracellular vesicles, the detection of extracellular vesicle subtypes expressing a certain specific membrane protein, and the detection of extracellular vesicle subtypes expressing two specific membrane proteins at the same time.

[0060] (2) The detection probe provided by the present application can be used not only for extracellular vesicle typing detection, but also for other bacterial, viral and micro-nano biological particle detection, and has a wide application prospect.

[0061] (3) The detection method provided by the present application does not need to separate EVs, and has the advantages of simple operation, rapid detection, high sensitivity, not easy to be affected by fluorescence intensity fluctuation and fluorescence bleaching, etc. Compared with other various signal amplification detection methods for EVs, the operation steps are simple, and only simple sample mixing is needed for determination, without separation, washing, and without the need for enzymes and nanomaterials for signal amplification, and the cost is low.

[0062] (4) The detection method provided by the present application still has high sensitivity and high specificity when used for the detection of complex clinical samples, and has a good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0063] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of preferred embodiments and are not meant to limit the present application. In the drawings:

[0064] Figure 1 Figure 2 shows the schematic diagram of the detection principle of extracellular vesicles and its subtypes in the separation-free system according to the embodiments of the present application; (A) is for detecting the total amount of EVs; (B) is for detecting EVs expressing specific proteins; (C) is for detecting EVs subtypes expressing two proteins simultaneously.

[0065] Figure 2 Figure 3 shows the working curve of detecting total extracellular vesicles by fluorescence polarization technology according to the embodiments of the present application;

[0066] Figure 3 Figure 4 shows the working curve of detecting EVs expressing specific proteins by fluorescence polarization technology according to the embodiments of the present application;

[0067] Figure 4 Figure 5 shows the working curve of detecting EVs subtypes expressing two specific proteins simultaneously by fluorescence polarization technology according to the embodiments of the present application;

[0068] Figure 5 Figure 6 shows the detection results of multiple EVs subtypes in EVs of different cell lines by fluorescence polarization technology according to the embodiments of the present application;

[0069] Figure 6 Figure 7 shows the detection results of different EVs subtypes in clinical plasma samples by fluorescence polarization technology according to the embodiments of the present application; (A) is for EVs subpopulation expressing CD63 protein and EpCAM protein simultaneously; (B) is for EVs subpopulation expressing PDL1 protein and EpCAM protein simultaneously; (C) is for EVs subpopulation expressing TβRII protein and EpCAM protein simultaneously; (D) is for EVs subpopulation expressing PDL1 protein and TβRII protein simultaneously. DETAILED DESCRIPTION

[0070] Exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure can be more thoroughly and completely conveyed to those skilled in the art.

[0071] ​​​​​​​​​​​​​​​​​​​​​​Fluorescence polarization (FP) or fluorescence anisotropy (FA) assay is a fluorescence labeling detection technique, which has the characteristics of good reproducibility, sensitivity, simple operation and easy high-throughput analysis. The fluorescence polarization and fluorescence anisotropy values are dimensionless ratios, so they are not affected by the fluctuations of fluorescence intensity, and the determination has good accuracy and reproducibility. In FP analysis, fluorescent dye-labeled molecules are often used. When the molecular rotation changes in the molecular interaction or reaction, the fluorescence polarization signal and the fluorescence anisotropy signal will change, and this change is often closely related to the specific reaction system. For small molecules, metal ions or some proteins with small molecular weight, the change of fluorescence polarization value is small, which affects the detection sensitivity.

[0072] Aptamer (also known as aptamer, adapter) is a single-stranded oligonucleic acid molecule (ssDNA or ssRNA) that can bind to a certain biological target with high affinity and high specificity. Aptamer is a single-stranded DNA / RNA that can bind to a target molecule with high specificity, which is obtained by screening from an artificially synthesized DNA / RNA library by Systematic Evolution of Ligands by Exponential enrichment (SELEX). It has been reported that the targets of aptamer include metal ions, small organic molecules, polypeptides, proteins, cells and even tissues. The molecular recognition function of aptamer is similar to that of antibody, and it has a target recognition ability comparable to or even stronger than that of antibody molecule. However, compared with antibody, aptamer has many excellent properties, such as small molecular weight, batch production, non-immunogenicity, easy synthesis and labeling, rapid tissue penetration, good metabolic kinetics, no difference between different batches of products, and good chemical stability. It has important application prospects in the fields of biological detection, disease diagnosis and treatment.

