Detection method for multiple membrane proteins of tumor-derived exosome and application

Abstract

The invention relates to the technical field of biology, in particular to a method for detecting multiple membrane proteins of tumor-derived exosomes and application of the method. The method comprises the following steps: designing and preparing a corresponding target DNA sequence according to a target protein; designing a DNA probe according to the target DNA sequence, and fixing the DNA probe on the surface of a sensing area of a two-dimensional material sensor; and dropping clear liquid containing the target DNA sequence into a sensing area of the two-dimensional material sensor, and then detecting. The method can realize simultaneous detection of various membrane proteins of TDEVs at low concentration, has the advantages of high sensitivity and strong specificity, avoids the problems of sensitivity reduction and false positive increase caused by non-specific amplification in the traditional nucleic acid detection amplification process, and has the technical advantages of rapidness, simplicity, convenience, accuracy and low cost. Meanwhile, the technology can avoid the overlapping problem of multicolor fluorescence spectrums, and multi-channel mutually independent specific sensing is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting multiple membrane proteins in tumor-derived exosomes and applications thereof. Background Art

[0002] Exosomes refer to extracellular vesicles with a size of 30 to 150 nm in body fluids. In the field of tumors, as a natural carrier of intercellular communication, tumor-derived exosomes (TDEVs) have a highly stable phospholipid bilayer structure. Its formation and secretion are extremely time-sensitive, and its quantity can effectively reflect the progression and activity level of in situ tumors, which is conducive to the real-time monitoring of tumor status. Therefore, TDEVs are considered to be ideal tumor markers, which have important research significance and application value for tumor diagnosis and early screening.

[0003] TDEVs carry a variety of parental cell characteristic molecules and have complex and diverse membrane proteins on their surface. The detection of specific TDEVs membrane proteins has unique advantages in tumor diagnosis. Specifically, TDEVs membrane proteins can exist stably in exosomes and have a long half-life. At the same time, they can further clarify the specificity of tumors by providing rich, stable, sensitive and unique information, thereby more accurately and sensitively reflecting the real-time status of tumors. Therefore, the accurate detection of multiple membrane proteins of TDEVs and their use as a basis for evaluating characteristics such as tumor type and degree of progression will help improve the accuracy and reliability of tumor diagnosis.

[0004] Enzyme-Linked Immunosorbent Assays (ELISA), Western Blot (WB), Mass Spectrometry (MS) and Immunofluorescence (IF) are currently commonly used methods for specific protein detection. ELISA uses specific antibodies to recognize target proteins and quantifies protein expression levels by detecting product signals from reactions with enzyme substrates. WB relies on protein extraction, separation, transfer and other operations to determine the presence of proteins by detecting the binding of antibodies to target proteins. MS identifies and quantifies proteins by measuring the mass and charge ratio of proteins or peptides, and has the characteristics of high sensitivity and high throughput. IF uses fluorescently labeled secondary antibodies for detection, which can show the localization of proteins in cells and their relative expression levels.

[0005] Although the above-mentioned technical methods can specifically detect the target protein, they are unable to achieve the simultaneous detection of multiple membrane proteins of TDEVs, and the detection objects are mostly limited to certain specific protein types. Moreover, the various membrane proteins on the surface of TDEVs are often interrelated. Only by achieving the simultaneous detection of multiple membrane proteins can a detection model based on tumor information be constructed from multiple aspects and dimensions. At the same time, TDEVs are extremely rare in patients, especially in patients with early tumors, and the content of related membrane proteins is even lower. The simultaneous detection of multiple membrane proteins in TEDVs has extremely high requirements for specificity and sensitivity. Although the CRISPR / Cas9 technology with dual precise recognition of EpCAM antibodies and aptamers can increase the number of detectable membrane proteins by utilizing the targeted cutting characteristics of Cas9, the concentration of DNA products produced by the shearing of the CRISPR / Cas system is extremely low, and it is necessary to rely on amplification and other amplification methods for signal recognition, among which non-specific amplification will lead to decreased sensitivity and increased false positives. In addition, the amplified DNA is mostly labeled with fluorescent groups for the final signal readout, and the overlap of multi-color fluorescence spectra also limits the number of proteins that can be detected simultaneously.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method and application for detecting multiple membrane proteins of tumor-derived exosomes. The method for detecting multiple membrane proteins of tumor-derived exosomes can detect and identify multiple exosome membrane proteins at low concentrations, and has high sensitivity and specificity.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] One aspect of the present invention relates to a method for detecting multiple membrane proteins in tumor-derived exosomes, comprising the following steps:

