Tumor exosome fluorescence detection method based on tandem hybrid chain reaction and selective coordination

Abstract

The invention provides a tumor exosome (TDEs) fluorescence detection method based on tandem hybrid chain reaction (HCR) and selective coordination, and aims to combine an aptamer, a tandem enzyme-free amplification technology and selective coordination of Cu < 2 + > to analyze a clinical lung cancer blood sample. The programmed cell death ligand 1 (PD-L1) biomarker overexpressed on the surface of the TDEs is selected as a target for detecting the exosome. The HCR can easily generate a large amount of double-stranded DNA, so that the HCR can be used as a means for signal amplification. The high affinity of the aptamer to the protein will affect the tandem HCR reaction and then modulate the HCR product. Cu < 2 + > and a double-stranded DNA product are utilized to form a compound, and a fluorescence signal of calcein can be accurately adjusted, so that accurate fluorescence quantification is performed on the concentration of TDEs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical diagnostic technology, and in particular to a method for detecting tumor exosome fluorescence based on tandem hybridization chain reaction and selective coordination. Background Art

[0002] Lung cancer is one of the most common malignant tumors in the world. Its early symptoms are not obvious and are easily overlooked. Commonly used diagnostic methods in clinical practice, such as clinical imaging examinations and pathological biopsies, have certain limitations in terms of detection sensitivity, false positive results, and invasiveness. Tumor liquid biopsy is a technology that can replace tissue biopsy, which can achieve non-invasive sampling and effective detection, thereby realizing early diagnosis and dynamic monitoring of tumors.

[0003] Tumor exosomes (TDEs) are ideal biomarkers for liquid biopsy because they are secreted into the blood relatively early, with higher abundance and greater stability compared to other markers such as circulating tumor DNA and cells. However, TDEs are at low concentrations in the early stages of tumorigenesis and are easily interfered by a large number of exosomes from normal host cells. Therefore, traditional methods, such as precipitation, nanoparticle tracking analysis, and Western blotting, are limited in sensitivity and specificity in isolating and detecting exosomes. Therefore, there is a need to further develop simplified and sensitive exosome quantitative detection technologies.

[0004] In recent years, a lot of efforts have been made to develop ultrasensitive detection methods for exosomes, mainly focusing on more effective specific recognition, signal amplification and detection technologies. Peptidomers can bind to targets efficiently and specifically, and are the first choice for identifying exosomes. In addition, compared with antibodies, aptamers are easier to chemically synthesize and have lower immunogenicity and toxicity. More importantly, single-stranded DNA aptamers can modify DNA without affecting target binding and combine with various DNA-based amplification reactions. There are currently a variety of signal amplification technologies that can improve the sensitivity of TDEs detection, including DNA polymerase / ligation endonuclease-assisted exponential amplification reactions, rolling circle amplification (RCA) and hybridization chain reaction (HCR). Among them, isothermal, enzyme-free single-stranded DNA amplification using HCR is a more stable and simpler reaction system that can reduce background noise. Other signal analysis techniques, such as surface-enhanced Raman spectroscopy, fluorescence, colorimetry, surface plasmon analysis, electrochemistry, etc., can also be used for sensitive detection of exosomes. However, these methods inevitably rely on large-scale precision equipment, professional operations and high-cost detection materials. Therefore, it is necessary to develop efficient, low-cost, and reproducible detection methods to effectively apply TDEs to early cancer diagnosis.

[0005] About DNA and metal ions (Hg 2+ 、Ag + , K+ 、Na + , Cu 2+ The research on building complex functional nanostructures will help develop new TDEs detection methods based on single-chain aptamers. + and Hg 2+ Metal ions and their mediated base pairs (C-Ag + -C and T-Hg 2+ -T) is often used to construct recognition elements and cascade non-enzymatic signal amplification elements. + Need to avoid light, introduce Hg 2+ G-quadruplexes are often used to construct recognition elements, but due to the stability and conformational issues of the G-quadruplex structure, and the need to introduce K + and Na + , which limits its application in biological analysis. 2+ Nanomaterials are cheaper and less toxic, so Cu 2+ A new option can be provided. In this case, Cu 2+ Combined with HCR, it helps to develop a new aptamer-based TDEs detection method. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for fluorescent detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination to solve the technical problems of the defects of the prior art recorded in the background technology.