[0073] FP has important applications in intermolecular interaction analysis, clinical drug detection, drug screening, ligand screening, etc. FP is mainly used for the detection of single targets such as nucleic acids, small molecules and free proteins in analysis and detection. In the previous study of the present application, FP was successfully used for the detection of exosomes, which showed the superiority of FP in the non-separation system, and good sensitivity and specificity were obtained (Nanoscale, 2019, 11, 10106). However, this method still has some shortcomings: due to the charge effect, the fluorescent dye will interact with the nucleic acid sequence, causing the change of the fluorescence polarization value (Talanta, 2016, 154, 567), and the different nucleic acid aptamer sequences will interact with the fluorescent dye, which will greatly affect the fluorescence polarization change value of the nucleic acid aptamer modified with the fluorescent dye after the target protein on the exosome is combined with the nucleic acid aptamer. This situation will cause the fluorescence polarization change value of some target proteins on the exosome to be large, and the fluorescence polarization change value of some target proteins to be small, or even negative, thus causing difficulty in developing a universal extracellular vesicle detection method.

[0074] In view of this, the present application combines the fluorescence polarization method with the joint footing mode, and for the first time proposes a fluorescence polarization method suitable for all recognition units, without difference and simple design. The method introduces a "T" type design to separate the recognition sequence and the signal reporting sequence, which can further improve the fluorescence polarization signal amplification specificity without changing the sequence modified with the fluorescent dye. Only by simply changing the sequence for recognizing the target can the "T" type structure be formed by the joint footing effect under the condition that the target exists, so that the dye molecules and biotin are close to each other. The large size of the extracellular vesicle can greatly improve the fluorescence polarization signal of the dye molecules. In the preferred embodiment, the combination of streptavidin and biotin finally causes the dye molecules to be close to the streptavidin molecules, and the proximity effect of streptavidin further helps to improve the specific fluorescence polarization signal. Combined with the above two aspects of fluorescence polarization signal amplification effect, the present application provides a specific, high-sensitivity and non-separation extracellular vesicle subpopulation detection method suitable for complex clinical samples.

[0075] It should be noted that according to the technical scheme of the present application, the recognition unit can be selected as a molecule for recognizing biological membranes (such as cholesterol), or a nucleic acid aptamer for recognizing specific membrane proteins, or an antibody for recognizing specific membrane proteins, or a recognition unit for recognizing specific polysaccharides, or a recognition unit for recognizing certain lipids, etc. The detection of the target can be realized by simply changing the recognition unit.

[0076] In the following examples, the cholesterol and the nucleic acid aptamer that can specifically recognize the extracellular vesicle marker protein are taken as examples. Specifically, the nucleic acid aptamer Apt CD63 (Angew. Chem. Int. Ed. 2017, 56, 11916-11920), the nucleic acid aptamer Apt EpCAM (Anal. Chem. 2013, 85, 4141-4149; Angew. Chem. Int. Ed. 2017, 56, 11916-11920), the nucleic acid aptamer Apt PDL1 , the nucleic acid aptamer Apt TβRII , and the nucleic acid aptamer Apt

[0077] It should be noted that the specific sequences provided in the present disclosure are to better illustrate that the technical scheme of the present application has universal significance in the selection of nucleic acid aptamers. The selection of nucleic acid aptamers can be any nucleic acid aptamer that binds to a characteristic protein on the outer membrane of a cell extracellular vesicle, and is suitable for the technical scheme of the present application. But it is not limited to the specific sequences described in the present disclosure. The fluorescent dye for modifying nucleic acid is only used to provide a fluorescence signal for fluorescence polarization, so any molecule that can provide a fluorescence signal is suitable for the present application. The fluorescent dye for modifying nucleic acid is only used to provide a fluorescence signal for fluorescence polarization, so any labeling position connected to the nucleic acid is suitable for the present application. The following is the sequence used in the examples:

[0078] III Chol-C The sequence is: 5'-GTCACTGAGACGACGTTAGA(T) 30 -cholesterol-3';

[0079] II Fam-F The sequence is: 5'-TCTAACGTCGTCACTCC-fluorescent dye-3', wherein the fluorescent dye is Fam;

[0080] I biotin-Chol The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 -3';

[0081] I biotin-AptX The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20- AptX-3', wherein AptX is the nucleic acid aptamer corresponding to the target.

