CN110108885B

CN110108885B - Method for detecting PD-L1 protein on surface of extracellular vesicle by aptamer of programmed death receptor-ligand 1

Info

Publication number: CN110108885B
Application number: CN201910308759.9A
Authority: CN
Inventors: 杨朝勇; 黄梦娇; 宋彦龄; 王腾; 陈小锋; 朱志
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2019-04-17
Filing date: 2019-04-17
Publication date: 2020-10-09
Anticipated expiration: 2039-04-17
Also published as: CN110108885A

Abstract

The invention discloses a method for detecting PD-L1 protein on the surface of an extracellular vesicle by using a nucleic acid aptamer of programmed death receptor-ligand 1(PD-L1), which comprises the following steps: 1) preparing a mixture of PD-L1 aptamer with constant concentration and extracellular vesicle samples with different concentrations; 2) the sample mixture was placed in a microcalorimetric Thermophoresis (MST) device for measurement; 3) and measuring the fluorescence of the sample mixture of each different extracellular vesicle concentration, plotting the obtained normalized fluorescence with respect to time, fitting the MST track line, and then plotting the normalized fluorescence of the MST track line with respect to the concentration of the extracellular vesicles to fit a binding curve of the extracellular vesicle concentration and the normalized fluorescence, thereby realizing the detection of the PD-L1 protein on the surface of the extracellular vesicles. Compared with the prior art, the method has the advantages of high sensitivity, simple and convenient operation, rapid and efficient detection, small sample consumption, economical price and wide popularization potential.

Description

Method for detecting PD-L1 protein on surface of extracellular vesicle by aptamer of programmed death receptor-ligand 1

Technical Field

In recent years, liquid biopsy technology for precision medicine has been widely used in the fields of cancer early screening, auxiliary diagnosis, drug efficacy prediction and monitoring. Accurate prediction and dynamic monitoring of the efficacy of PD-1/PD-L1 inhibitors have become a major concern in the clinical community. The invention relates to a method for detecting PD-L1 protein on the surface of an extracellular vesicle by using a nucleic acid aptamer of programmed death receptor-ligand 1, which is helpful for advancing a new method for detecting the expression level of PD-L1.

Background

With the development of science and technology, as a new revolutionary technology for tumor therapy, immunotherapy, especially immune checkpoint therapy, develops rapidly, becoming one of the important directions in the field of tumor research at present. Among them, immunotherapy based on the PD-1/PD-L1 antibody is a research hotspot in the field of tumor immunotherapy nowadays, is concerned, and opens a new mode of tumor therapy.

Immunotherapy based on the PD-1/PD-L1 antibody has been approved for the treatment of a variety of advanced tumors, and while some tumor patients benefit from it, not all tumor patients benefit from it. For patients who do not respond to the drug, it is important to know which patients will have the effect, because the treatment is not effective, but also suffers from the side effects of the treatment and the expensive burden of the treatment. Studies have shown that PD-L1 is highly expressed on tumor cells, and thus, high expression of PD-L1 means that when the binding of PD-1 to PD-L1 is blocked, the effect of the PD inhibitor is better and the patient's benefit is more likely. Therefore, the expression level of tumor cell PD-L1 is considered as a prediction index of the curative effect of PD-1/PD-L1 antibody drug treatment, and the detection of the PD-L1 level of a patient before drug administration can guide whether the drug treatment is needed. Therefore, the use of PD-1/PD-L1 antibody drugs requires additional detection of the expression of PD-L1, and then selection of whether the drug can be administered or not is made according to the patient's own condition.

The determination of the expression level of tumor cell PD-L1 by immunohistochemistry can directly reflect the response rate of cancer patients to PD inhibitors. However, the expression level of tumor cell PD-L1 is not constant, but dynamically changing. Chemotherapy, radiotherapy and the like can affect the expression of the tumor PD-L1. Therefore, the expression level of the PD-L1 of the tumor cells cannot be used as the only prediction index of the curative effect of the PD-1/PD-L1 antibody medicine.

