CN110954703A

CN110954703A - Method for simultaneously detecting protein in exosome, RNA and exosome membrane protein

Info

Publication number: CN110954703A
Application number: CN201911377286.4A
Authority: CN
Inventors: 曾恒山
Original assignee: Hangzhou Dixiang Industrial Co ltd
Current assignee: Hangzhou Dixiang Industrial Co ltd
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-03
Also published as: WO2021128678A1

Abstract

A method for simultaneously detecting protein, RNA and exosome membrane protein in exosome belongs to the technical field of exosome detection. The method comprises capturing exosomes in a sample; detecting the corresponding membrane protein; preparing nanoparticles; fusing the nanoparticle with exosomes; and (4) taking a picture and imaging by using the total internal reflection fluorescence microscope, and analyzing and processing data. The invention respectively verifies the aspects of exosome characteristic detection, cell supernatant exosome membrane protein and mRNA expression comparison experiment, human plasma exosome specificity detection, human plasma exosome membrane protein comparison experiment, multiple samples of human plasma exosome membrane protein, RNA and membrane protein detection, and compares the experiments with the existing acknowledged exosome detection technology, so that the consistency of the detection result of the same marker detected by the technology of the invention for the same sample and the acknowledged technology is shown, and the technology can simultaneously detect exosome protein, RNA and exosome membrane protein and judge the difference of the samples.

Description

Method for simultaneously detecting protein in exosome, RNA and exosome membrane protein

Technical Field

The invention belongs to the technical field of exosome detection, and particularly relates to a method for simultaneously detecting proteins in exosomes, RNA (ribonucleic acid) and exosome membrane proteins.

Background

Exosomes (exosomes), a tiny membrane vesicle secreted by most cells, with a diameter of about 30-150nm, have a lipid bilayer membrane structure, and protect its coating well. The micro-vacuole contains specific proteins, nucleic acids and lipids derived from host cells, can be used as signal molecules to be transmitted to other cells, is an important medium for communication between cells, and can change various biological functions of receptor cells. Exosomes are produced by all cells, but secreted by different cells in different compositions and amounts, and specific gene products are selectively loaded into exosomes to participate in the regulation of biological functions of recipient cells by transferring bioactive molecules between different cells. One of the most useful properties of exosomes, their abundance, specific and stable inclusion source, is becoming the focus of cell biology research.

Exosomes are one of three fields of liquid biopsy, and potential biological targets of exosomes are numerous, but a detection system capable of simultaneously using trace nucleic acid and protein, particularly protein contained in exosomes for in-situ detection as targets is not reported.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of a method for simultaneously detecting proteins in exosomes, RNA and exosome membrane proteins.

The invention is realized by the following technical scheme:

the method for simultaneously detecting the protein in the exosome, RNA and the exosome membrane protein, wherein the RNA comprises mRNA, miRNA, lncRNA and CircRNA, and is characterized by comprising the following steps of:

1) taking crude extracellular vesicles obtained by a precipitation method, and directly capturing exosomes in a sample by adopting an exosome in-situ capturing chip under a direct-current electric field of 15-90V;

2) combining a target antibody on the exosome membrane captured in the step 1), and emitting labeled A fluorescent signals through the combination of an antigen and the antibody to detect corresponding membrane proteins;

3) preparing nanoparticles, and wrapping RNA molecular beacons of targets to be detected and fluorescent monoclonal antibodies by the nanoparticles;

4) fusing the nanoparticles coated with the RNA molecular beacon and the fluorescent monoclonal antibody obtained in the step 3) with the exosomes obtained in the step 2), enabling the RNA molecular beacon and the fluorescent monoclonal antibody to enter the exosomes, combining the RNA molecular beacon with corresponding target genes, opening a beacon stem-loop structure, emitting marked B fluorescent signals under the excitation of laser, and detecting corresponding RNA; combining the antigen and the antibody, emitting labeled C fluorescent signals under the excitation of laser, and detecting corresponding proteins;

5) and (4) taking a picture and imaging by using the total internal reflection fluorescence microscope, and analyzing and processing data.

The method for simultaneously detecting the protein in the exosome, RNA and the membrane protein of the exosome is characterized in that the chip for capturing the exosome in situ in the step 1) is as follows: and the in-situ capture chip is coated with the exosome specificity capture antibody marked by avidin and biotin.