[0082] I biotin-AptY The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20-AptY-3', where AptY is the nucleic acid aptamer corresponding to the target.

[0083] III AptX The sequence is: 5'-GTCACTGAGACGACGTTAGA(T) 20- AptX-3', where AptX is the nucleic acid aptamer corresponding to the target.

[0084] Example 1: Preparation of Standard Solution of Extracellular Vesicles from Cell Lines

[0085] (1) Extraction of extracellular vesicles from cell lines

[0086] Cell culture medium was centrifuged at 3000g at room temperature to remove cells and large debris, yielding a supernatant. This supernatant was centrifuged at 100,000g for 120 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS buffer (pH 7.4). The precipitate was then centrifuged again at 100,000g for 120 minutes, and the supernatant was discarded. The precipitate was resuspended in 100 μL of PBS buffer (pH 7.4) to obtain extracellular vesicles of the cell line. The concentration of the obtained extracellular vesicles was determined by nanoflow cytometry, and the vesicles were aliquoted and stored at -80℃ for subsequent construction of a standard curve.

[0087] (2) Preparation of EV-free plasma

[0088] Normal human plasma samples were centrifuged at 100,000g overnight, and the supernatant was collected to obtain EV-free plasma.

[0089] (3) Preparation of standard solutions for cell line EVs

[0090] Different concentrations of cell line EVs were added to exosome-free plasma to obtain a series of EV standard solutions of different concentrations.

[0091] Example 2: Construction of a working curve for detecting total EVs in MCF7 tumor cells using fluorescence polarization method.

[0092] The EVs obtained in Example 1 were subjected to fluorescence polarization detection (the principle is as follows). Figure 1 A). I biotin-Chol II Fam-F III Chol-C The three strands were mixed in the sample dilution buffer at a 1:1:1 ratio to obtain the reaction solution, with a final concentration of 2 nM. 50 μL of the reaction solution was then mixed with 50 μL of MCF7 cell line EV standard solutions of different concentrations (EV final concentration: 1.25 × 10⁻⁶). 2 cells / μL, 1.25×10 3 cells / μL, 1.25×10 4 cells / μL, 1.25×10 5cells / μL, 1.25×10 6 cells / μL, 1.25×10 7 The reaction mixture was incubated at room temperature in the dark for 50 minutes, followed by the addition of 1 μL of 1 mg / mL streptavidin and incubation at room temperature in the dark for 10 minutes. After the reaction, the reaction solution was added to a black microplate, and the fluorescence polarization value was measured using a multi-mode microplate reader (Infinite M 1000 PRO, Tecan, Switzerland). For Fam fluorescent dye-modified aptamers, the excitation filter was 475 nm, and the emission filter was 518 nm.

[0093] The results are as follows Figure 2 As shown in the figure, the change in fluorescence polarization signal increases with the increase of EV concentration. Using lg[EV concentration] as the X-axis and ΔFP (the difference between the fluorescence polarization value corresponding to the presence of the analyte and the fluorescence polarization value corresponding to the blank sample without the analyte) as the Y-axis, the working curve is obtained by fitting the line Y = aX + b.

[0094] Example 3: Construction of working curves for detecting EVs expressing a specific protein in MCF7 tumor cells using fluorescence polarization method.

[0095] Ⅰ biotin-AptCD63 The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 CACCCCACCTCGCTCCCGTGACACTAATGCTA-3'.

