Amphiphilic polypeptide for capturing and detecting exosome

Abstract

The invention discloses an amphiphilic polypeptide for capturing and detecting exosomes, and relates to the technical field of biology. The sequence of the amphiphilic polypeptide is RKTFNKVLGHVNKFYTKILPKFLGGIVRALGSFRKDC, and the sequence of the amphiphilic polypeptide is shown in the description. The amphiphilic polypeptide provided by the invention can be used for capturing and detecting the exosome, and compared with traditional hydrophobic alkyl capture, the amphiphilic polypeptide has stronger specificity, can effectively avoid non-specific binding, and is used for quantifying the exosome and specific protein on the exosome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an amphipathic polypeptide for capturing and detecting exosomes. Background Art

[0002] As a key medium for intercellular communication, the unique properties and functions of exosomes have attracted widespread attention from the scientific community in recent years. These small membrane-enclosed vesicles, with diameters ranging from 30 to 150 nanometers, not only carry a wealth of biomolecular information such as proteins, RNA, and lipids, but also play an indispensable role in intercellular communication, material transport, and immune system regulation. With the deepening of research, the application potential of exosomes in the field of in vitro diagnosis has become increasingly prominent, especially in the early diagnosis of diseases, cancer prognosis assessment, and the detection of infectious diseases, showing great clinical value.

[0003] Exosomes can carry specific molecules that reflect the physiological or pathological state of cells. These molecules can be used as biomarkers for diseases, providing an important basis for early detection of diseases and the formulation of treatment strategies. In the field of cancer, tumor-related proteins, miRNAs, and even gene mutation information in exosomes have become powerful tools for monitoring tumor occurrence, development, and metastasis. For example, exosomes released by specific types of cancer cells may be rich in specific tumor markers, such as PCA3mRNA in prostate cancer and HER2 protein in breast cancer. The detection of these markers can help achieve early screening of cancer and the formulation of personalized treatment plans. In addition, exosomes are also involved in the immune escape mechanism of tumors. The level of PD-L1 protein on their surface is closely related to the prognosis of patients with gastric cancer, head and neck squamous cell carcinoma, etc., providing an important reference for evaluating the treatment effect and formulating subsequent treatment strategies.

[0004] In the study of neurodegenerative diseases, exosomes also show great potential. The pathogenesis of diseases such as Alzheimer's disease and Parkinson's disease is complex and difficult to diagnose early. However, specific proteins and RNA molecules in exosomes, such as β-amyloid protein, tau protein and related miRNA, provide a new perspective for the early diagnosis and monitoring of these diseases. By analyzing the changes in the composition of exosomes in patients' body fluids (such as blood and cerebrospinal fluid), dynamic monitoring of the disease process can be achieved, which helps to take timely intervention measures to delay disease progression.

[0005] Exosomes also have broad application prospects in the diagnosis of infectious diseases. After pathogens infect cells, their genetic material or proteins can be encapsulated in exosomes and reach remote tissues or organs through body fluid circulation, so that they can be detected in the early stages of infection. This characteristic makes exosomes a potential biomarker for early diagnosis of infectious diseases. For example, in the study of infectious diseases such as HIV and Mycobacterium tuberculosis, pathogen-related molecules carried by exosomes have been found, providing a new method for rapid and accurate pathogen detection and typing.

[0006] Although exosomes have shown great potential in in vitro diagnosis, current enrichment and detection technologies still face many challenges. Ultracentrifugation, as the gold standard for exosome enrichment, can effectively separate exosomes, but the operation is complicated, time-consuming (usually 3 to 4 hours), and easily leads to sample loss, which limits its widespread application in clinical practice. In addition, ultracentrifugation also has a certain impact on the purity, integrity and accuracy of subsequent analysis of exosomes. Therefore, the development of efficient, rapid and highly specific exosome enrichment and detection technology has become a hot spot and difficulty in current research. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention utilizes an amphipathic polypeptide that can specifically bind to exosomes, thereby achieving rapid enrichment of exosomes while reducing sample loss. The amphipathic polypeptide can be used to modify the surface of a surface plasmon resonance detection chip or an ELISA plate, thereby achieving exosome enrichment, exosome detection, and detection of specific proteins on exosomes; the amphipathic polypeptide can also be used to modify the surface of magnetic beads, thereby achieving a large amount of exosome enrichment.

[0008] In particular, compared with the long-chain alkane-based exosome capture method, this amphipathic peptide has better specificity and is less likely to interact with antibodies or other proteins, facilitating further characterization of exosomes after capture.

[0009] The invention discloses an amphipathic polypeptide for capturing and detecting exosomes, wherein the sequence of the amphipathic polypeptide is RKTFNKVLGHVNKFYTKILPKFLGGIVRAMLGSFRKDC.