The method for simultaneously detecting the protein, RNA and the exosome membrane protein in the exosome is characterized in that the avidin and biotin-labeled specific capture antibody is recognized as marker antibodies CD63 and CD9 on the exosome membrane.

The method for simultaneously detecting the protein in the exosome, RNA and the protein of the exosome membrane is characterized in that the target antibody in the step 2) is a specific antibody capable of binding to an antigen on the exosome membrane.

The method for simultaneously detecting the protein in the exosome, the RNA and the exosome membrane protein is characterized in that the nanoparticle in the step 3) is prepared by the following steps:

1) fully and uniformly mixing DOTMA, DSPE-PEG-2000 and cholesterol according to the ratio of 3:1:4, carrying out ultrasonic treatment for 5min, and standing at room temperature for 2 hours to obtain a stable cationic liposome;

2) fully and uniformly blowing a proper amount of molecular beacon, a fluorescence monoclonal antibody and the cationic liposome in PBS, and performing ultrasonic treatment for 5 min;

3) quickly injecting the mixed solution obtained in the step 1) into an EP tube containing a proper amount of PBS, carrying out vortex for 10s, carrying out ultrasonic treatment for 5min, and standing at room temperature for 2 hours to obtain stable nanoparticles.

The method for simultaneously detecting the protein in the exosome, RNA and the membrane protein of the exosome is characterized in that the molar ratio of the molecular beacon to the fluorescent monoclonal antibody in the nanoparticles in the step 3) is 1: 2-1: 5.

The method for simultaneously detecting the protein in the exosome, the RNA and the exosome membrane protein is characterized in that in the step 3), the RNA molecular beacon is that a 5 ' end stem and a ring are completely complementary with a target gene, a 3 ' end stem is partially complementary with a 5 ' end stem, the 5 ' end and the 3 ' end are respectively modified by a fluorescent group and a quenching group, and partial basic groups on the ring are modified by locked nucleic acid.

The method for simultaneously detecting the protein in the exosome, RNA and the membrane protein of the exosome is characterized in that the fluorescent monoclonal antibody in the step 3) is a fluorescein-labeled monoclonal antibody aiming at a specific target protein in the exosome.

The method for simultaneously detecting the protein in the exosome, RNA and the membrane protein of the exosome is characterized in that the fusion condition in the step 4) is a principle of heterogeneous charge attraction and membrane fusion, and the fusion is carried out under a direct current field of 15-90V and at the temperature of 4-60 ℃.

Compared with the prior art, the invention has the following beneficial effects:

1) the invention can realize the simultaneous detection of the protein in the exosome, RNA and the exosome membrane protein (three-in-one) on the same technical platform, only needs 6 hours from the sample processing to the detection, the analysis and the report, can be manufactured into the exosome in-situ capture chip with various specifications such as 96 holes, 384 holes and the like which is universal in the biological industry, and has high efficiency, economy, convenience and quickness. Exosome is one of three fields of liquid biopsy, and the potential biological targets of exosome are numerous, but a detection system capable of simultaneously using trace nucleic acid and protein as targets is not reported. Nucleic Acid (RNA) and protein under the protection of exosome are very stable and are very suitable for being used as detection targets, the two biomacromolecules are organically combined, the state of a specimen can be reflected, and the specificity and the sensitivity of detection are further improved. The existing method needs 3 technologies to realize the classification detection of proteins in exosomes, RNA and exosome membrane proteins: usually, immunoblotting (Western Blot, WB for short) is adopted for detecting the exosome protein, which is time-consuming and labor-consuming, requires 1-3 days, and cannot strictly distinguish whether the protein is in exosome or on membrane; the detection of the exosome membrane protein usually adopts a flow cytometer, special magnetic beads are required to be used, and consumables and instruments are expensive; the detection of the exosome nucleic acid is usually carried out by a fluorescent quantitative PCR technology, the RNA extraction process is complex and tedious, amplification is needed, a sample is easy to pollute, and the result is unstable.