[0096] The EVs obtained in Example 1 were subjected to fluorescence polarization detection (the principle is as follows). Figure 1 B). I biotin-AptCD63 II Fam-F III Chol-C The three strands were mixed in the sample dilution buffer at a ratio of 1:1:1 to obtain the reaction solution, with a final concentration of 2 nM. 50 μL of the reaction solution was then mixed with 50 μL of MCF7 cell line EV standard solutions at different concentrations (EVs final concentration: 12.5 cells / μL, 1.25 × 10⁻⁶). 2 cells / μL, 1.25×10 3 cells / μL, 1.25×10 4 cells / μL, 1.25×10 5 cells / μL, 1.25×10 6 cells / μL, 1.25×10 7μL) and incubated at room temperature for 50 min in the dark, followed by the addition of 1 μL of 1 mg / mL streptavidin and incubation at room temperature for 10 min in the dark. After the reaction, the reaction solution was added to a black microplate, and the fluorescence polarization value was measured using a multifunctional microplate reader (infinite M 1000 PRO, Tecan, Switzerland). For the Fam fluorescent dye-modified nucleic acid aptamer, the excitation filter was 475 nm, and the emission filter was selected as 518 nm.

[0097] The results are shown in Figure 2. As can be seen from the figure, the change in fluorescence polarization signal increases with the increase in the concentration of EVs. The working curve was obtained by using lg [EVs concentration] as the X axis, using △FP (the difference between the fluorescence polarization value corresponding to the presence of the test substance and the fluorescence polarization value corresponding to the blank sample without the test substance) as the Y axis, and using the straight line Y = aX + b for fitting. Figure 3

[0098] Example 4 Construction of the working curve for detecting EVs subtypes expressing two specific proteins of tumor cell MCF7 simultaneously by fluorescence polarization method

[0099] The technical solution of the present application can not only detect EVs subtypes expressing the marker protein CD63, but also detect tumor-related protein positive EVs subtypes. This embodiment is demonstrated by taking the EpCAM protein widely expressed on tumor cells as an example.

[0100] (1) Extraction of EpCAM positive exosomes

[0101] It has been reported in the literature that human breast cancer cells MCF7 and MCF7 exosomes both highly express EpCAM protein (Journal of Nanobiotechnology, 2018, 16, 65; Anal. Chem. 2018, 90, 14402-14411), so the MCF7 cell line is used to prepare EpCAM positive exosomes.

[0102] (2) Ⅰ biotin-AptCD63 The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 CACCCCACCTCGCTCCCGTGACACTAATGCTA-3';

[0103] Ⅲ AptEpCAM The sequence is: 5'-GTCACTGAGACGACGTTAGA(T) 30 CACTACAGA GGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'.

[0104] ​The EVs obtained in Example 1 were subjected to fluorescence polarization detection (principle as shown in Figure 1 C). The I biotin-AptCD63 , II Fam-F , III AptEpCAM triplex was mixed at 1:1:1 in sample diluent to obtain a reaction solution with a final concentration of 2 nM. 50 μL of the reaction solution was mixed with 50 μL of different concentrations of MCF7 cell line EVs standard solution (EVs final concentration: 1.25 x 10 2 / μL, 1.25 x 10 3 / μL, 1.25 x 10 4 / μL, 1.25 x 10 5 / μL, 1.25 x 10 6 / μL, 1.25 x 10 7 / μL) respectively, and reacted at room temperature in the dark for 50 minutes, followed by the addition of 1 μL of 1 mg / mL streptavidin, and reacted at room temperature in the dark for 10 minutes. After the reaction was completed, the reaction solution was added to a black microplate, and the fluorescence polarization value was measured using a multifunctional enzyme label instrument (infinite M 1000 PRO, Tecan, Switzerland). For the Fam fluorescent dye modified nucleic acid aptamer, the excitation filter was 475 nm, and the emission filter was selected as 518 nm.

[0105] The results are shown in Figure 4 The figure shows that the change in fluorescence polarization signal increases with the increase in EVs concentration. Using lg[EVs concentration] as the X axis, using △FP (the difference between the fluorescence polarization value corresponding to the presence of the test substance and the fluorescence polarization value corresponding to the blank sample without the test substance) as the Y axis, and using the straight line Y = aX + b to fit the working curve.