[0010] The present invention also provides a detection product, which includes a polypeptide modification layer, wherein the polypeptide modification layer is modified on the surface of a carrier and is used to capture exosomes;

[0011] The polypeptide in the polypeptide modification layer is the amphiphilic polypeptide.

[0012] Exosomes are tiny vesicles secreted by cells, and their range of existence is quite wide. Specifically, exosomes are present in a variety of biological fluids, including but not limited to blood, urine, saliva, breast milk, cerebrospinal fluid, and cell culture medium. These exosomes play an important physiological function in the body, mainly by releasing bioactive molecules such as proteins, nucleic acids and lipids to communicate with other cells. In the embodiments of the present invention, serum is selected for experiment as an example.

[0013] Furthermore, the carrier of the detection product is any one of a surface plasmon resonance detection chip, an ELISA plate or magnetic beads.

[0014] Specifically, when the carrier of the detection product is a surface plasmon resonance detection chip, the method for modifying the polypeptide on the surface of the surface plasmon resonance detection chip includes:

[0015] Activating the carboxyl groups on the surface of the surface plasmon resonance detection chip by using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride reagents, causing the activated carboxyl groups to undergo a condensation reaction with the amino groups of the amphiphilic polypeptide, thereby covalently linking the amphiphilic polypeptide to the surface of the surface plasmon resonance detection chip;

[0016] Or:

[0017] N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride reagents were used to activate the carboxyl groups on the surface of the surface plasmon resonance detection chip;

[0018] fixing 2-(2-pyridyldithio)ethylamine hydrochloride onto the activated surface plasmon resonance detection chip;

[0019] The thiol group on the amphiphilic polypeptide undergoes a condensation reaction with 2-(2-pyridyldithio)ethylamine hydrochloride, thereby covalently connecting the amphiphilic polypeptide to the surface of a surface plasmon resonance detection chip.

[0020] The present invention also provides a method for quantitatively detecting exosomes, using the detection product to capture exosomes and perform quantitative detection.

[0021] Specifically, the method for quantitative detection of exosomes comprises the following steps:

[0022] When the carrier of the detection product is a surface plasmon resonance detection chip, the sample to be detected is passed into the surface plasmon resonance detection chip modified with the amphiphilic polypeptide to capture exosomes, and the signal response value of the binding process is detected by surface plasmon resonance technology to achieve quantitative detection of exosomes;

[0023] Or when the carrier of the detection product is an ELISA plate, the sample to be detected is added to the well of the ELISA plate modified with the amphiphilic polypeptide for incubation and capture, and then a ligand with a labeled exosome constitutive protein is added to the ELISA plate for capturing exosomes, and the absorbance is detected by enzyme-linked immunosorbent assay to achieve quantitative detection of exosomes;

[0024] Or when the carrier of the detection product is a magnetic bead, the sample to be detected is incubated and captured with the magnetic beads modified with the amphiphilic polypeptide, and the eluate is eluted and collected after multiple washings, and the protein concentration in the eluate is determined by the bicinchoninic acid method (BCA) to achieve quantitative detection of exosomes.

[0025] Specifically, the target protein detected by the enzyme-linked immunosorbent assay can be CD63.

[0026] Furthermore, the present invention also provides a method for quantitatively detecting specific proteins on the surface of exosomes, and uses the detection product to capture exosomes and detect proteins.

[0027] The quantitative detection method of specific proteins on the surface of exosomes includes the following steps:

[0028] When the carrier of the detection product is a surface plasmon resonance detection chip, the sample to be detected is passed into the surface plasmon resonance detection chip modified with the amphiphilic polypeptide to capture exosomes, and then the ligand of the specific protein is passed into the surface plasmon resonance detection chip for capturing exosomes, and the change of the signal response value of the binding process is detected by surface plasmon resonance technology to achieve quantitative detection of specific proteins on exosomes;

[0029] Or when the carrier of the detection product is an ELISA plate, the sample to be detected is added to the wells of the ELISA plate modified with the amphiphilic polypeptide for incubation and capture, and then a ligand with a labeled specific protein is added to the ELISA plate for capturing exosomes, and the absorbance is detected by enzyme-linked immunosorbent assay to achieve quantitative detection of specific proteins on exosomes.

[0030] In an embodiment of the present invention, the specific protein is PD-L1 protein.

[0031] Beneficial effects of the present invention:

[0032] The amphiphilic polypeptide provided by the present invention can be used for the capture and detection of exosomes. Compared with the traditional hydrophobic alkyl capture, the amphiphilic polypeptide has stronger specificity, can effectively avoid nonspecific binding, and is used for the quantification of exosomes and specific proteins on exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the non-specific binding between the detection chip modified with hydrophobic alkyl and the protein.