[0007] In order to achieve the above-mentioned object, the present invention provides a method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination, the method comprising a specific recognition reaction between the membrane protein PD-L1 overexpressed on tumor exosomes and the PD-L1 aptamer probe, and a double-stranded DNA product generated by a tandem hybridization chain reaction between the PD-L1 aptamer probe and a bidirectionally extendable hairpin DNA and a Cu 2+ The fluorescence signal of calcein is effectively changed, and a single target is quantified based on the fluorescence signal of calcein, wherein the target is a membrane protein PD-L1 or a tumor exosome.

[0008] According to an optional embodiment, when the Cu 2+ When the calcein coordinates with the base pairs and the phosphate backbone in the double-stranded DNA product to form a complex and the fluorescence signal of the calcein does not change, the target does not exist.

[0009] According to an optional embodiment, when the PD-L1 aptamer probe and the bidirectionally extendable hairpin DNA do not undergo a tandem hybridization chain reaction and do not generate a double-stranded DNA product, the Cu 2+ When recognized by calcein, the fluorescence signal of calcein decreases, indicating the presence of the target.

[0010] According to an optional embodiment, the bidirectionally extendable hairpin DNA comprises H1, H2, H3 and H4;

[0011] The sequence of H1 is: ATC AAT GAG TTG ATG TGG CCC GTC TAA GAA CGGGCC ACA TCA ACT;

[0012] The sequence of H2 is: ACG GGC CAC ATC AAC TCA TTG ATA GTT GAT GTGGCC CGT TCT TAG;

[0013] The H3 sequence is: ACG GGC AGT GGA CGC ATT GTC TTC ATA CAG ACAATG CGT CCA CT;

[0014] The H4 sequence is: AGA CAA TGC GTC CAC TGC CCG TAG TGG ACG CATTGT CTG TAT GA.

[0015] According to an optional embodiment, the PD-L1 aptamer probe sequence is: ACG GGC CACATC AAC TCATTG ATA GAC AAT GCG TCC ACT GCC CGT.

[0016] According to an optional embodiment, the tumor exosomes are derived from lung cancer A549 cells using differential ultracentrifugation.

[0017] According to an alternative embodiment, the lung cancer A549 cells can be expanded to other circulating tumor cells expressing mucin 1.

[0018] According to an optional embodiment, the calcein can be replaced by quantum dots, carbon dots, N-methylporphyrin dipropionic acid IX or other Cu-recognizable 2+ signaling molecules.

[0019] The method for detecting tumor exosome fluorescence based on tandem hybridization chain reaction and selective coordination provided by the present invention has the following technical effects:

[0020] This method of fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective ligand is designed to combine aptamers, tandem enzyme-free amplification technology and Cu 2+ The selective ligand of aptamers was used to analyze clinical lung cancer blood samples. The programmed cell death ligand 1 (PD-L1) biomarker overexpressed on the surface of TDEs was selected as the target for detection of exosomes. Since HCR can easily generate a large amount of double-stranded DNA, it can be used as a means of signal amplification. The high affinity of the aptamer to the protein affects the tandem HCR reaction and subsequently regulates the HCR product. 2+ The formation of a complex with the double-stranded DNA product can precisely modulate the fluorescence signal of calcein, thereby accurately quantifying the concentration of TDEs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 is a schematic diagram of the enrichment and determination of exosomes;

[0023] Figure 2 It is Cu 2+ The coordination with double-stranded DNA modulates the fluorescence signal of calcein;