[0106] Example 5 Detection of multiple EVs subtypes in different cell line EVs in plasma samples

[0107] This example can detect multiple EVs subtypes in different cell line EVs by selecting different EVs targets and using the reaction solution of Example 2, Example 3, and Example 4, and can be performed in clinical samples (plasma). By replacing the nucleic acid aptamer sequence corresponding to a specific protein, not only can the exosome marker CD63 and the tumor marker EpCAM be recognized, but also other types of target proteins (this example uses immunotherapy related PDL1 and TβRII proteins as an example for demonstration).

[0108] (1) Extraction of EpCAM positive EVs

[0109] It has been reported that both human breast cancer cell MCF7 and MCF7 exosomes highly express EpCAM protein (Journal of Nanobiotechnology, 2018, 16, 65; Anal. Chem. 2018, 90, 14402-14411), so MCF7 cell line is used to prepare EpCAM positive EVs.

[0110] (2) Extraction of PDL1 and TβRII positive EVs

[0111] It has been reported that EVs produced by human breast cancer cell MDA-MB-231 highly express PDL1 and TβRII proteins, so MDA-MB-231 cell line is used to prepare PDL1 and TβRII positive EVs.

[0112] (3) I biotin-AptCD63 The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 CACCCCACCTCGCTCCCGTGACACTAATGCTA-3';

[0113] I biotin-AptPDL1 The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-3';

[0114] I biotin-AptTβRII The sequence is: 5'-Biotin-GGAGTGATCTCAGTGAC(T) 20 ACATTG CTGCGTGATCGCCTCACATGGGTTTGTCTGGTCGATTTGGAGGTGGTGGGT GGC-3';

[0115] III AptEpCAM The sequence is: 5'-GTCACTGAGACGACGTTAGA(T) 30 CACTACAG AGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3';

[0116] III AptTβRII The sequence is: 5'-GTCACTGAGACGACGTTAGA(T) 30 ACATTGCTGC GTGATCGCCTCACATGGGTTTGTCTGGTCGATTTGGAGGTGGTGGGTGGC -3';

[0117] (4) Preparation of artificial vesicles

[0118] A 2 mM lipid solution was prepared by mixing phosphatidylcholine: sphingomyelin: cholesterol at a molar ratio of 7:1.5:1.5. The lipid solution was then repeatedly frozen and thawed by 3-5 cycles. Finally, artificial vesicles were obtained by using a Liposofast LF-1 extruder through a porous membrane with an average pore size of 50 nm. The concentration of the obtained artificial vesicles was measured by NTA.

[0119] Cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibo). To obtain MCF7 exosomes, MCF7 cells were cultured in serum-free DMEM medium for 48 h. After collecting the cell culture medium, the cells and their fragments were removed by centrifugation at 2000 g, and the supernatant was collected. Then, the supernatant was centrifuged at 10000 g for 45 min, and the filtrate was collected through a 0.20 μm filter membrane. The filtered solution was centrifuged at 100000 g for 120 min, and the supernatant was discarded and the precipitate was collected. The precipitate was resuspended with 100 μL of PBS buffer at pH 7.4 to obtain the exosomes of MCF7 cells. The obtained exosomes were divided and stored at -80°C for use after determining their concentration by NTA.

[0120] First, the following six reaction solutions were prepared with a buffer containing 1% EV-free plasma.

[0121] Reaction solution one contains: I biotin-Chol , II Fam-F , III Chol-C The three strands were mixed at a ratio of 1:1:1 in sample diluent to obtain a reaction solution with a final concentration of 2 nM.

[0122] Reaction solution two contains: I biotin-AptCD63 , II Fam-F , III Chol-C The three strands were mixed at a ratio of 1:1:1 in sample diluent to obtain a reaction solution with a final concentration of 2 nM.

[0123] Reaction solution three contains: I biotin-AptCD63 , II Fam-F , III AptEpCAM The three strands were mixed at a ratio of 1:1:1 in sample diluent to obtain a reaction solution with a final concentration of 2 nM.

[0124] Reaction solution four contains: I biotin-AptPDL1 , II Fam-F , III Chol-C The three strands were mixed at a ratio of 1:1:1 in sample diluent to obtain a reaction solution with a final concentration of 2 nM.