[0034] Figure 2 The binding process of different concentrations of exosomes in serum with the detection chip after amino modification of amphiphilic peptides (A) and the change of the final response value (B). ① Exosome sample diluted 100 times; ② Exosome sample diluted 200 times; ③ Exosome sample diluted 500 times; ④ Exosome sample diluted 1000 times; ⑤ Exosome sample diluted 2000 times; ⑥ Exosome sample diluted 5000 times.

[0035] Figure 3 It is a detection chip for amino-modified amphiphilic peptides and non-specific adsorption of proteins.

[0036] Figure 4 The binding process of different concentrations of exosomes in serum with the detection chip after amphiphilic peptide thiol modification (A) and the final response value change (B). ① Exosome sample 100-fold dilution; ② Exosome sample 200-fold dilution; ③ Exosome sample 500-fold dilution; ④ Exosome sample 1000-fold dilution; ⑤ Exosome sample 2000-fold dilution; ⑥ Exosome sample 5000-fold dilution.

[0037] Figure 5 It is a detection chip for thiol-modified amphiphilic peptides and non-specific adsorption of proteins.

[0038] Figure 6 The binding process of different types of exosomes in serum with the detection chip after amino modification of amphiphilic peptides (A) and the final response value change (B). ①H1299: A549 = 100: 0; ②H1299:

[0039] A549=50:50;③H1299:A549=20:80;④H1299:A549=10:90;⑤

[0040] H1299:A549=5:95.

[0041] Figure 7 To capture the binding process of different types of exosome samples with anti-PD-L1 antibodies (A) and the final response value changes (B) (amino modification). ①H1299: A549 = 100: 0; ②H1299:

[0042] A549=50:50;③H1299:A549=20:80;④H1299:A549=10:90;⑤

[0043] H1299:A549=5:95.

[0044] Figure 8The binding process of different types of exosomes in serum with the detection chip after amphiphilic peptide thiol modification (A) and the final response value change (B). ①H1299: A549 = 100: 0; ②H1299:

[0045] A549=50:50;③H1299:A549=20:80;④H1299:A549=10:90;⑤

[0046] H1299:A549=5:95.

[0047] Fig. 9 To capture the binding process of different types of exosome samples with anti-PD-L1 antibodies (A) and the final response value changes (B) (thiol modification). ①H1299: A549 = 100: 0; ②H1299:

[0048] A549=50:50;③H1299:A549=20:80;④H1299:A549=10:90;⑤

[0049] H1299:A549=5:95. DETAILED DESCRIPTION

[0050] Example 1: Chips modified with hydrophobic alkyl groups have strong nonspecific binding

[0051] (1) Antibody preparation

[0052] Antibodies or proteins purchased from different companies were diluted at a ratio of 1:100 using PBS buffer and filtered using a 0.22 μm PES filter for later use.

[0053] (2) Surface plasmon resonance detection

[0054] Surface plasmon resonance detection was performed using a Biacore T200 molecular interaction analyzer. Before the detection, a Series S L1 sensor chip was loaded. The surface of the chip was modified with hydrophobic long-chain alkanes and can be used for the analysis of lipids and exosomes.

[0055] The running buffer was PBS buffer; the sample loading flow rate was 30 μL / min, and the time was 2 min; the regeneration solution was 50 mM NaOH:isopropanol (volume ratio 3:2), the regeneration flow rate was 30 μL / min, and the regeneration time was 30 s.

[0056] (3) Experimental results

[0057] Binding of different antibodies to Series S L1 sensor chip Figure 1As shown in the figure, there is strong non-specific binding between the antibody and the Series SL1 sensor chip, which may affect the detection of exosomes and exosomal proteins.

[0058] Example 2: Screening of amphiphilic peptides

[0059] (1) Peptide selection and synthesis

[0060] The target polypeptide was artificially synthesized by solid phase synthesis, and the polypeptide sequence is shown in Table 1 below.

[0061] Table 1 Amphiphilic polypeptide sequences

[0062]

[0063] (2) Detection chip surface modification

[0064] The amphiphilic peptides were coupled to the carboxyl groups of the Series S CM5 sensor chip by amino modification, thereby preparing a surface plasmon resonance detection chip modified with amphiphilic peptides. The specific process is as follows:

[0065] 1) Weigh 1 mg of peptide into a sample bottle, dissolve it completely with 100 μL of dimethyl sulfoxide, and then dilute it with 900 μL of ultrapure water;

[0066] 2) diluting the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0067] 3) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip into the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0068] 4) Mix 0.1 mol / L N-hydroxysuccinimide (NHS) and 0.4 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in a volume ratio of 1:1;

[0069] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0070] 6) Passing the polypeptide solution described in step 2) at a flow rate of 10 μL / min for 14 minutes;

[0071] 7) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block excess carboxyl groups.

[0072] (3) Exosome extraction

[0073] 40 mL of complete cell culture medium was added to several T225 culture flasks with cells growing on the bottom, and the flasks were placed in a cell culture incubator for 48 hours (37° C., 5% CO 2 ), and then the cell supernatant culture fluid was collected.