[0010] (a) Capturing the target protein in the tumor-derived exosome sample and preparing the corresponding target DNA sequence according to the target protein design;

[0011] (b) designing a DNA probe according to the target DNA sequence, and fixing the DNA probe on the surface of the sensing area of ​​the two-dimensional material sensor;

[0012] (c) Dropping a clear solution containing the target DNA sequence into the sensing area of ​​the two-dimensional material sensor for detection.

[0013] The method for detecting multiple membrane proteins of tumor-derived exosomes modifies the DNA chain capture probes corresponding to the target proteins in different regions of the two-dimensional material sensor, and realizes the simultaneous detection of multiple membrane proteins of TDEVs at low concentrations. It has the advantages of high sensitivity and strong specificity, and avoids the problems of decreased sensitivity and increased false positives caused by nonspecific amplification in the amplification process of traditional nucleic acid detection. It has the technical advantages of being fast, simple, accurate, and inexpensive. At the same time, this technology can avoid the overlap of multi-color fluorescence spectra and realize multi-channel independent specific sensing.

[0014] The tumor-derived exosome samples mentioned in the present invention are usually obtained from clinical cancer patients, and the sampling site is adjusted according to different cancer types. For example, the tumor-derived exosome samples of lung cancer patients are usually obtained from the pleural effusion of lung cancer patients.

[0015] The present invention does not specifically limit the method for capturing the target protein, and conventional capture methods in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the target protein is captured in a tumor-derived exosome sample by magnetic adsorption.

[0016] In some specific embodiments, carboxyl magnetic beads are used to separate and capture TDEVs in tumor-derived exosome samples, and aptamer-labeled magnetic beads are used to separate and capture target proteins. The carboxyl magnetic beads are used to capture tumor-derived exosomes by modifying targeted binding substances related to tumor-derived exosomes on carboxyl magnetic beads. For each target protein, corresponding aptamer-labeled magnetic beads are designed and prepared, and the aptamer can specifically bind to the target protein.

[0017] The target DNA sequence mentioned in the present invention is a DNA sequence obtained by reverse deducing the sequence of the target protein based on the central dogma.

[0018] Furthermore, the sgRNA is programmed to guide the CRISPR / Cas9 protein to cut the double-stranded nucleic acid substrate to produce the target DNA sequence. The concentration of the DNA product produced by CRISPR / Cas protein shearing is extremely low, and it is necessary to rely on amplification and other amplification methods for signal recognition, among which non-specific amplification will lead to decreased sensitivity and increased false positives. In addition, the amplified DNA is mostly labeled with fluorescent groups for the final signal readout, and the overlap of multi-color fluorescence spectra also limits the number of proteins that can be detected simultaneously. Combining CRISPR / Cas technology with two-dimensional material sensors can overcome the technical problems of low sensitivity, high false positives, and inability to detect multiple membrane proteins simultaneously in CRISPR / Cas technology.

[0019] The present invention does not specifically limit the method for fixing the DNA probe on the surface of the two-dimensional material sensor, and conventional DNA probe connection methods in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the DNA probe is fixed on the surface of the sensing area of ​​the two-dimensional material sensor by surface amino modification.

[0020] In some specific embodiments, surface amino modification specifically comprises the following steps:

[0021] 1. Change the hydroxyl groups on the surface of the sensing area into amino groups: soak the MoS2-TFT in a 5% APTES solution and incubate it in a shaker at 25°C and 180 r / min for 1 h.