[0125] Reaction solution five contains: I biotin-AptTβRII , II Fam-F , III Chol-CThe three strands are mixed in a sample diluent at 1:1:1 to obtain a reaction solution, and the final concentration is 2 nM.

[0126] The reaction solution six comprises: I biotin-AptPDL1 , II Fam-F , III AptTβRII The three strands are mixed in a sample diluent at 1:1:1 to obtain a reaction solution, and the final concentration is 2 nM.

[0127] Sample one (MCF7 EVs), sample two (MDA-MB-231 EVs) and sample three (artificial vesicles) are prepared with a buffer solution containing 1% EV-free plasma. 50 μL of each sample is mixed with 50 μL of reaction solution, and the mixture is reacted at room temperature for 50 minutes in the dark, followed by the addition of 1 μL of 1 mg / mL streptavidin, and the mixture is reacted at room temperature for 10 minutes in the dark. After the reaction is completed, the reaction solution is added to a black microplate, and the fluorescence polarization value is measured using a multifunctional enzyme marker (infinite M 1000 PRO, Tecan, Switzerland). For the Fam fluorescent dye-modified aptamer, the excitation filter is 475 nm, and the emission filter is selected as 518 nm.

[0128] The results are shown in Figure 5 , using ΔFP (the difference between the fluorescence polarization value corresponding to the presence of the test substance and the fluorescence polarization value corresponding to the blank sample without the test substance) as the Y-axis. As can be seen from the figure, the change in fluorescence polarization signal is significantly different in EVs from different sources: artificial vesicles have a significant positive signal only in reaction solution one; EVs from cell lines express CD63, so they have a significant positive signal in reaction solution two; and EpCAM, PDL1 and TβRII proteins are all related to the cell lines from which the EVs are derived, and different cell lines express different proteins. This result shows that the detection method provided by the present application can characterize the content of different EVs subtypes from different cells, and has good specificity and sensitivity when used for clinical samples (plasma).

[0129] Example 6: Detection of multiple EVs subtypes in clinical plasma using fluorescence polarization method

[0130] (1) Preparation of clinical plasma samples

[0131] The plasma of 3 healthy volunteers and 6 tumor patients was collected, centrifuged at 3000g at room temperature for 10 min, and centrifuged twice to remove cells and their fragments, and the supernatant was collected and stored at -80°C for use.

[0132] (2) Preparation of reaction solution

[0133] Four kinds of reaction solutions were prepared using a buffer solution.

[0134] The reaction solution one comprises: Ibiotin-AptCD63 , I Fam-F , II Apt-EpCAM , III Mix the three strands in 1:1:1 in sample dilution buffer to get reaction solution, final concentration is 2nM.

[0135] Reaction solution two contains: I biotin-AptPDL1 , II Fam-F , III Apt-EpCAM Mix the three strands in 1:1:1 in sample dilution buffer to get reaction solution, final concentration is 2nM.

[0136] Reaction solution three contains: I biotin-AptTβRII , II Fam-F , III Apt-EpCAM Mix the three strands in 1:1:1 in sample dilution buffer to get reaction solution, final concentration is 2nM.

[0137] Reaction solution four contains: I biotin-AptPDL1 , II Fam-F , III Apt-TβRII Mix the three strands in 1:1:1 in sample dilution buffer to get reaction solution, final concentration is 2nM.

[0138] (3) Detection of clinical plasma samples

[0139] Mix 0.5μL of plasma sample treated in step (1) with 49.5μL of reaction solution prepared in step (2) uniformly, and react for 50 minutes at room temperature in the dark, then add 0.5μL of 1mg / mL streptavidin, and react for 10 minutes at room temperature in the dark. After the reaction is completed, add the reaction solution to a black microplate, and measure the fluorescence polarization value by using a multifunctional enzyme label instrument (infinite M 1000PRO, Tecan, Switzerland). For the nucleic acid aptamer modified with Fam fluorescent dye, the excitation filter is 475nm, and the emission filter is selected as 518nm.