[0074] Take 150mL of cell supernatant culture medium and centrifuge at 3000×g for 20 minutes at 4℃, and take the supernatant and centrifuge at 7000×g for 30 minutes at 4℃. Place the cell supernatant culture medium after centrifugation in a 20mL protein concentrator tube and centrifuge at 4000-4700×g for 10 minutes at 4℃, repeat several times, and concentrate to less than 60mL. Take the supernatant from the ultrafiltration tube, place it in an ultracentrifuge tube, centrifuge at 110000g for 70 minutes at 4℃, remove the supernatant (at this time, the exosome particles are in the precipitate), resuspend with 1×PBS, and repeat the ultracentrifugation step. After the ultracentrifugation, resuspend the precipitate in 0.1mL 1×PBS. The extracted exosomes can be stored at 4℃ for subsequent experiments or stored in a -80℃ refrigerator for long-term storage.

[0075] (4) Exosome detection

[0076] The exosomes were introduced into the detection chips with different peptides on the surface, with a flow rate of 10 μL / min and a time of 7 min. The changes in the detection signals before and after the introduction of the exosomes are shown in Table 2. The results show that Seq1 can effectively capture exosomes to the surface of the detection chip.

[0077] Table 2 Characteristics of amphiphilic peptides and exosome capture efficiency (signal response changes)

[0078] Seq ID Hydrophobicity Hydrophobic polarity Charge number Signal response changes Seq_1 0.560 0.534 +8 1331.21 Seq_2 0.267 0.495 +2 344.55 Seq_3 0.479 0.455 +2 97.93 Seq_4 0.440 0.526 +1 94.00 Seq_5 0.546 0.530 0 0.68 Seq_6 0.026 0.421 +5 20.60 Seq_7 0.440 0.526 +1 -11.84 Seq_8 0.631 0.442 +3 30.81 Seq_9 0.300 0.480 -1 9.37 Seq_10 0.101 0.460 +4 -17.59 Seq_11 0.394 0.466 +3 520.33 Seq_12 0.634 0.447 +1 —19.99 Seq_13 0.331 0.840 +13 -36.71 Seq_14 0.380 0.522 +3 -28.22 Seq_15 0.759 0.456 +4 -24.11 Seq_16 0.559 0.072 +3 46.90 Seq_17 0.518 0.446 -1 6.51 Seq_18 0.764 0.344 0 9.97 Seq_19 0.338 0.511 +2 -9.53 Seq_20 0.617 0.483 +5 99.03

[0079] Example 3: Signal response of amphiphilic peptides capturing exosomes of different concentrations - surface plasmon resonance method (amino group connection)

[0080] (1) Preparation of peptide solution

[0081] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0082] (2) Detection chip surface modification

[0083] 1) Dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0084] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip into the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0085] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a volume ratio of 1:1;

[0086] 4) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0087] 5) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0088] 6) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block excess carboxyl groups.

[0089] (3) Exosome extraction

[0090] The exosome extraction steps were the same as those described in Example 2.

[0091] (4) Exosome detection

[0092] The extracted exosomes were diluted appropriately with fetal bovine serum (exosome-free) in proportions (100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, and 5000-fold), and then the exosome samples of different concentrations were passed into the detection chip with Seq1 peptide modified on the surface at a flow rate of 10 μL / min for 7 min. Figure 2 As shown, the change in signal response was linearly correlated with the exosome dilution factor.

[0093] (5) Protein nonspecific adsorption detection

[0094] Antibodies or proteins purchased from different companies were diluted in a ratio of 1:100 using PBS buffer and filtered with a 0.22μm PES filter for later use. Non-specific adsorption detection of the detection chip with Seq1 peptide modified on the surface, the running buffer was PBS buffer; the sample loading flow rate was 30μL / min, the time was 2min; the regeneration liquid was 0.04mol / L n-octyl β-D-glucopyranoside, the regeneration flow rate was 30μL / min, and the regeneration time was 30s. The test results are as follows Figure 3 As shown, the nonspecific adsorption was significantly lower than that of the detection chip modified with hydrophobic alkyl.

[0095] Example 4: Signal response of amphiphilic peptides capturing exosomes of different concentrations - surface plasmon resonance method (thiol linkage)

[0096] (1) Preparation of peptide solution

[0097] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0098] (2) Detection chip surface modification

[0099] 1) Dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0100] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip into the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0101] Mix 0.1mol / L NHS and 0.4mol / L EDC in a ratio of 1:1 and use immediately after preparation;

[0102] 4) Mix 0.12 mol / L 2-(2-pyridinyldithio)ethaneamine hydrochloride (PDEA) and 0.15 mol / L sodium borate buffer (pH 8.5) in a ratio of 2:1 and use immediately after preparation;

[0103] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0104] 6) Pass PDEA working solution at a flow rate of 10 μL / min for 4 min;

[0105] 7) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0106] 8) 0.05 mol / L L-cysteine, 1 mol / L sodium chloride, and 0.1 mol / L sodium acetate buffer (pH 4.0) were introduced at a flow rate of 10 μL / min for 4 min to block excess thiol groups.