Extracellular Vesicles (EVs) are a subcellular component produced by paracrine secretion, and are essentially a group of nanoscale particles including exosomes (exosomes), membrane Microparticles (MPs), Microvesicles (MVs), and the like. Almost all cells produce extracellular vesicles containing various bioactive components derived from the mother cell such as lipids, proteins, and nucleic acids (DNA, mRNA, microRNA, lncRNA, circRNA, etc.), and these information substances are encapsulated in the vesicles or carried on the membrane. The extracellular vesicles of specific types are expected to become new molecular markers for assisting disease diagnosis and prognosis judgment, and have wide prospects in anti-tumor treatment, regenerative medicine, immune regulation and other aspects. In a new study, it was found that the exosome surface released by tumor cells also expressed PD-L1, and also bound T cells directly and inhibited the anti-cancer activity of these T cells. Therefore, the identification that exosomes secreted by tumor cells also express PD-L1 provides new insights into tumor immune escape and immune checkpoint mechanisms. Therefore, monitoring the expression level of PD-L1 in exosomes may become another way for cancer patients to predict the therapeutic efficacy of PD-1/PD-L1 antibody drugs.

Currently, the commonly used PD-L1 detection methods include two methods, namely Immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and the like, wherein IHC is most widely used in membrane type PD-L1(mPD-L1) detection, but has disadvantages, such as diversity of antibodies to be stained, and non-uniformity of staining techniques and conditions; the result interpretation is mainly determined by people and has certain subjectivity; while ELISA has a dominant position in soluble form PD-L1(sPD-L1), the method is complex and time-consuming in operation and large in sample consumption, and is not an economical and practical choice. In addition, the above two methods use antibodies, and have the disadvantages of instability, easy inactivation and high cost, so that the detection of the expression level of PD-L1 by using a more stable, more effective and lower-cost binding ligand of PDL1 is needed, and the detection method of PD-L1 is in need of further innovation and popularization.

Microcalorimetry (MST) is a new technology for measuring biomolecular interactions that has emerged in recent years. Based on the directional movement of the fluorescence labeling molecules in the temperature gradient, the method can sensitively and rapidly measure the interaction between the biomolecules in a homogeneous solution. The change of the charge, the mass, the conformation and the hydration layer of the biological molecules can change the movement rate of the biological molecules in the temperature gradient field, and the interaction between the biological molecules is analyzed through the change, so that the method is suitable for the interaction research of various substances such as proteins, nucleic acids, small molecules and the like. It has the advantages that: the test mode is superior to the traditional technology, can be applied to any buffer solution for operation, and does not need surface fixation; a small amount of material is used, and only 4 mu L of sample is needed at minimum; the method is more efficient, is simple and convenient to operate, and does not need to purify a sample; the application range is wide, the interaction between any biomolecules can be detected, and the kit is also suitable for unstable samples with large volume, such as liposomes, outer vesicles and exosomes.

The aptamer (aptamer) is a functional single-stranded oligonucleotide obtained by Exponential Enrichment of ligand Systematic Evolution (SELEX), and can be combined with a target with high affinity and high specificity. Compared with an antibody, the aptamer has the advantages of chemical synthesis, small batch difference, small molecular weight, high thermal stability and the like, is a molecular detection tool with excellent performance, and has important application value in the field of biochemical analysis and detection.

With the continuous development of precise medicine, the expression detection of PD-L1 in extracellular vesicles is likely to be a marker for concomitant or auxiliary diagnosis, which will help clinicians to effectively screen patients for potential benefit of PD-1/PD-L1 treatment. The detection method of PD-L1 is continuously innovative, which will be a great progress in precision medicine and personalized medicine, but still needs more research.

Disclosure of Invention

The invention mainly aims to overcome the problems of uncertainty, incompleteness, complex detection method steps, complex and time-consuming operation, large sample consumption, high price and the like of detecting the expression level of tumor cell PD-L1 in the prior art, and provides a method for detecting PD-L1 protein on the surface of an extracellular vesicle by using a aptamer of programmed death receptor-ligand 1, wherein the PD-L1 expressed on the surface of the extracellular vesicle is detected by using the aptamer of PD-L1 with high specificity and high affinity, and the combination of the PD-L1 and the PD-L is detected by using microcalorimetric electrophoresis equipment.

The invention adopts the following technical scheme:

a method for detecting PD-L1 protein on the surface of an extracellular vesicle by using aptamer of programmed death receptor-ligand 1, which comprises the following steps:

1) preparing a PD-L1 aptamer with constant concentration and extracellular vesicles with different concentrations, and respectively mixing the PD-L1 aptamer with constant concentration and the extracellular vesicles with different concentrations to prepare a series of sample mixtures;

2) the series of sample mixtures was placed in a microcale Thermophoresis (MST) apparatus for measurement;

3) and measuring the fluorescence of the sample mixture of each different extracellular vesicle concentration, plotting the obtained normalized fluorescence with respect to time, fitting the MST track line, and then plotting the normalized fluorescence of the MST track line with respect to the concentration of the extracellular vesicles to fit a binding curve of the extracellular vesicle concentration and the normalized fluorescence, thereby realizing the detection of the PD-L1 protein on the surface of the extracellular vesicles.