2) The invention can realize the simultaneous detection of trace nucleic acid and protein multi-targets of exosomes by respectively adopting the nanoparticle molecular beacon and the specific fluorescent monoclonal antibody for exosomes separated and purified from the same sample, the exosomes are captured and then membrane-fused with the nanoparticles to form a lipid membrane complex, the fused exosomes are mixed with the contents of the nanoparticles, the specific molecular beacon is hybridized with the target RNA thereof, the stem-loop structure is opened, and the fluorescence is separated from a quenching group; meanwhile, the original inner protein and membrane protein of the exosome are redistributed, but the antigenicity of the exosome is not influenced, after the exosome and the specific fluorescent monoclonal antibody are subjected to warm bath, the two are subjected to specific combination, the molecular beacon and the fluorescent monoclonal antibody emit respective specific fluorescent signals under the excitation of laser, a Total Internal Reflection Fluorescence Microscope (TIRFM) technology is used for realizing high-resolution microscopic imaging, the direct imaging of the nano-scale vesicle of the exosome and the semi-quantitative determination of a target are realized by utilizing the characteristics of ultramicro imaging and hypersensitive to the fluorescent signals, and the sample difference is analyzed, detected and judged through the signal intensity of the distribution of the target protein related to the exosome and the membrane surface. The single-hole single-sample exosome trace nucleic acid and protein multi-target simultaneous detection and multi-dimensional analysis judgment greatly improve the accuracy of specimen detection. Compared with the conventional means such as high-throughput sequencing NGS, quantitative PCR and the like, the method not only reduces the detection cost of a single sample and multiple targets, but also further improves the sensitivity to the detection of the specimen.

3) The invention adopts a specific exosome extraction, purification and capture method to realize high-sensitivity trace in-situ capture of exosomes, can keep the original form of exosomes, obtains high-purity exosomes, improves the efficiency, realizes high flux and reduces the exosome extraction cost. Moreover, on the basis of meeting the requirement of a chip for detecting a trace amount of exosomes, the exosome purity is improved to a new level, the fluorescence signal-to-noise ratio of a specific target is further increased, and the detection sensitivity is improved. The existing methods for extracting, purifying and capturing exosome mainly comprise the following steps: the overspeed and density gradient centrifugation method has low flux, long time consumption (only sample treatment needs 3 hours), and great influence on the physical performance of exosome; the precipitation method based on the polymer has more impurities and great damage to the effective components of exosomes; the immuno-separation method has low flux and is not suitable for a large number of samples. Furthermore, isolated vesicles may lose functional activity.

4) The nanoparticle which is provided with positive charges and can be fused with exosomes with negative charges comprises the molecular beacon and the fluorescent monoclonal antibody, and through reasonable nanoparticle structure design and proper molar ratio of the fluorescent monoclonal antibody to the molecular beacon, protein and RNA in a single exosome can be directly combined in situ and subjected to fluorescence imaging, so that the specificity is stronger, RNA extraction and amplification are not needed, the structure of the exosome is prevented from being cracked and damaged, and the nanoparticle is more accurate, more convenient and higher in cost performance.

5) The invention adopts the ultra-thin (the thickness is 0.17-0.19mm), ultra-low fluorescence and ultra-high light transmission special glass, the geometric mean roughness (RMS roughnesss) is less than 100nm, the glass surface is coated with avidin, biotinylation and antibodies, the exosome in the sample can be captured in situ uniformly and efficiently through the LINKER molecular high affinity combination and the multistage amplification effect, and the load concentration of the molecular beacon and the fluorescence monoclonal antibody in the nanoparticle can be adjusted according to the experimental requirement, thereby realizing the high-efficiency detection of the protein, RNA and membrane protein in the exosome.