[0107] (3) Exosome extraction

[0108] The exosome extraction steps were the same as those described in Example 2.

[0109] (4) Exosome detection

[0110] The extracted exosomes were diluted appropriately with fetal bovine serum (exosome-free) in proportions (100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, and 5000-fold), and then exosomes of different concentrations were passed into the detection chip with Seq1 peptide modified on the surface at a flow rate of 10 μL / min for 7 min. Figure 4 As shown, the change in signal response was linearly correlated with the exosome dilution factor.

[0111] (5) Protein nonspecific adsorption detection

[0112] Antibodies or proteins purchased from different companies were diluted in a ratio of 1:100 using PBS buffer and filtered with a 0.22μm PES filter for later use. Non-specific adsorption detection of the detection chip with Seq1 peptide modified on the surface, the running buffer was PBS buffer; the sample loading flow rate was 30μL / min, the time was 2min; the regeneration liquid was 0.04mol / L n-octyl β-D-glucopyranoside, the regeneration flow rate was 30μL / min, and the regeneration time was 30s. The test results are as follows Figure 5 As shown, the nonspecific adsorption was significantly lower than that of the detection chip modified with hydrophobic alkyl.

[0113] Example 5: Signal response of amphiphilic peptides capturing exosome PD-L1 at different concentrations - surface plasmon resonance method (carboxyl linkage)

[0114] (1) Preparation of peptide solution

[0115] The peptide was synthesized by solid phase synthesis, and the peptide sequence was shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, and then diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0116] (2) Detection chip surface modification

[0117] 1) Dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0118] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip into the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0119] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a ratio of 1:1;

[0120] 4) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0121] 5) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0122] 6) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block excess carboxyl groups.

[0123] (3) Exosome extraction

[0124] The exosome extraction step is the same as that described in Example 2. Exosomes were extracted using the cell culture supernatants of H1299 cells and A549 cells, respectively. The extracted exosomes were analyzed by western blot. The exosomes extracted from the H1299 cell culture supernatant contained PD-L1 protein, while the exosomes extracted from the A549 cell culture supernatant did not contain PD-L1 protein.

[0125] (4) Exosome capture

[0126] First, the H1299 exosomes and A549 exosomes samples were diluted to similar concentrations using fetal bovine serum that had been depleted of exosomes. Then, the H1299 exosomes and A549 exosomes were mixed in different proportions (H1299:A549=100:0, 50:50, 20:80, 10:90, 5:95) to obtain a series of samples to be analyzed with similar exosome concentrations and different PD-L1 protein concentrations. These samples were then passed through a detection chip with a surface modified with Seql polypeptide at a flow rate of 10 μL / min for 7 minutes. The test results are shown in Figure 2. Figure 6 As shown, the signal response changes of different samples to be analyzed are similar.

[0127] (5) PD-L1 protein analysis

[0128] On the basis that the detection chip modified with Seq1 peptide on the surface has captured exosomes, anti-PD-L1 monoclonal antibody was introduced into the system at an antibody concentration of 10 μg / mL, a flow rate of 10 μL / min, and a time of 7 minutes. Figure 7 As shown, the change in signal response was linearly correlated with the PD-L1 protein content.

[0129] Example 6: Signal response of amphiphilic peptides capturing exosome PD-L1 at different concentrations - surface plasmon resonance method (thiol linkage)

[0130] (1) Preparation of peptide solution

[0131] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0132] (2) Detection chip surface modification

[0133] 1) Dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0 and prepare it for use immediately;

[0134] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip into the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0135] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a ratio of 1:1;

[0136] 4) Mix 0.12 mol / L PDEA and 0.15 mol / L sodium borate buffer (pH 8.5) in a ratio of 2:1 and use immediately after preparation;

[0137] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0138] 6) Pass PDEA working solution at a flow rate of 10 μL / min for 4 min;

[0139] 7) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0140] 8) 0.05 mol / L L-cysteine, 1 mol / L sodium chloride, and 0.1 mol / L sodium acetate buffer (pH 4.0) were introduced at a flow rate of 10 μL / min for 4 min to block excess thiol groups.

[0141] (3) Exosome extraction

[0142] The exosome extraction step is the same as that described in Example 2. Exosomes were extracted using the cell culture supernatants of H1299 cells and A549 cells, respectively. The extracted exosomes were analyzed by western blot. The exosomes extracted from the H1299 cell culture supernatant contained PD-L1 protein, while the exosomes extracted from the A549 cell culture supernatant did not contain PD-L1 protein.