Wherein the PD-L1 aptamer has a sequence shown in SEQ ID No.1 and is:

5'-TACAGGTTCTGGGGGGTGGGTGGGGAACCTGTT-3'。

preferably, the PD-L1 nucleic acid aptamer sequence may be labeled 5' with CY 5.

Preferably, the extracellular vesicles are secreted by the tumor cell line to include exosomes.

Preferably, when the extracellular vesicles are exosomes, the concentration of exosomes in the sample mixture can be in the range of 22.2-668.3 μ g/mL.

Preferably, the concentration of the PD-L1 aptamer in the sample mixture can be 0.05-0.2 nM.

Preferably, the cell may be a tumor cell.

Preferably, the tumor may be metastatic melanoma, non-small cell lung cancer, gastric cancer, breast cancer, or the like.

Preferably, the microcalorimetric electrophoresis device may be Monolith nt.115.

Preferably, the sample mixture is tested without requiring excessive incubation, and the test is performed immediately, the whole test process is about 10min, and the required volume is at least 4 μ L.

As can be seen from the above description of the invention, compared with the prior art, the method for detecting the PD-L1 protein on the surface of the extracellular vesicle by using the aptamer of the programmed death receptor-ligand 1 has the advantages of high sensitivity, simple operation, rapid and efficient detection, small sample consumption, low price, economy and practicability, and wide popularization potential.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a representative Transmission Electron Microscope (TEM) image of exosomes secreted from melanoma cell line A375. Scale bar, 100 nm.

FIG. 2 is a graph of the particle size of exosomes secreted by melanoma cell line A375 as characterized by a Nanoparticle Tracking Analyzer (NTA). In fig. 2, the ordinate is the exosome concentration and the abscissa is the particle size.

FIG. 3 is a Western Blot analysis (Western Blot) of a cell lysate (W) microvesicle (M) and an exosome (E) of melanoma cell line A375, which analyzes three proteins, PD-L1, CD63 and Actin, wherein PD-L1 is programmed death receptor-ligand 1, CD63 is transmembrane protein which is rich in exosome transport on an exosome membrane, namely exosome marker, and Actin is Actin and is an endoglin. And all lanes contained the same amount of total protein.

FIG. 4 shows the flow cytometry investigation of the expression of two proteins, PD-L1 and CD63, secreted by melanoma cell line A375, wherein panel A shows the binding of CD63 antibody to exosome, panel B shows the binding of PD-L1 antibody to exosome, panel C shows the binding of CY 5-labeled PD-L1 aptamer to exosome, and exosome is conjugated to latex aldehyde beads through adsorption. In fig. 4, the abscissa represents the fluorescence intensity.

FIG. 5 shows that the binding between the CY 5-labeled PD-L1 aptamer and the exosome PD-L1 protein is analyzed by examining the expression of the exosome PD-L1 protein secreted by the melanoma cell line A375 through a laser confocal fluorescence microscope. And exosomes are conjugated to latex aldehyde beads by adsorption.

FIG. 6 shows the binding of PD-L1 aptamer to the exosome PD-L1 protein secreted by melanoma cell line A375 detected by microcale thermolysis (MST). Wherein graph A is a measure of each differenceFluorescence within the capillary at the exosome concentration and normalized fluorescence in the heating spot was plotted against time to give the MST trace. In the graph A, the ordinate represents the fluorescence intensity, and the abscissa represents time. Panel B is a plot of relative normalized fluorescence of the MST traces (Δ Fnorm) versus exosome concentration fitted to obtain binding curves correlating exosome concentration to Δ Fnorm, with the markers on the right side of the plot being 668, 579, 490, 401, 312, 223, 134, 89, 67, 22, 0 μ g/mL, from top to bottom, respectively. In panel B, the ordinate is relative normalized fluorescence and the abscissa is exosome concentration. The concentration of CY 5-labeled PD-L1 aptamer remained constant at 0.1nM, while the exosome concentration ranged from 668.25 to 22.275. mu.g/mL. By Fnorm ═ F_hot/F_coldNormalized fluorescence (Fnorm) was calculated. Δ Fnorm ═ Fnorm_{(high concentration of exosomes)}-Fnorm_(blank)In which F is_hotAnd F_coldMean fluorescence intensity at defined time points representing MST traces.