6) The invention relates to high-definition total internal reflection fluorescence microscope imaging, which is based on an artificial intelligence efficient algorithm and fluorescence image analysis processing. The high-resolution microscopic imaging has the characteristics of ultramicro and super-sensitivity to fluorescence signals, can realize dynamic observation of a nano vesicle surface substance such as an exosome, high-resolution direct imaging of a micro structure, a monomolecular structure and a cell structure and content determination of a target thereof, and analyzes and detects the signal intensity of the distribution of targets related to the membrane surface in the exosome and judges the sample difference. Through the classification and marking of massive pictures obtained by a completed large sample experiment, more than 1000000 (512x512 pixels/piece) fluorescence image classification databases are established, and the databases are used for training a picture intelligent screening module based on deep learning, so that the early-stage screening of abnormal fluorescence images is realized. In addition, the method of region splitting and merging, GraphCut and the like is adopted to further effectively distinguish the fluorescence signal and impurity interference of the sample in the image; on the basis of the modules, a set of comprehensive optimization programs of the sample images, including abnormal image screening, sample phosphor form separation and optimal threshold selection, are developed by combining optimization algorithms such as PSO (particle Swarm optimization), DPSO (Darwinian PSO), Fo-DPSO (Fractional-Order DPSO) and the like.

Drawings

FIG. 1 is a diagram of putative exosome proteins TSG101, HSP70, HIF-1a, AFP and representative RNAs: the detection principle schematic diagram takes miR-21, AFPmRNA, exosome membrane protein CD63 and PD-L1 as examples;

FIGS. 2,3, 4, 5, 6 and 7 are graphs showing the results of detection in the examples.

As shown in fig. 1: the invention relates to a brand-new in-situ capture and detection technology of exosomes, in particular to a technology for simultaneously detecting in-vivo proteins, RNA and exosome membrane proteins (three-in-one). The activated ultra-low fluorescence and ultra-high light transmittance special glass coated with exosome specific capture antibodies CD63 and CD9 is used as a carrier, exosomes in various biological samples can be captured in situ, and a fluorescent monoclonal Antibody in a solution is combined with exosome membrane proteins CD63, PD-L1(Antibody, AB for short) and the like to emit A kinds of labeled fluorescent signals; after the cationic lipid nanoparticles wrapped with the specific fluorescent monoclonal antibody and the Molecular beacon (self-designed) for specifically recognizing the target gene are fused with the exosome with negative charge captured by the chip, the Molecular beacon (MB for short) is combined with the target gene miR-21, AFP mRNA and the like to emit B kinds of labeled fluorescent signals. Meanwhile, the specific fluorescent monoclonal Antibody is combined with target proteins TSG101, HSP70, HIF-1a, AFP (Antibody, AB for short) and the like in a fusion body, C kinds of marked fluorescent signals are emitted to generate fluorescent signals under the excitation of laser, the fluorescent signals are detected by TIRFM, and the signal intensity is in direct proportion to the content of corresponding targets, so that the difference of samples is judged.

Detailed Description

The present invention will be further described with reference to examples.

Example 1: exosome in-situ capture chip processing

1) Adopting ultrathin (thickness is 0.17-0.19mm), ultralow fluorescence, ultrahigh light transmittance, roughness geometric mean value (RMS roughnesss) <100nm special glass, using high-purity electronic grade alcohol, repeatedly cleaning under the clean condition of 24-200 ℃, and then blowing and drying by using high-purity nitrogen;

2) repeatedly cleaning with piranha solution, and keeping clean and dry;

3) placing glass surface and organic siloxane with functional group (organic siloxane with functional groups) in vacuum container, vacuum degree should be less than 10mmHg, and vapor-depositing at 24-180 deg.C for 6-24 hr;

4) mixing avidin and biotin in a proper ratio of 1: 7-1: 30, mixing evenly at low speed, standing for 30-60 minutes, and adding PBS and BupH^TMA buffer solution prepared from Phosphate Buffered Saline and ultrapure water (about 18 megaohms) and 0.01-1% Tween 20 are repeatedly cleaned and then placed in an EP tube, so that the combination efficiency is improved, and the EP tube is placed in a refrigerator for cold storage for 6-12 hours at 4 ℃ in a dark place;

5) taking out the liquid combined with biotin and avidin from the EP tube, and mixing the biotin marked with the CD9 or CD63 antibody in a proper ratio of 1: 5-1:25, uniformly mixing at a low speed, uniformly coating on the treated glass, slightly vibrating, fully combining, keeping out of the sun, and incubating for 1-3 hours at the temperature of 24-60 ℃;

6) under the reaction conditions, the LINKER molecule presents high affinity binding and multi-stage amplification effect to capture the exosome in situ by using the coated glass slide, biotin, avidin and exosome capture specific antibody CD9 or CD 63.