[0143] (4) Exosome capture

[0144] First, the H1299 exosomes and A549 exosomes samples were diluted to similar concentrations using fetal bovine serum without exosomes, and then the H1299 exosomes and A549 exosomes were mixed in different proportions to obtain a series of samples with similar exosome concentrations and different PD-L1 protein concentrations. These samples were then passed into the detection chip with Seq1 peptide modified on the surface at a flow rate of 10 μL / min for 7 minutes. The test results are shown in Figure 2. Figure 8 As shown, the signal response changes of different samples to be analyzed are similar.

[0145] (5) PD-L1 protein analysis

[0146] On the basis that the detection chip modified with Seq1 peptide on the surface has captured exosomes, anti-PD-L1 monoclonal antibody was introduced into the system at an antibody concentration of 10 μg / mL, a flow rate of 10 μL / min, and a time of 7 minutes. The detection results are shown in Figure 9, and the signal response change is linearly correlated with the PD-L1 protein content.

[0147] Example 7: Signal response of amphiphilic peptides capturing exosomes of different concentrations - ELISA

[0148] (1) Preparation of peptide solution

[0149] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0150] (2) ELISA plate coating and blocking

[0151] 1) Dilute the above polypeptide stock solution at a ratio of 1:100, and the dilution buffer is 0.05 mol / L sodium bicarbonate buffer (pH 9.6), and prepare it for use immediately;

[0152] 2) Add 100 μL of diluted peptide solution to each well of the ELISA plate, cover the plate with appropriate plastic products, and incubate overnight at 4°C

[0153] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper. Then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper. Repeat 3-5 times.

[0154] 4) Add 200 μL of PBS buffer containing 3% BSA to each well and incubate at 37°C for 90 min;

[0155] 5) Repeat step 3).

[0156] (3) Exosome extraction and preparation

[0157] The exosome extraction steps were the same as those described in Example 2.

[0158] (4) Exosome capture

[0159] 1) The extracted exosomes were appropriately diluted using fetal bovine serum from which exosomes were removed to obtain exosome samples of different concentrations.

[0160] 2) Add 100 μL of diluted exosome sample to each well and incubate at 37°C for 60 min.

[0161] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper. Then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper. Repeat 3-5 times.

[0162] (5) Exosome detection

[0163] 1) Dilute the HRP-labeled anti-CD63 monoclonal antibody at a ratio of 1:1000 in a PBS buffer containing 3% BSA. CD63 is widely present in various exosomes and is a marker protein of exosomes;

[0164] 2) Add 100 μL of diluted antibody to each well and incubate at 37°C for 60 min;

[0165] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper. Then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper. Repeat 3-5 times;

[0166] 4) Add 100 μL TMB color development working solution to each well and incubate at 37°C in the dark for 30 min. After the reaction is completed, add 100 μL of reaction stop solution to each well immediately;

[0167] 5) Within 30 minutes after the reaction is terminated, measure and record the absorbance at a wavelength of 450 nm on an enzyme reader.

[0168] (6) Experimental results

[0169] The experimental results are shown in Table 3 below. The exosome concentration is linearly correlated with the test results.

[0170] Table 3 Detection of exosome concentration using enzyme-linked immunosorbent assay

[0171] Exosome dilution factor <![CDATA[OD 450 ]]> 100 2.432 200 1.294 500 0.563 1000 0.337 2000 0.190 5000 0.059

[0172] Example 8: Signal response of amphiphilic peptides capturing exosome PD-L1 at different concentrations - ELISA

[0173] (1) Preparation of peptide solution

[0174] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0175] (2) ELISA plate coating and blocking

[0176] 1) Dilute the above polypeptide stock solution at a ratio of 1:100, and the dilution buffer is 0.05 mol / L sodium bicarbonate buffer (pH 9.6), and prepare it for use immediately;

[0177] 2) Add 100 μL of the diluted peptide solution to each well of the ELISA plate, cover the plate with appropriate plastic products, and incubate overnight at 4°C;

[0178] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper. Then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper. Repeat 3-5 times;

[0179] 4) Add 200 μL of PBS buffer containing 3% BSA to each well and incubate at 37°C for 90 min;

[0180] 5) Repeat step 3).

[0181] (3) Exosome extraction

[0182] The exosome extraction step is the same as that described in Example 2. Exosomes were extracted using the cell culture supernatants of H1299 cells and A549 cells, respectively. The extracted exosomes were analyzed by western blot. The exosomes extracted from the H1299 cell culture supernatant contained PD-L1 protein, while the exosomes extracted from the A549 cell culture supernatant did not contain PD-L1 protein.