Detailed Description

The present invention will be described in detail with reference to the following examples:

example 1 differential centrifugation method for extracting exosomes secreted by melanoma cell line A375

For exosomes extracted from melanoma cell line a375 cell culture supernatant, interference from exosomes in fetal bovine serum was avoided, and bovine serum (FBS) without bovine exosomes was obtained by centrifuging fetal bovine serum overnight at 100000 × g to remove exosomes. Then, the cells were cultured at 37 ℃ in medium supplemented with 10% exosome-depleted FBS and 1% (v/v) penicillin-streptomycin. After 60 hours of incubation, cell culture supernatants were collected and centrifuged at 2000 × g for 20 minutes at 4 ℃ (Eppendorf, 5424R) to remove cells and cell debris, and supernatants were collected and centrifuged at 16500 × g for 45 minutes at 4 ℃ to remove a large number of microvesicles (Eppendorf, 5424R). The microvesicle pellet was resuspended in PBS and stored in the refrigerator at 4 ℃. The supernatant was centrifuged at 100000g for 2 hours at 4 ℃ and the exosomes were collected (Beckman Coulter, Optima XPN-90). The precipitated exosomes were suspended in PBS and collected by ultracentrifugation at 100000 × g for 2 hours, repeated once, and finally suspended in PBS and stored at 4 ℃.

Example 2 morphological characterization of exosomes secreted by melanoma cell line A375

In order to identify exosomes, morphological analysis of exosomes is required. Then, in order to characterize the purified vesicles as exosomes, they can be characterized by transmission electron microscopy and nanoparticle tracking analyzer. Firstly, characterizing the appearance and size of exosomes by a transmission electron microscope, and specifically operating the following steps: exosomes were dropped onto carbon-coated copper grids, left to stand for 1 minute (the mesh was held lightly with tweezers to prevent the mesh from breaking), stained with 2% uranyl acetate at room temperature for 1 minute, then the stain was blotted dry along the edges with filter paper, the mesh was baked under a lamp for 10 minutes, and photographed by observation using a transmission electron microscope (Tecnai, G2spirit FEI, USA). Whereas the concentration and particle size distribution of exosomes were measured by nanoparticle tracking analyzer (particle metric, Zeta View, Germany).

Results as shown in fig. 1 to 2, fig. 1 is a representative transmission electron microscope image of exosomes secreted from melanoma cell line a 375. Scale bar, 100 nm. From the figure, it is clear and intuitive that the outer vesicle of the bilayer membrane is a typical structure of exosome. Fig. 2 shows the particle size of exosomes secreted by melanoma cell line a375 characterized by a nanoparticle tracking analyzer. The average particle size of the exosome can be seen to be 134.4nm, which is consistent with the 30-150 nm reported in the literature. The above two results indicate that exosomes have been successfully extracted.

Example 3 validation of secretion of melanoma cell line A375 by exosomes expressing PD-L1

Following successful extraction of exosomes, it is important to characterize the proteins expressed by exosomes, such as CD63 and PD-L1, as identifiable by western blotting. Using 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to analyze, loading cell lysate (W) microvesicles (M) and exosomes (E) respectively, wherein the total amount of loaded protein is equal, and three pieces of gel run in parallel; after that and transferred to nitrocellulose membrane, blots were blocked with blocking buffer (Beyotime Biotechnology) for 1 hour at room temperature, washed and then incubated with PD-L1, CD63 and Actin primary antibody respectively overnight at 4 ℃ and washed and then incubated with HRP conjugated secondary antibody (Cell signaling technology) for 2 hours at room temperature; after washing, the blot on the membrane was developed with ECL detection reagent (Pierce), and photographed by chemiluminescence imager. Wherein CD63 is used as an exosome marker. Actin is used as an internal reference protein.