Example 2: nanoparticle preparation and antibody coating

1) Fully and uniformly mixing DOTMA (trimethyl-2, 3-dioleyloxypropyl ammonium chloride), DSPE-PEG-2000(1, 2-distearoyl-SN-glycerin-3-phosphoethanolamine-polyethylene glycol 2000) and cholesterol according to the ratio of 3:1:4, performing ultrasonic treatment for 5min, and standing at room temperature for 2 hours to obtain a stable cationic liposome;

2) a proper amount of molecular beacon, fluorescent monoclonal antibody (RNA molecular beacon and antibody can be mixed and wrapped with nanoparticles according to a molar ratio of 1: 2-1: 5 on the basis of excess) and the cationic liposome are fully blown, beaten and uniformly mixed in PBS, and then are subjected to ultrasound for 5min, wherein the molecular beacon is a 5 ' end stem and a ring which are completely complementary with a target gene, a 3 ' end stem is partially complementary with a 5 ' end stem, the 5 ' end and the 3 ' end are respectively modified by a fluorescent group and a quenching group, a part of basic groups on the ring are modified by locked nucleic acid, and the fluorescent monoclonal antibody is a fluorescein-labeled monoclonal antibody aiming at a specific target protein in a secretion body;

Example 3: method for simultaneously detecting protein in exosome, RNA and exosome membrane protein

1) Taking crude extracellular vesicles obtained by a precipitation method, and directly capturing exosomes in a sample by adopting an exosome in-situ capturing chip (prepared by the method in example 1) under a direct-current electric field of 15-90V;

3) taking nanoparticles (prepared by the method of example 2) wrapping the RNA molecular beacon and the fluorescent monoclonal antibody of the target to be detected;

4) fusing the nanoparticles coated with the RNA molecular beacon and the fluorescent monoclonal antibody obtained in the step 3) with the exosomes captured in the step 1), enabling the RNA molecular beacon and the fluorescent monoclonal antibody to enter the exosomes, enabling the RNA molecular beacon to be combined with corresponding target genes, opening a beacon stem-loop structure, emitting marked B fluorescent signals under the excitation of laser, and detecting corresponding RNA; combining the antigen and the antibody, emitting labeled C fluorescent signals under the excitation of laser, and detecting corresponding proteins;

5) and (4) photographing and imaging by using a Total internal reflection fluorescence microscope (TIRFM for short), and analyzing and processing data.

Example 4: detection test (taking the existing recognized targets of exosome proteins TSG101, HSP70, HIF-1a, AFP and representative RNA: miR-21, AFP mRNA, exosome membrane proteins CD63 and PD-L1 as examples respectively)

First, sample pretreatment

1. Taking 200ul of sample (cell culture supernatant, isolated experimental animal plasma, serum, isolated human plasma, serum, urine and other body fluids or excrement samples), centrifuging at room temperature of 200 Xg for 10min, and removing cells;

2. centrifuging the supernatant at room temperature of 2000 Xg for 10min, and removing debris;

3. centrifuging the supernatant at 12000 Xg for 30min at room temperature to remove platelets and large vesicles;

4. filtering the supernatant with a filter with a pore diameter of 0.45um at medium speed;

5. adding an exosome precipitation reagent, uniformly mixing, and standing for 1 hour at 4 ℃;

6. centrifuging at 12000 Xg for 10min, and discarding the supernatant;

7. resuspend the pellet with 1 × PBS for use.

Second, exosome capture

1. Taking out an exosome-capturing chip (the chip is prepared in example 1, each well of the chip is coated with exosome-specific capture antibodies CD63 and CD9), and adding the obtained sample to be tested into a sample well;

2. adding negative and positive control (the negative and positive control is exosome separated from supernatant of PD-L1 and CD63 over-expressed H1299 cells, or a sample of plasma exosome of healthy and patients with known expression abundance) into a subsequent sample well;

3. after incubation for 2 hours at 37 ℃, the plates were washed 3 times with 1 × PBS;

4. exosome capture conditions: a 15-90V DC electric field.