[0183] (4) Exosome capture

[0184] 1) First, the H1299 exosomes and A549 exosomes samples were diluted to similar concentrations using exosome-depleted fetal bovine serum, and then the H1299 exosomes and A549 exosomes were mixed in different proportions to obtain a series of samples to be analyzed with similar exosome concentrations and different PD-L1 protein concentrations;

[0185] 2) Add 100 μL of diluted exosome sample to each well and incubate at 37°C for 60 min.

[0186] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper. Then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper. Repeat 3-5 times.

[0187] (5) Target protein detection

[0188] 1) dilute the HRP-labeled anti-PD-L1 monoclonal antibody at a ratio of 1:1000 in PBS buffer containing 3% BSA;

[0189] 2) Add 100 μL of diluted antibody to each well and incubate at 37°C for 60 min;

[0190] 3) Discard the liquid in the ELISA plate and pat it dry on absorbent paper; then add 200 μL PBS buffer to each well, let it stand at room temperature for 3 minutes, then discard the liquid in the ELISA plate and pat it dry on absorbent paper, repeat 3-5 times;

[0191] 4) Add 100 μL TMB color development working solution to each well and incubate at 37°C in the dark for 30 min. After the reaction is completed, add 100 μL of reaction stop solution to each well immediately;

[0192] 5) Within 30 minutes after the reaction is terminated, measure and record the absorbance at a wavelength of 450 nm on an enzyme reader.

[0193] (6) Experimental results

[0194] The experimental results are shown in Table 4 below. The PD-L1 protein content is linearly correlated with the test results.

[0195] Table 4 Detection of PD-L1 protein concentration on exosomes using enzyme-linked immunosorbent assay

[0196] Exosome ratio (H1299: A549) OD450 100：0 2.028 50：50 1.006 20：80 0.490 10：90 0.296 5：95 0.192

[0197] Example 9: Signal response of amphiphilic peptides capturing exosomes at different concentrations - immunoprecipitation

[0198] (1) Preparation of peptide solution

[0199] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0200] (2) Modification of immunoprecipitation magnetic beads

[0201] 1) Dilute the above polypeptide stock solution at a ratio of 1:10, and the dilution buffer is 0.05 mol / L boric acid buffer (pH 8.5), which is prepared and used immediately;

[0202] 2) Equilibrate the magnetic beads with NHS groups on the surface to room temperature, take an appropriate amount of magnetic beads, fix the magnetic beads and remove the supernatant buffer;

[0203] 3) Add an appropriate amount of 1 mmol / L hydrochloric acid solution, vortex mix for 15 seconds, then fix the magnetic beads and remove the supernatant buffer;

[0204] 4) Add an appropriate amount of the peptide dilution described in step 1), vortex mix for 30 seconds, incubate at 4°C overnight, flip and mix during the process, then fix the magnetic beads and remove the supernatant;

[0205] 5) Add an appropriate amount of 0.1 mol / L glycine buffer (pH 2.0), vortex mix for 15 seconds, then fix the magnetic beads and remove the supernatant buffer;

[0206] 6) Repeat step 5) once;

[0207] 7) Add appropriate amount of ultrapure water, vortex mix for 15 seconds, then fix the magnetic beads and remove the supernatant buffer;

[0208] 8) Add an appropriate amount of 3 mol / L ethanolamine buffer (pH 9.0), vortex mix for 30 seconds, incubate at 37°C for 2 hours, flip and mix during the period, then fix the magnetic beads and remove the supernatant;

[0209] 9) Add appropriate amount of ultrapure water, vortex mix for 15 seconds, then fix the magnetic beads and remove the supernatant buffer;

[0210] 10) Add an appropriate amount of 0.05 mol / L boric acid buffer (pH 8.5), vortex mix for 15 seconds, then fix the magnetic beads and remove the supernatant buffer;

[0211] 11) Repeat step 10) twice, and then store at 4°C until use.

[0212] (3) Exosome extraction

[0213] The exosome extraction steps were the same as those described in Example 2.

[0214] (4) Exosome capture

[0215] 1) Using fetal bovine serum from which exosomes have been removed, the extracted exosomes were appropriately diluted in proportions ((100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold and 5000-fold)) to obtain exosome samples of different concentrations;

[0216] 2) taking an appropriate amount of magnetic beads modified with the amphiphilic peptide Seq1, then fixing the magnetic beads and removing the supernatant buffer;

[0217] 3) Add appropriate amount of exosome sample, incubate at 37°C for 2 hours, flip and mix, then fix the magnetic beads and remove the supernatant;

[0218] 4) Add an appropriate amount of 1× PBS buffer, mix gently, fix the magnetic beads and remove the supernatant;

[0219] 5) Repeat step 4) once;

[0220] 6) Add appropriate amount of ultrapure water, mix gently, fix the magnetic beads and remove the supernatant;

[0221] 7) Add an appropriate amount of elution buffer (0.04 mol / L n-octyl β-D-glucopyranoside), vortex mix for 30 seconds, and then collect the eluate for testing.