The results are shown in FIG. 3, which is a Western blot analysis of microvesicles (M) and exosomes (E) of cell lysate (W) of melanoma cell line A375, and three proteins PD-L1, CD63 and Actin were analyzed. From the figure, it can be seen that only exosomes have CD63 expression, further confirming the successful extraction of exosomes. For PD-L1, cell lysate, microvesicles and exosomes are expressed, and the expression of PD-L1 on the extracted exosomes is successfully verified. In addition, Actin is used as an internal reference protein, and cell lysate, microvesicles and exosomes are expressed, so that the experiment is proved to have no problems.

Exosomes are too small to be reliably analyzed directly by flow cytometry and confocal imaging. To overcome this problem, exosomes were conjugated to latex aldehyde beads of approximately 3 μm size by adsorption. Latex aldehyde bead-exosome complexes were then detected with fluorescently labeled antibodies and analyzed by flow cytometry and confocal imaging. This facilitates rapid semi-deterministic characterization of exosome surface proteins. First, 10 μ g of exosomes quantified by a dihydrochlorinated formic acid assay (BCA) were incubated with 5 μ L of latex aldehyde beads in a 1.5mL microcentrifuge tube for 15 minutes, followed by addition of PBS to a final volume of 1mL and incubation for 2 hours. Next, 100. mu.L of a blocking solution containing 1M glycine and 20% BCA (w/v) was added and incubated for 30 minutes. Finally, centrifugation was carried out at 5000rpm for 3 minutes, the supernatant was removed, the latex aldehyde bead-exosome complex pellet was resuspended and washed twice by centrifugation, and finally resuspended in 50 μ L PBS (0.5% BSA) and stored at 4 ℃. All steps were performed at room temperature. Next, 5. mu.L of latex aldehyde bead-exosome complex were incubated with 5. mu.L of exosome protein antibody (PD-L1 and CD63 antibody) and 200nM CY 5-labeled PD-L1 aptamer (PD-L1 aptamer shown as SEQ ID No. 1) in 100. mu.L PBS (0.5% BSA) for 40 minutes at room temperature, respectively. Centrifugation was carried out at 5000rpm for 3 minutes, the supernatant was removed, the latex aldehyde bead-exosome complex pellet was resuspended and washed twice by centrifugation, resuspended in 50. mu.L of PBS (0.5% BSA), and finally the fluorescence intensity of the latex aldehyde bead-exosome-antibody or aptamer complex was measured using a flow cytometer (Beckman Coulter, CytoFLEX) and the fluorescence signal of the latex aldehyde bead-exosome-aptamer complex was detected by a confocal laser fluorescence microscope. Irrelevant isotype-matched primary antibody (Control) and Random Sequence (RS) served as negative controls.

The results are shown in fig. 4 to fig. 5, fig. 4 shows the expression of two proteins, PD-L1 and CD63, secreted by melanoma cell line a375, by flow cytometry, wherein fig. a shows the binding of the CD63 antibody to exosomes, fig. B shows the binding of the PD-L1 antibody to exosomes, and fig. C shows the binding of the CY 5-labeled PD-L1 aptamer to exosomes. As can be seen in panel a, the CD63 antibody was more biased relative to the control antibody, indicating that exosomes expressed CD63, again supplementing the demonstration of successful exosome extraction. As can be seen in FIG. B, the PD-L1 antibody is more biased relative to the control antibody, again supplementing the evidence that PD-L1 is expressed on the extracted exosomes. As can be seen from FIG. C, the PD-L1 aptamer was more shifted from the random sequence, demonstrating that the PD-L1 aptamer can specifically bind to PD-L1 on the surface of exosomes, i.e., the PD-L1 protein on the surface of exosomes can be recognized and detected by the PD-L1 aptamer. FIG. 5 shows that the binding between the CY 5-labeled PD-L1 aptamer and the exosome PD-L1 protein is analyzed by examining the expression of the exosome PD-L1 protein secreted by the melanoma cell line A375 through a laser confocal fluorescence microscope. As can be seen from the figure, the random sequence has no fluorescence or very weak fluorescence, while the PD-L1 aptamer has stronger fluorescence compared with the random sequence, and the PD-L1 aptamer is proved to recognize, bind and detect PD-L1 on the surface of the exosome again.

Example 4 detection of exosome PD-L1 protein secreted by melanoma cell line A375 by the PD-L1 aptamer through microcalorimetric electrophoresis

First, a dilution of CY 5-labeled PD-L1 nucleic acid aptamer (PD-L1 nucleic acid aptamer is shown in SEQ ID No. 1) was prepared. First, the optimal concentration of the PD-L1 aptamer was determined to be 0.1nM by the NanoTemper analysis software. However, in preparing the final reaction mixture in binding buffer, mixing with serial dilutions of exosomes was required to be 1:1(v/v) to reduce the PD-L1 aptamer concentration by 50% in each dilution step, so the CY 5-labeled PD-L1 aptamer should be prepared at 0.2 nM.