[0022] 2. Use anhydrous ethanol to soak and repeatedly rinse the surface of the MoS2-TFT sensor to wash away the high concentration of solution residues. After rinsing, use nitrogen to quickly blow dry to prevent the residual APTES from forming white crystals on the device surface, which will affect the sensitivity of the device and the detection results.

[0023] 3. Place the blow-dried device in an oven at 110°C for 30 minutes for surface silanization to make the amino groups just grown on the surface of the MoS2-TFT sensing area more solid.

[0024] 4. Use 10mmol / L (pH 7.4) PBS solution to prepare 1mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide sodium salt (sulfo-SMCC). Immerse the device in the prepared solution and incubate it on a shaker at 180r / min for 2h. The amino groups in the sensing area of ​​the device form a stable amide bond with sulfo-SMCC.

[0025] 5. After taking out the device, rinse it with 10mmol / L (pH 7.4) PBS solution and blow dry it with nitrogen gas.

[0026] 6. Use 10 mmol / L PBS (pH 7.4) to dissolve tri-(2-formylethyl)phosphine hydrochloride particles, prepare a 10 mmol / L solution to dissolve the DNA probe powder, and incubate it in a shaker for 1 hour.

[0027] 7. Drop the DNA probe solution into the sensing area of ​​the device and incubate it in a wet box at 4°C for 12 hours. The sulfhydryl group at the 5' end of the DNA probe can form a stable thioether bond with sulfo-SMCC, thereby firmly connecting the DNA probe to the sensing area of ​​the device.

[0028] 8. After the probe incubation is completed, remove the device, rinse the unbound probes with 10mmol / L (pH 7.4) PBS solution, and blow dry with nitrogen. At this point, the DNA probe has been firmly connected to the surface of the sensing area through a stable chemical bond, and the detection can be carried out after dropping the DNA liquid sample.

[0029] In biosensor technology, DNA probe is a key molecular recognition element used to detect specific DNA sequences or target molecules. DNA probe can be complementary paired with specific target DNA sequences, and accurately recognize and bind to target molecules through the principle of base complementarity. This highly specific recognition ability makes DNA probe an ideal recognition element in biosensors. In the present invention, a DNA probe is fixedly connected to the surface of the sensing area of ​​the two-dimensional material sensor, and the DNA probe can specifically recognize the target DNA sequence.

[0030] Furthermore, the two-dimensional material sensor includes: a molybdenum disulfide thin film field effect transistor sensor and / or a graphene thin film field effect transistor sensor.

[0031] Two-dimensional material sensors have high sensitivity and can detect non-amplified nucleic acids, avoiding the problem of non-specific amplification in the traditional nucleic acid detection amplification process; two-dimensional material sensors are easy to form arrays, and by modifying different DNA probes for each detection unit, multiple DNA fragments can be detected simultaneously, and they are not easily interfered with each other.

[0032] Two-dimensional materials represented by MoS2 have the characteristics of high specific surface area, which can modify more recognition molecules per unit area and capture biological molecules on the surface of two-dimensional materials through specific binding properties, making it easy to detect concentrated samples.

[0033] Furthermore, in the clear solution containing the target DNA sequence, the concentration of the target DNA sequence is ≥10 -20 mol / L. The detection method of membrane protein of tumor-derived exosomes provided by the present invention has a minimum detection limit of 10 -20 mol / L, which can directly detect the DNA products obtained by CRISPR / Cas technology, which not only improves the sensitivity and specificity of the detection, but also simplifies the detection method.

[0034] Furthermore, in step (c), the amount of the clear solution containing the target DNA sequence added is 2 to 3 μL. The membrane protein detection method only requires a relatively small amount of liquid sample to complete the detection.