[0024] Figure 3 is the analytical performance of PD-L1;

[0025] Figure 4 It is the optimization of HCR conditions;

[0026] Figure 5 It is the optimization of conditions for analyzing PD-L1;

[0027] Figure 6 are the characteristics and analytical performance of exosomes;

[0028] Figure 7 This is a flow chart and results of testing blood samples from lung cancer patients. DETAILED DESCRIPTION

[0029] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0030] The principle of the analytical method of the present invention is as follows:

[0031] like Figure 1 The figure shows the schematic diagram of the enrichment and determination of exosomes. Exosomes were initially isolated from the supernatant of lung cancer A549 cells or clinical blood samples by differential ultracentrifugation. The principle of the exosome detection strategy is as follows Figure 1 As shown. The membrane protein PD-L1 overexpressed on tumor exosomes was selected as the specific detection target of tumor exosomes. PD-L1 aptamer can be used as a recognition probe to specifically recognize and bind to PD-L1 protein (K d =4.7nM).

[0032] When only exosomes from normal host cells are present in the detection system, the aptamer probe remains free and undergoes tandem HCR reaction with bidirectionally extendible hairpin DNA (H1, H2, H3, H4), producing a large amount of double-stranded DNA products derived from the aptamer. 2+ , a high fluorescence signal can be obtained because Cu 2+ It can form a complex with the base pairs and phosphate backbone in DNA, so it will not be recognized by the signaling molecule calcein.

[0033] However, in the presence of TDEs, the binding of the aptamer to PD-L1 on the exosome surface hindered the HCR reaction, thereby allowing more of the remaining Cu 2+ The calcein is recognized by the calcein, which reduces the fluorescence signal of the calcein. In this case, the intensity of the fluorescence signal of the calcein is measured to distinguish between negative and positive samples.

[0034] The technical solution of the present invention is specifically verified below in conjunction with embodiments.

[0035] 1. Materials and Reagents

[0036] All oligonucleotides with different sequences were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. ( Table 1 );

[0037] 3-Mercaptopropionic acid (MPA) and copper sulfate pentahydrate (CuSO4·5H2O) were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China);

[0038] NaNO3, HCl, and NaOH were purchased from Kelon Chemical Reagent Co., Ltd. (Chengdu, China);

[0039] Agarose and PD-L1 protein were purchased from Shanghai Sangon Biotechnology Co., Ltd.;

[0040] IgG, interferon-γ (IFN-γ), alpha-fetoprotein (AFP), tetanus toxoid, papain, prostate-specific antigen (PSA), lysozyme, human serum albumin (HSA), and transferrin were purchased from Sigma-Aldrich (St. Louis, MO, USA);

[0041] 3-(N-Morpholinyl)propanesulfonic acid (MOPS) was purchased from Sora Biotechnology Co., Ltd. (Beijing, China);

[0042] All working solutions were prepared with MOPS buffer (10 mM, 100 mM NaNO , pH 7.6);

[0043] Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin / streptomycin, phosphate-buffered saline (PBS), and syringe filters (0.22 μm) were purchased from Gibco Invitrogen (CA, USA);

[0044] Anti-CD9, anti-CD63, anti-CD81, and anti-PD-L1 antibodies were purchased from R&D systems (Minneapolis, USA);

[0045] Ultraclean centrifuge tubes (25 × 89 mm, 38.5 mL, sterile, open-cap thin-walled) were purchased from Beckman Coulter (Indianapolis, IN, USA);

[0046] Human lung cancer cells (A549) were provided by the core facility of West China Hospital;

[0047] The water used in all experiments was purified by a water purification system (Chengdu Ultrapure Technology Co., Ltd., Chengdu, China) with a resistivity of 18.25 MΩ·cm;

[0048] All reagents used in the work were of analytical grade or higher without further purification;

[0049] Healthy human serum and whole blood samples from lung cancer patients were donated by West China Hospital of Sichuan University (Chengdu, China, approval number: 20191045);

[0050] All solutions were stored in a refrigerator at 4°C before use.