Nanoparticle fusion and target detection

1. Adding cationic nanoparticles wrapped with target proteins TSG101, HSP70, HIF-1a and AFP fluorescent monoclonal antibodies aiming at exosome and miR-21 and AFPmRNA molecular beacons aiming at exosome into all sample wells;

2. after incubation for 4 hours at 37 ℃, the plates were washed 3 times with 1 × PBS;

3. blocking with 5% BSA at room temperature for 30 min;

4. adding CD63 and PD-L1 fluorescent monoclonal antibody diluent, and incubating for 2 hours at 37 ℃;

5. washing the plate with 1 × PBS for 3 times, and collecting a fluorescence picture by using a TIRF microscope;

6. and (4) analyzing the picture by using DXimageV1 software, automatically setting cut-off value, and automatically interpreting the result of the sample to be detected.

Design of specific molecular beacon (taking target miR-21 and AFP mRNA as examples)

The design of a specific molecular beacon for detecting a target gene is important for detecting specific nucleic acid by an exosome capture pore plate or a chip. For this reason, in combination with the characteristics of the target gene, the applicant designs a molecular beacon with a special stem-loop structure, wherein a 5 ' end stem and a loop are completely complementary with the target gene, a 3 ' end stem is partially complementary with the 5 ' end stem, the 5 ' end and the 3 ' end are respectively modified by a fluorescent group and a quenching group, a part of bases on the loop are modified by locked nucleic acid, specific sequences of the specific miR-21 and PD-L1 mRNA molecular beacon are shown in Table 1, and the base modification mode of the sequence shown in SEQ ID No.1 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 36 th base, wherein the base modification mode of the sequence shown in SEQ ID No.2 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.3 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th and 25 th bases and BHQ1 modification of 34 th base, wherein the base modification mode of the sequence shown in SEQ ID No.4 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.5 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.6 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 36 th base, wherein the base modification mode of the sequence shown in SEQ ID No.7 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 36 th base, wherein the base modification mode of the sequence shown in SEQ ID No.8 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 36 th base, wherein the base modification mode of the sequence shown in SEQ ID No.9 is as follows: base 6FAM modifications at position 1, LNA modifications at positions 10, 13, 16, 19, 22, 25 and 28, and BHQ1 modifications at position 36. The specific sequence of the specific PD-L1 molecular beacon is shown in Table 2, and the base modification mode of the sequence shown in SEQ ID No.10 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.11 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th, 28 th and 31 th bases and BHQ1 modification of 38 th base, wherein the base modification mode of the sequence shown in SEQ ID No.12 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th and 25 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.13 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th, 28 th and 31 th bases and BHQ1 modification of 40 th base, wherein the base modification mode of the sequence shown in SEQ ID No.14 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th, 28 th and 31 th bases and BHQ1 modification of 38 th base, wherein the base modification mode of the sequence shown in SEQ ID No.15 is as follows: 6FAM modification of 1 st base, LNA modification of 10 th, 13 th, 16 th, 19 th, 22 th, 25 th and 28 th bases and BHQ1 modification of 35 th base, wherein the base modification mode of the sequence shown in SEQ ID No.16 is as follows: base 6FAM modifications at position 1, LNA modifications at positions 10, 13, 16, 19, 22, 25 and 28, and BHQ1 modifications at position 36.

The specific molecular beacon designed by the invention improves the specificity of the combination of the molecular beacon and the target gene to the maximum extent, and simultaneously reduces the background fluorescence intensity of the reaction. After synthesis of molecular beacons, in order to verify the specificity of binding with the corresponding target genes and the optimal working temperature, we designed the following table 3, and selected the optimal molecular beacons and their working temperatures according to the highest snr.

TABLE 1 miR-21 Probe sequences

TABLE 2 AFP mRNA Probe sequences

Fifthly, fusing the nanoparticles wrapped with the RNA molecular beacon and the fluorescent monoclonal antibody with the exosomes obtained in the step 2): and (3) carrying out fusion under the condition of a direct current field of 5-90V and at the temperature of 4-60 ℃.

Sixthly, detection results

The invention respectively verifies the aspects of exosome characteristic detection, cell supernatant exosome membrane protein and mRNA expression comparison experiment, human plasma exosome specificity detection, human plasma exosome membrane protein comparison experiment, multi-sample human plasma exosome in-membrane protein, RNA and membrane protein detection and carries out comparison experiment with the existing acknowledged detection technology.