[0140] The detection results are shown in Figure 6 , in which H1, H2, H3 are healthy people, and P1, P2, P3, P4, P5, P6 are patients. For different EVs subtypes, the fluorescence polarization values of tumor patients can be divided into three groups of high, medium and low (circled in Figure 6 ). However, the patient individuals in the three groups of high, medium and low in different EVs subgroups are not completely the same, which indicates the heterogeneity of tumor patient EVs. The fluorescence polarization values of tumor patients in all EVs subgroups are at low values and comparable to those of healthy people. The above results show that for tumor patients, not all EVs subgroups are higher than those of healthy people, and it is more meaningful to focus on the EVs subgroups higher than normal ones, which can be used for tumor diagnosis or drug guidance. The above results also show the importance and significance of EVs subpopulation characterization.

[0141] The above description is only preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any changes or replacements within the technical range disclosed by the present application can be easily thought by those skilled in the art, and should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims. SEQUENCE LISTING <110> INSTITUTE OF CHEMISTRY, CHINESE ACADEMY OF SCIENCES <120> A method for detecting extracellular vesicles and subtypes thereof in a separation-free system and applications thereof <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <400> 1 ggagtgatct cagtgac 17 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <400> 2 tctaacgtcg tcactcc 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gtcactgaga cgacgttaga 20 <210> 4 <211> 50 <212> DNA <213> Artificial Sequence <400> 4 gtcactgaga cgacgttaga tttttttttt tttttttttt tttttttttt 50 <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <400> 5 ggagtgatct cagtgacttt tttttttttt ttttttt 37 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence <400> 6 ggagtgatct cagtgacttt tttttttttt ttttttt 37 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <400> 7 gtcactgaga cgacgttaga tttttttttt tttttttttt 40 <210> 8 <211> 69 <212> DNA <213> Artificial Sequence <400> 8 ggagtgatct cagtgacttt tttttttttt tttttttcac cccacctcgc tcccgtgaca 60 ctaatgcta 69 <210> 9 <211> 98 <212> DNA <213> Artificial Sequence <400> 9 gtcactgaga cgacgttaga tttttttttt tttttttttt tttttttttt cactacagag 60 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttgttgcgtctg tcccacgttg tcatgggggg ttggcctg 98 <210> 10 <211> 70 <212> DNA <213> Artificial Sequence <400> 10 ggagtgatct cagtgacttt tttttttttt ttttttttac aggttctggg gggtgggtgg 60 ggaacctgtt 70 <210> 11 <211> 97 <212> DNA <213> Artificial Sequence <400> 11 ggagtgatct cagtgacttt tttttttttt tttttttaca ttgctgcgtg atcgcctcac 60 atgggtttgt ctggtcgatt tggaggtggt gggtggc 97 <210> 12 <211> 110 <212> DNA <213> Artificial Sequence <400> 12 gtcactgaga cgacgttaga tttttttttt tttttttttt tttttttttt acattgctgc 60 gtgatcgcct cacatgggtt tgtctggtcg atttggaggt ggtgggtggc 110

Claims

1. A detection probe, characterized in that, It includes probe I, probe II, and probe III; one end of probe I is labeled with biotin, and the other end is labeled with a first recognition unit; one end of probe II is labeled with a fluorescent dye; and one end of probe III is labeled with a second recognition unit. The probe I and the probe II partially pair up, so that the biotin and the fluorescent dye are close to each other; under the condition of detecting the target, the probe I and the probe II partially pair up with the probe III, so that the probe I, probe II and probe III hybridize to form a T-shaped structure; The first recognition unit specifically recognizes the cell membrane, the first target protein, the first target polysaccharide, or the first target lipid; The second recognition unit specifically recognizes the cell membrane, the second target protein, the second target polysaccharide, or the second target lipid.

2. The detection probe as described in claim 1, characterized in that, The first recognition unit is cholesterol, a first nucleic acid aptamer, or a first antibody.

3. The detection probe as described in claim 1, characterized in that, The second recognition unit is cholesterol, a second nucleic acid aptamer, or a second antibody.

4. The detection probe as described in claim 1, characterized in that, In probe I, one end marked with the first identification unit has a first unpaired sequence, which cannot be complementary to other sequences in the detection probe. In the probe III, one end marked with the second identification unit has a second unpaired sequence, which cannot be complementary to other sequences in the detection probe.