[0051] Table 1. Sequences of oligonucleotides used in the study

[0052]

[0053] 2. Exosome Analysis Steps

[0054] The exosome samples were diluted 10 5 Double before use.

[0055] 10 μL of 1 μM aptamer-DNA and 40 μL of exosomes of different concentrations were added to 70 μL MOPS buffer (10 mM, 100 mM NaNO3, pH 7.6) and reacted at room temperature for 40 min.

[0056] Subsequently, 10 μL of 4 μM H1, H2, H3, and H4-DNA chains were added respectively and reacted at room temperature for 2 h to complete the HCR reaction.

[0057] Then, 10 μL of 6 μM CuSO 4 solution was added and reacted for 20 minutes.

[0058] Finally, 9 μL of 10 μM calcein disodium salt was added, and the fluorescence value was detected after 2 minutes of reaction.

[0059] 3. Pre-processing steps for clinical samples

[0060] Clinical peripheral blood samples (West China Hospital, Sichuan University, approval number: 20191045) were collected using a disposable closed ethylenediaminetetraacetic acid (EDTA) anticoagulation vacuum blood drawer to extract 4 mL of whole blood. The samples were centrifuged at 2,500 g for 15 min at 25°C within 2 hours after collection to precipitate red blood cells, white blood cells, and platelets.

[0061] The plasma was then centrifuged under the same conditions to separate platelet-poor plasma. The resulting plasma sample was diluted 1:20 with ice-cold PBS solution and then centrifuged at 10,000 rpm for 30 minutes at 4°C to remove large extracellular vesicles. The supernatant produced in the previous step was filtered through a 0.22 μm polyethersulfone filter to minimize microparticle contamination.

[0062] Subsequently, the filtered samples were subjected to ultracentrifugation at 150,000 g for 2 h at 4 °C, and the resulting pellets were washed with PBS and then centrifuged under the same conditions.

[0063] Finally, the isolated exosomes were resuspended in 200 μL of PBS and stored at −80 °C for further use.

[0064] 4. Cell Recovery, Culture and Exosome Extraction

[0065] Cell recovery: Take out A549 cells from the liquid nitrogen tank, shake rapidly in a 37°C water bath for 2 minutes, and then transfer to a centrifuge tube. Add 5 mL of RPMI 1640 medium containing 10% (v / v) fetal bovine serum, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, add 5 mL of RPMI 1640 medium, and then transfer to a culture dish. The cells are cultured in a cell culture incubator at 37°C and 5% CO2.

[0066] Cell culture: A549 cells were cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin solution at 37°C and 5% CO2. When the cells grew to 70-80% confluence, they were washed twice with PBS. The cell culture supernatant was then collected and centrifuged to extract exosomes.

[0067] Exosome preparation: The collected cell culture supernatant was centrifuged at 500g for 10 minutes at 4°C to discard cells and cell debris. The supernatant was then centrifuged at 2,000g for 20 minutes at 4°C to remove apoptotic bodies. The resulting liquid was centrifuged at 10,000g for 30 minutes at 4°C to remove large extracellular vesicles. The supernatant from the previous step was treated with a filter membrane (0.22μm) to minimize microparticle contamination. It was then ultracentrifuged at 150,000g for 2 hours at 4°C. The resulting pellet was resuspended in PBS and centrifuged under the same conditions. Finally, the isolated exosomes were suspended in PBS and stored at -80°C until use.

[0068] 5.Cu 2+ Coordination with DNA modulates the fluorescence signal of calcein

[0069] Figure 2 a shows Cu 2+ The phenomenon of binding to calcein to inhibit fluorescence signals and forming a complex with base pairs and phosphate backbones in DNA. Figure 2 b shows the characteristic ultraviolet absorption peak of calcein at a wavelength of 480 nm.