[0222] (5) Exosome detection

[0223] The protein concentration in the eluate was detected by the BCA method at a detection wavelength of 562 nm. The protein concentration indicated the exosome concentration, and the detection results are shown in Table 5 below.

[0224] Table 5 Exosome samples enriched by immunoprecipitation

[0225] Exosome dilution factor <![CDATA[OD 562nm ]]> 100 2.882 200 1.434 500 0.630 1000 0.284 2000 0.160 5000 0.148

[0226] The exosome protein content captured by magnetic beads is proportional to the exosome concentration in the loaded sample.

Claims

1. An amphiphilic polypeptide for exosome capture and detection, characterized in that: The sequence of the amphipathic polypeptide is RKTFNKVLGHVNKFYTKILPKFLGGIVRAMLGSFRKDC.

2. A detection product, characterized in that: The detection product includes a polypeptide modification layer, which is modified on the surface of the carrier and is used to capture exosomes; The polypeptide in the polypeptide modification layer is the amphiphilic polypeptide according to claim 1.

3. The detection product according to claim 2, characterized in that: The carrier of the detection product is any one of a surface plasmon resonance detection chip, an ELISA plate or magnetic beads.

4. The detection product according to claim 3, characterized in that: When the carrier of the detection product is a surface plasmon resonance detection chip, the method for modifying the polypeptide on the surface of the surface plasmon resonance detection chip includes: The carboxyl groups on the surface of the surface plasmon resonance detection chip are activated by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride reagents, and the activated carboxyl groups are condensed with the amino groups of the amphiphilic polypeptides to covalently link the amphiphilic polypeptides to the surface of the surface plasmon resonance detection chip.

5. The detection product according to claim 3, characterized in that: When the carrier of the detection product is a surface plasmon resonance detection chip, the method for modifying the polypeptide on the surface of the surface plasmon resonance detection chip comprises: using N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride reagents to activate the carboxyl groups on the surface of the surface plasmon resonance detection chip; fixing 2-(2-pyridyldithio)ethylamine hydrochloride onto the activated surface plasmon resonance detection chip; The thiol group on the amphiphilic polypeptide undergoes a condensation reaction with 2-(2-pyridyldithio)ethylamine hydrochloride, thereby covalently connecting the amphiphilic polypeptide to the surface of a surface plasmon resonance detection chip.

6. A method for quantitative detection of exosomes, characterized in that: Use the detection product according to any one of claims 2 to 5 to capture exosomes and conduct quantitative detection.

7. The method for quantitative detection of exosomes according to claim 6, characterized in that: The following steps are involved: When the carrier of the detection product is a surface plasmon resonance detection chip, the sample to be detected is passed into the surface plasmon resonance detection chip modified with the amphiphilic polypeptide to capture exosomes, and the change in the signal response value of the binding process is detected by surface plasmon resonance technology to achieve quantitative detection of exosomes; Or when the carrier of the detection product is an ELISA plate, the sample to be detected is added to the well of the ELISA plate modified with the amphiphilic polypeptide for incubation and capture, and then a ligand with a labeled exosome constitutive protein is added to the ELISA plate for capturing exosomes, and the absorbance is detected by enzyme-linked immunosorbent assay to achieve quantitative detection of exosomes; Or when the carrier of the detection product is a magnetic bead, the sample to be detected is incubated and captured with the magnetic beads modified with the amphiphilic polypeptide, and the eluate is eluted and collected after multiple washings, and the protein concentration in the eluate is determined by the bicinchoninic acid method to achieve quantitative detection of exosomes.

8. The method for quantitative detection of exosomes according to claim 7, characterized in that: The target protein detected by the enzyme-linked immunosorbent assay is CD63.

9. A method for quantitatively detecting specific proteins on the surface of exosomes, characterized in that: Use the detection product according to any one of claims 2 to 5 to perform exosome capture and protein detection.

10. The method for quantitative detection of exosomes according to claim 9, characterized in that: The following steps are involved: When the carrier of the detection product is a surface plasmon resonance detection chip, the sample to be detected is passed into the surface plasmon resonance detection chip modified with the amphiphilic polypeptide to capture exosomes, and then the ligand of the specific protein is passed into the surface plasmon resonance detection chip for capturing exosomes, and the change of the signal response value of the binding process is detected by surface plasmon resonance technology to achieve quantitative detection of specific proteins on exosomes; Or when the carrier of the detection product is an ELISA plate, the sample to be detected is added to the wells of the ELISA plate modified with the amphiphilic polypeptide for incubation and capture, and then a ligand with a labeled specific protein is added to the ELISA plate for capturing exosomes, and the absorbance is detected by enzyme-linked immunosorbent assay to achieve quantitative detection of specific proteins on exosomes.

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