[0035] Furthermore, the detection result of step (c) is analyzed using a semiconductor analyzer. A semiconductor analyzer is a test instrument that integrates multiple measurement and analysis functions, and can accurately perform current-voltage measurement, capacitance measurement, etc., and quickly and easily analyze the measurement results to complete semiconductor parameter testing. The present invention can directly use a semiconductor analyzer to analyze the detection results, and the analysis process is no longer limited by the overlap of multi-color fluorescence spectra, so that multiple membrane proteins can be analyzed and detected, and the analysis process is short and accurate.

[0036] Furthermore, the target protein includes: several tumor-derived exosome membrane proteins. The present invention can simultaneously detect multiple tumor-derived exosome membrane proteins to improve detection efficiency.

[0037] The present invention does not specifically limit the tumor type, and common tumor-derived exosome membrane proteins can be detected by the detection method provided by the present invention. In some specific embodiments, the tumor includes at least one of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, cervical cancer or prostate cancer.

[0038] Another aspect of the present invention also relates to a method for evaluating tumor progression, including the method for detecting multiple membrane proteins in tumor-derived exosomes.

[0039] Accurate detection of multiple membrane proteins of TDEVs can be used as an evaluation basis for determining tumor type, degree of progression and other characteristics, which is beneficial to improving the accuracy and reliability of tumor diagnosis.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The method for detecting multiple membrane proteins of tumor-derived exosomes provided by the present invention modifies the DNA chain capture probes corresponding to the target protein in different regions of the two-dimensional material sensor, thereby realizing the simultaneous detection of multiple membrane proteins of TDEVs at low concentrations. It has the advantages of high sensitivity and strong specificity, avoids the problems of decreased sensitivity and increased false positives caused by nonspecific amplification in the traditional nucleic acid detection amplification process, and has the technical advantages of being fast, simple, accurate and inexpensive.

[0042] (2) The method for detecting multiple membrane proteins of tumor-derived exosomes provided by the present invention can avoid the overlap problem of multi-color fluorescence spectra and realize multi-channel independent specific sensing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 MoS2-TFT sensor before and after ssDNA probe modification I d -V g Curve changes;

[0045] Figure 2 MoS2-TFT sensor I for A1 chain sample d -V g curve;

[0046] Figure 3 I is the A1 chain sample d -V g The linear region of the curve;

[0047] Figure 4 is the response curve of A1 chain sample at different concentrations;

[0048] Figure 5 The fitting results of the linearity parameter test of the A1 chain sample response;

[0049] Figure 6 MoS2-TFT sensor I for A3 chain sample d -V g curve;

[0050] Figure 7 I is the A3 chain sample d -V g The linear region of the curve;

[0051] Figure 8 is the response curve of A3 chain sample at different concentrations;

[0052] Fig. 9 The fitting results of the response linearity parameter test for the A3 chain sample;

[0053] Fig.10 The Id-Vg curve of the MoS2-TFT sensor of the supernatant sample containing only the A1 chain after CRISPR / Cas9 technology treatment;

[0054] Fig.11 I is the supernatant sample containing only A1 chain after CRISPR / Cas9 technology treatment d -V g The linear region of the curve;

[0055] Fig.12 The Id-Vg curve of the MoS2-TFT sensor containing only the A3 chain supernatant sample after CRISPR / Cas9 technology treatment;

[0056] Fig.13 I is the supernatant sample containing only the A3 chain after CRISPR / Cas9 technology treatment d -V g The linear region of the curve;

[0057] Fig.14 For the supernatant sample with both A1 and A3 chains, the A1 capture probe sensor modified with TFT device was used for the detection of I d -V g curve;

[0058] Fig.15 For the supernatant sample with both A1 and A3 chains, the A3 capture probe sensor modified with TFT device was used for the detection of I d -V g curve. DETAILED DESCRIPTION

[0059] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0060] Example 1 Construction of CRISPR / Cas9 reaction system and verification of probe modification

[0061] The detection targets are two tumor-related proteins in lung cancer exosomes, including the following steps:

[0062] 1. Design and prepare carboxyl magnetic beads (MNPs1) for specific capture of TDEVs; design and prepare aptamer-labeled magnetic beads corresponding to two target proteins (MNPs2 A1 and MNPs2 A3), and use MNPs1, MNPs2 A1 and MNPs2 A3 to capture target proteins in tumor-derived exosome samples;

[0063] 2. Program the small guide RNA (sgRNA) to guide the CRISPR / Cas9 protein to cut the double-stranded nucleic acid substrate to produce two different target DNA sequence fragments A1 and A3 (A1: 5'-ACTAGGGACACACGACTCT-3'; A3: 5'-ATGAGGGCGGTGTGTGAGT-3') according to the captured target protein, converting the exosome membrane protein detection into DNA detection;

[0064] 3. Using magnetic separation technology, the supernatant containing two different DNA sequence fragments in the reaction system after step 2 is taken out for later use;

[0065] 4. Based on the two target DNA fragments, the corresponding DNA probes were designed respectively (A1 probe: 5'-AGAGTCGTGTGTCCCTAGT-3'; A3 probe: 5'-ACTCACACACCGCCCTCAT-3'), and the preparation method of substrate surface amino modification was adopted to firmly connect the DNA probes to the surface of MoS2-TFT sensing area through stable chemical bonds;

[0066] 5. Drop the reserved supernatant into the sensing area of ​​the MoS2-TFT chip for detection; use a semiconductor analyzer to verify the probe modification effect, such as Figure 1 shown.

[0067] It can be seen from the voltammetric characteristic curve that after the APTES-SMCC modification step, I d -V g The curve hardly changes. After modifying the ssDNA capture probe, the capture probe introduces additional electrons, which leads to I d The ssDNA probe was successfully connected.

[0068] Example 2 Highly sensitive detection of single component DNA standard samples

[0069] We purchased single-component standard samples of two single-stranded DNA aptamers, A1 and A3, respectively, and prepared a series of standard samples with different concentrations of target DNA sequences and conducted experiments to obtain a set of voltammetric characteristic curves. The experimental process includes:

[0070] 1. Add a sample of a certain concentration to the thin film sensor, let it stand for about 1 minute, and measure its voltammetric curve using a semiconductor analyzer;

[0071] 2. Add another concentration sample to the next sensing area and repeat the above process;

[0072] 3. Similarly, the volt-ampere characteristic curves of samples of all concentrations are obtained through testing.

[0073] Extract the voltammetric characteristic curve parameters and plot the changing trend with sample concentration to evaluate the sensitivity, linearity and dynamic stability of the thin film field effect transistor. Figure 2 , Figure 3 , Figure 4 and Figure 5 shown.

[0074] The concentration used is 10 -20 mol / L~10 -6 mol / L A1 chain standard sample was used for detection. As the concentration of the standard sample increased, the number of additional electrons introduced increased, thus I d The curve shifts to the right, proving that the MoS2-TFT sensor can achieve high sensitivity detection of the concentration of the A1 chain. By fitting the data of the standard sample concentration gradient detection results, it is proved that the MoS2-TFT sensor has good linearity for the detection of different concentrations of the A1 chain standard samples.

[0075] Similarly, the A3 chain sensing data based on MoS2-TFT chip is as follows Figure 6 , Figure 7 , Figure 8 and Fig. 9 shown.

[0076] The concentration used is 10 -20 mol / L~10 -6 mol / L A3 chain standard sample was tested. As the concentration of the standard sample increased, the number of additional electrons introduced increased, thus I d The curve shifts to the right, proving that the MoS2-TFT sensor can achieve high sensitivity detection of the concentration of the A3 chain. By fitting the data of the standard sample concentration gradient detection results, it is proved that the MoS2-TFT sensor has good linearity for the detection of A3 chain standard samples with different concentrations.

[0077] A horizontal comparison of the data of A1 and A3 shows that the MoS2-TFT sensor can not only detect the standard sample of DNA chain, but also detect different types of DNA samples. d -V g The curves showed different trends with sample concentration, which was reflected in the response linearity parameters.