Detection of exosome characteristics

The invention adopts the existing accepted characterization mode of exosome: dynamic Light Scattering (DLS) method.

As shown in fig. 2: the plasma exosome obtained by the treatment of the invention is detected by DLS dynamic light scattering and TEM transmission electron microscope technology, the particle size range is 101.8 +/-41 nm, zeta potential measurement results show that the plasma exosome is negatively charged, the charged charge is-3.25 mV, and electron microscope pictures show that the plasma exosome is of a cup-shaped or bowl-shaped structure, which is basically consistent with the literature reports.

(II) comparative experiment of expression of exosome membrane protein and mRNA in cell supernatant

The invention adopts the existing recognized immunoblotting (Western Blot, WB for short) and fluorescence quantitative PCR technology for detecting the protein and RNA of the exosome, and simultaneously detects the expression of PD-L1 protein and mRNA in the exosome from the cell supernatant of the transfected H1299 lung cancer cell line as a comparison experiment.

As shown in fig. 3: DLS particle size analysis results show that the particle size range of exosomes in the supernatant of transfected H1299 cells is about 205.3 +/-69.6 nm, and zeta potential measurement results show that EVs is negatively charged and the charge is 13.0 mV.

As shown in fig. 4: a is PCR, B is WB, C and D are the results of the experiments on the chip of the invention. And (3) transfecting the H1299 lung cancer cell line with the control expression plasmid and the PD-L1 expression plasmid respectively, extracting total RNA and protein of the cells, and detecting by fluorescence quantitative PCR and WB technology, wherein compared with a control group, both the PD-L1 mRNA and the protein realize overexpression. Exosomes obtained by extracting and purifying the cell culture supernatant through the steps are added to an exosome in-situ capture chip, and the exosome in-situ capture chip is detected by molecular beacon PD-L1 mRNA coated by the nanoparticles and membrane protein thereof, and TIRFM imaging shows that the expression of PD-L1 mRNA and protein in the exosome from the cell culture supernatant is obviously improved compared with that of a control group. The comparison experiment shows that the invention is consistent with the results of western blot (WB for short) and fluorescence quantitative PCR technology for detecting exosome protein and RNA for the same marker PDL1 membrane protein and mRNA simultaneously detected by the same sample.

(III) detection of specificity of human plasma exosomes

The invention uses PBS, pure exosome (blank control, EX only) separated from human plasma, isotype control (EXisotype) and antibody (EX abs) to respectively detect the well-known exosome proteins TSG101, HSP70, membrane protein CD63 and PD-L1.

As shown in fig. 5: exosomes obtained by separating human plasma through the method of the invention are added on an exosome in-situ capture chip, and are detected by targets of subsequent membrane proteins CD6 (figure C)3 and PD-L1 (figure D), exosome in-vivo protein TSG101 (figure A) and HSP70 (figure B), and TIRFM imaging shows that compared with PBS, blank control, isotype control and antibody, the specificity of the fluorescent monoclonal antibody of CD63 and PD-L1 is better.

(IV) human plasma exosome membrane protein comparison experiment

The present invention uses the current accepted flow cytometry for the detection of plasma exosome membrane proteins CD63 and PD-L1 in healthy humans (Normal) and lung cancer patients (Patient) as a comparative experiment.

As shown in fig. 6: the exosomes in the plasma of the healthy control and the lung cancer patient are captured by using the magnetic beads coated by the CD63 monoclonal antibody, the CD63-AF488 and PD-L1-AF647 fluorescent monoclonal antibodies are combined with the CD63 and PD-L1 membrane proteins on the exosome membrane, and the number of the lung cancer patient plasma exosomes and the expression quantity of the CD63 and PD-L1 proteins on the membrane are found to be remarkably increased compared with those of the healthy control group through flow cytometry detection.