Then, exosome serial dilutions were prepared. For this experiment, 12 concentrations of exosomes were detected, so 12 200 μ L centrifuge tubes were prepared, labeled from 1 to 12; then 20 μ L of exosome stock was added to

tube

1, 10 μ L of binding buffer was added to centrifuge tubes 2-12, respectively, 10 μ L of exosome stock from tube 1 was transferred to tube 2 and mixed well, 10 μ L was transferred to the next tube and the dilution was repeated for the remaining tubes, and finally 10 μ L was discarded from tube 12. Thus, 12 10 μ L exosome dilution series were finally obtained.

Then, a final reaction mixture, namely a mixed solution of the CY 5-labeled PD-L1 aptamer and exosome, is prepared. To minimize pipetting errors, the optimal volume for individual binding reactions was 20 μ L (10 μ L aptamer solution +10 μ L exosome dilution). Therefore, 10. mu.L of 0.2nM aptamer working solution was added to 10. mu.L of each exosome dilution, respectively, and the samples were mixed well several times by pipetting up and down, and then the sample mixture was filled into a good quality capillary (MO-K025Premium Capillaries, Beijing, China). The capillaries were then placed on a capillary tray and then inserted into a MST apparatus (MicroScale Thermophoresis instrument Monolith NT.115, NanoTemper technologies, Munich, Germany) for measurement.

And finally, measuring the fluorescence in the capillary at each different exosome concentration, plotting the obtained normalized fluorescence with respect to time, fitting an MST track line, and then, according to the plotting of the normalized fluorescence of the MST track line with respect to the exosome concentration, fitting a binding curve of the exosome concentration and the normalized fluorescence, thereby realizing the detection of the exosome surface PD-L1 protein.

The results are shown in FIG. 6, and FIG. 6 shows the detection of the binding of the PD-L1 aptamer to the exosome PD-L1 protein secreted by melanoma cell line A375 by microcalorimetric electrophoresis. Where panel a is normalized fluorescence plotted against time, resulting in a MST trace. Panel B is a plot of the relative normalized fluorescence (Δ Fnorm) of the MST traces versus the exosome concentration, fitted to obtain a binding curve correlating exosome concentration with Δ Fnorm. As can be seen from Panel A, there is a good distribution of MST traces over the range of exosome concentrations, while the binding curve established from Panel B relating exosome concentrations to Δ Fnorm shows that the detectable exosome concentrations of PD-L1 aptamers range from 22.275 μ g/mL to 668.25 μ g/mL.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Sequence listing

<110> university of mansion

<120> method for detecting PD-L1 protein on surface of extracellular vesicle by aptamer of programmed death receptor-ligand 1

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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tacaggttct ggggggtggg tggggaacct gtt 33

Claims

1. A method for detecting extracellular vesicle surface PD-L1 protein with aptamers to programmed death receptor-ligand 1(PD-L1), said method being a method for non-disease diagnostic purposes comprising the steps of:

1) preparing a PD-L1 aptamer with constant concentration and extracellular vesicles with different concentrations, and respectively mixing the PD-L1 aptamer with constant concentration and the extracellular vesicles with different concentrations to prepare a series of sample mixtures; the PD-L1 aptamer has a sequence shown in SEQ ID No. 1;

2) placing the series of sample mixtures into a microcalorimetric electrophoresis (MST) device for measurement;

2. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the extracellular vesicles include exosomes.

3. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the extracellular vesicles are exosomes; in the sample mixture, the concentration range of exosomes is 22.2-668.3 mug/mL.

4. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the concentration of the PD-L1 aptamer in the sample mixture is 0.05-0.2 nM.

5. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the PD-L1 aptamer sequence is a 5' end CY5 marker.

6. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the cell is a tumor cell.

7. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 6, wherein: the tumor is metastatic melanoma, non-small cell lung cancer, gastric cancer or breast cancer.

8. The method for detecting the extracellular vesicle surface PD-L1 protein with aptamer of programmed death receptor-ligand 1(PD-L1) according to claim 1, wherein: the microcalorimetric electrophoresis device is Monolith NT.115.