[0078] Example 3 Highly specific detection of mixed samples of multiple DNA components

[0079] In the actual environment, the sequence fragments generated by CRISPR / Cas9 technology cutting double-stranded nucleic acid substrates are usually mixed samples of DNA with multiple components. In order to detect the target DNA chain content in the mixed sample, the sgRNA is programmed to induce CRISPR / Cas9 technology to cut different nucleic acid substrates, and after centrifugation, the supernatant obtained contains only A1 chain, only A3 chain, and both A1 chain and A3 chain. The supernatant is dripped into the sensing area after surface functionalization treatment to evaluate the DNA chain-specific detection ability of the TFT sensor.

[0080] First, the supernatant containing only A1 chains was dropped into the MoS2-TFT sensing area of ​​the surface-modified A1 capture probe. The voltammetric characteristic curve is shown in Fig.10 and Fig.11 shown.

[0081] From the experimental results, it can be seen that with the increase of A1 chain sample concentration, I d The curves shifted rightward successively, proving that the surface-functionalized MoS2-TFT sensor can be used to distinguish the differences in A1 chain content in the supernatant.

[0082] To further verify the specificity, the supernatant containing only A3 chains was dropped into the MoS2-TFT sensing area of ​​the surface-modified A3 capture probe. The voltammetric characteristic curves are shown in Figure 2. Fig.12 and Fig.13 shown.

[0083] From the experimental results, it can be seen that with the increase of A3 chain sample concentration, I d The curves shifted rightward successively, proving that the surface-functionalized MoS2-TFT sensor can be used to distinguish the differences in A3 chain content in the supernatant.

[0084] On this basis, the clinically obtained concentrations were 0, 10 -9 mol / L and 10 -8 After CRISPR / Cas9 treatment and centrifugation, the supernatant containing the mixed sample of A1 chain and A3 chain was obtained. The concentration of DNA chain was different. The supernatant was added to the surface of TFT sensor modified with A1 capture probe and A3 capture probe respectively. The sensing test results are shown in Figure 2. Fig.14 and Fig.15 shown.

[0085] It can be seen from the experimental results that, for supernatants with different concentrations of A1 and A3 chains coexisting, the TFT sensor device can recognize single DNA chains respectively and has good specific detection capability.

[0086] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. A method for detecting multiple membrane proteins in tumor-derived exosomes, characterized in that: The following steps are involved: (a) capturing a target protein in a tumor-derived exosome sample, and designing and preparing a corresponding target DNA sequence according to the target protein; (b) designing a DNA probe according to the target DNA sequence, and fixing the DNA probe on the surface of the sensing area of ​​the two-dimensional material sensor; (c) Dropping a clear solution containing the target DNA sequence into the sensing area of ​​the two-dimensional material sensor for detection.

2. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: The sgRNA is programmed to guide the CRISPR / Cas9 protein to cut the double-stranded nucleic acid substrate to produce the target DNA sequence.

3. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: The DNA probe is fixed on the surface of the sensing area of ​​the two-dimensional material sensor by modifying the surface amino groups.

4. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: The two-dimensional material sensor includes: a molybdenum disulfide thin film field effect transistor sensor and / or a graphene thin film field effect transistor sensor.

5. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: In the clear solution containing the target DNA sequence, the concentration of the target DNA sequence is ≥10 -20 mol / L.

6. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: In step (c), the amount of the supernatant containing the target DNA sequence added is 2 to 3 μL.

7. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: The detection result of step (c) is analyzed using a semiconductor analyzer.

8. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 1, characterized in that: The target proteins include: several tumor-derived exosome membrane proteins.

9. The method for detecting multiple membrane proteins of tumor-derived exosomes according to claim 8, characterized in that: The tumor includes at least one of lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, cervical cancer or prostate cancer.

10. A method for evaluating tumor progression, characterized in that: A method for detecting multiple membrane proteins in tumor-derived exosomes comprising the method according to any one of claims 1 to 9.

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