(V) detection of multiple samples of human plasma exosome in-membrane protein, RNA and membrane protein

By applying the invention, the generally recognized proteins HSP70, AFP, HIF-1a and representative RNA in the exosome are selected: miR-21, AFPmRNA, and exosome membrane protein PD-L1, which are detected by separating exosomes from 30 plasma samples of healthy human (Normal, shown as JK in the figure) and lung cancer patients (Patient, shown as PT in the figure) respectively, and are respectively as follows: lung cancer detection exosome proteins HSP70 (panel a), HIF-1a (panel B), exosome membrane proteins PD-L1 (panel D), miR-21 (panel E); liver cancer detection exosome protein AFP (graph C) and AFPmRNA (graph F), which are all detected at the same time as healthy controls.

As shown in fig. 7: by using the method for extracting and purifying exosomes, exosomes obtained by separating 30 plasma samples from healthy people (JK) and cancer Patients (PT) are added to an exosome in-situ capture chip, fused with nanoparticles of molecular beacons targeting afpmRNA and miR-21 by wrapping fluorescent monoclonal antibodies of proteins HSP70, HIF-1a and AFP in the exosome in-situ capture chip, combined with fluorescent monoclonal antibodies of targeting exosome membrane protein PD-L1, and TIRFM imaging shows that compared with a healthy control group, in plasma specimens of lung cancer and liver cancer patients, the protein HSP70 in exosome, AFP, HIF-1a and representative RNA: the total fluorescence intensity of miR-21, AFPmRNA and the exosome membrane protein PD-L1 is higher than that of healthy people, and the expression level is obviously increased.

The invention respectively verifies the aspects of exosome characteristic detection, cell supernatant exosome membrane protein and mRNA expression comparison experiment, human plasma exosome specificity detection, human plasma exosome membrane protein comparison experiment, multi-sample human plasma exosome membrane protein, RNA and membrane protein detection, and compares the experiments with the existing recognized immunoblotting (Western Blot, WB for short), fluorescent quantitative PCR technology and flow cytometry for detecting exosome protein, RNA and membrane protein, so that the consistency of the detection result of the same marker simultaneously detected by the same sample and the recognized technology is shown.

Claims

1. A method for simultaneously detecting protein and RNA in exosome and protein of exosome membrane, wherein the RNA comprises mRNA, miRNA, lncRNA and CircRNA, and the method is characterized by comprising the following steps:

2. The method for simultaneously detecting the protein in the exosome, the RNA and the exosome membrane protein according to claim 1, wherein the chip for in-situ capturing the exosome in the step 1) comprises: and the in-situ capture chip is coated with the exosome specificity capture antibody marked by avidin and biotin.

3. The method for simultaneously detecting protein in exosome and RNA as well as exosome membrane protein according to claim 2, wherein the avidin-biotin labeled specific capture antibody is recognized as marker antibodies CD63 and CD9 on exosome membrane.

4. The method for simultaneously detecting an exosome protein and an RNA, exosome membrane protein according to claim 1, wherein the target antibody in step 2) is a specific antibody capable of binding to an antigen on an exosome membrane.

5. The method for simultaneously detecting an exosome protein and an RNA, an exosome membrane protein according to claim 1, wherein the nanoparticle in the step 3) is prepared by the following steps:

6. The method for simultaneously detecting the protein, RNA and the membrane protein of the exosome according to claim 1 or 5, wherein the molar ratio of the molecular beacon to the fluorescent monoclonal antibody in the nanoparticle in the step 3) is 1: 2-1: 5.

7. The method for simultaneously detecting the protein, RNA and the exosome membrane protein in the exosome according to claim 1 or 5, wherein the RNA molecular beacon in the step 3) is that a 5 ' end stem and a loop are completely complementary with a target gene, a 3 ' end stem is partially complementary with a 5 ' end stem, a 5 ' end and a 3 ' end are respectively modified by a fluorescent group and a quenching group, and partial bases on the loop are modified by locked nucleic acid.

8. The method for simultaneously detecting proteins in exosomes and RNA, exosome membrane proteins according to claim 1 or 5, wherein the fluorescent monoclonal antibody in step 3) is a fluorescein-labeled monoclonal antibody directed against a specific exosome target protein.

9. The method for simultaneously detecting the protein in the exosome, the RNA and the membrane protein of the exosome according to claim 1, wherein the fusion condition in the step 4) is the principle of heterogeneous charge attraction and membrane fusion, and the method is carried out under the condition of a direct current field of 15-90V and at the temperature of 4-60 ℃.