5. The detection probe as described in claim 4, characterized in that, The lengths of the first unpaired sequence and the second unpaired sequence are each independently selected from 0-80 bp.

6. The detection probe as described in claim 4, characterized in that, The first unpaired sequence and the second unpaired sequence are each independently selected from oligothymidine nucleotide, oligoadenine nucleotide, oligocytosine nucleotide, oligoguanine nucleotide, and oligouracil nucleotide.

7. The detection probe as described in claim 1, characterized in that, The sequence lengths of partial pairings of probe I and probe II, partial pairings of probe I and probe III, and partial pairings of probe II and probe III are each independently selected from 5-30 bp.

8. A testing product, characterized in that, Includes the detection probe and buffer solution as described in any one of claims 1-7.

9. The testing product as described in claim 8, characterized in that, The testing products also include streptavidin.

10. The test product as described in claim 8, characterized in that, The buffer solution includes PBS buffer, Mg 2+ .

11. The test product as described in claim 8, characterized in that, In the detection product, the concentration of the detection probe is 0.5-5 nM.

12. The test product as described in claim 8, characterized in that, In the detection product, the molar ratio of probe I, probe II, and probe III is 1-1.2:1-1.2:1-1.

2.

13. The testing product as described in claim 9, characterized in that, In the tested product, the concentration of streptavidin is 2-20 μg / mL.

14. A reagent kit, characterized in that, Includes the detection probe as described in any one of claims 1-7 and / or the detection product as described in any one of claims 8-13.

15. Use of the detection probe according to any one of claims 1-7, the detection product according to any one of claims 8-13, or the kit according to claim 14 in the following (a), (b), and (c): (a) Detection of total extracellular vesicles; (b) Detection of extracellular vesicle subtypes by targeting a single protein; (c) Detection of extracellular vesicle subtypes by simultaneously targeting two target proteins.

16. The use as described in claim 15, characterized in that, The extracellular vesicles being tested were obtained from cell culture medium, urine, plasma, or tissue fluid samples.

17. A method for detecting total extracellular vesicles, characterized in that, Includes providing a detection probe as described in any one of claims 1-7, incubating the detection probe with a sample to be tested, and measuring the fluorescence anisotropy change value and / or fluorescence polarization change value; Both probe I and probe III are labeled with cholesterol.

18. The method for detecting total extracellular vesicles as described in claim 17, characterized in that, The incubation step and the assay step also include the following step: adding streptavidin to carry out the reaction.

19. The method for detecting total extracellular vesicles as described in claim 17, characterized in that, The incubation time is 20-90 minutes; the incubation temperature is 20-37°C.

20. The method for detecting total extracellular vesicles as described in claim 18, characterized in that, The reaction time after adding streptavidin is 5-30 minutes.

21. A method for detecting extracellular vesicle subtypes, characterized in that, Includes providing a detection probe as described in any one of claims 1-7, incubating the detection probe with a sample to be tested, and measuring the fluorescence anisotropy change value and / or fluorescence polarization change value; In this embodiment, one of probe I and probe III is labeled with cholesterol, and the other is labeled with a nucleic acid aptamer or antibody; the nucleic acid aptamer or antibody specifically binds to a target protein of the subtype; or, The probe I is labeled with a first nucleic acid aptamer or a first antibody, and the probe III is labeled with a second nucleic acid aptamer or a second antibody; the first nucleic acid aptamer / first antibody and the second nucleic acid aptamer / second antibody specifically bind to the two target proteins of the subtype, respectively.

22. The method for detecting extracellular vesicle subtypes as described in claim 21, characterized in that, The incubation step and the assay step also include the following step: adding streptavidin to carry out the reaction.

23. The method for detecting extracellular vesicle subtypes as described in claim 21, characterized in that, The incubation time is 20-90 minutes; the incubation temperature is 20-37°C.

24. The method for detecting extracellular vesicle subtypes as described in claim 22, characterized in that, The reaction time after adding streptavidin is 5-30 minutes.

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