[0070] In a specific concentration range, Cu 2+ The increase in concentration will gradually reduce the fluorescence signal of calcein ( Figure 2 c) On the contrary, at a given Cu 2+ The fluorescence signal of calcein increases with the increase of double-stranded DNA concentration, which means that Cu 2+ It forms a complex with double-stranded DNA, thereby preventing Cu 2+ Reacts with calcein ( Figure 2 de).

[0071] Then, Figure 2 f studied the HCR reaction of Cu2+ Influence of coordination phenomena with calcein.

[0072] The results showed that as the concentration of aptamer increased, the double-stranded DNA products formed by HCR increased, thereby consuming more Cu 2+ , the fluorescence signal of calcein gradually increases. Therefore, the fluorescence intensity can be measured by Cu 2+ The coordination between Cu and DNA is regulated, thus reflecting the results of HCR. In addition, the Zeta potential results also show that Cu 2+ Complements with double-stranded DNA.

[0073] 6. PD-L1 analysis performance

[0074] To validate the proposed strategy, gel electrophoresis experiments were performed.

[0075] Figure 3 a shows that when the aptamer and four hairpin DNAs are present at the same time, a high molecular weight product is produced, indicating that an HCR reaction occurs (lane 4). In addition, reducing the aptamer concentration reduces the product yield (lanes 5-6). When different concentrations of PD-L1 are introduced, the band intensity gradually increases (lanes 7-9), which means that the aptamer specifically binds to PD-L1, thereby affecting the HCR reaction.

[0076] Figure 4 , Figure 5 The experimental conditions optimized for best assay performance are shown. The amount of aptamer used was determined to be 1 μM ( Figure 4 a), the incubation time of PD-L1 and aptamer was 40 min ( Figure 4 b) If Figure 4 As shown in c and 4d, the use of 4 μM H1-H4 can achieve the greatest difference in fluorescence signal (blank group vs. 1 fg / mL PD-L1). An incubation period of 120 minutes is sufficient to complete HCR ( Figure 4 e) When Cu 2+ When the concentration is 6 μM, the difference in fluorescence signals between the two is the largest ( Figure 5 ab).Cu 2+ The coordination reaction time with double-stranded DNA is 20 minutes ( Figure 5 c). When the volume of calcein (10 μM) reached 9 μL, the relative signal difference reached the maximum value ( Figure 5 de). In addition, free Cu 2+ The reaction time with calcein was 2 minutes ( Figure 5 f).

[0077] Under optimal conditions, there was a strong linear relationship between the fluorescence signal of calcein and the logarithm of PD-L1 concentration in the range of 100 ag / mL to 5 fg / mL ( Figure 3 bd). The limit of detection (LOD) calculated based on a 3-fold signal-to-noise ratio was 30 ag / mL, which is lower than that in previously published studies. This suggests that the current assay may be suitable for accurate quantification of exosomes.

[0078] The specificity of current strategies was evaluated by detecting non-target proteins, including immunoglobulin G (IgG), human serum albumin (HSA), and several cancer biomarkers ( Figure 3 e). Compared with the blank group, the results showed that the impact of high concentrations of potential interfering proteins (100 fg / mL) in the detection system was negligible, while obvious fluorescence reactions could be observed even at low concentrations of PD-L1. This laid a solid foundation for the subsequent clinical TDEs detection.

[0079] 7. Analytical performance of A549 cells

[0080] Given that PD-L1 is overexpressed on the surface of tumor exosomes, exosomes extracted from A549 cells were used for analysis.

[0081] First, the cell supernatant and plasma were ultracentrifuged to extract exosomes. Transmission electron microscopy (TEM) images showed that both types of exosomes had a unique cup-shaped morphology ( Figure 6 ab). Nanoparticle tracking analysis (NTA) determined that the average concentration of exosomes in cell supernatant and plasma was 6.0×10 8 and 2.8×10 10 Particles / mL ( Figure 6 cd). Capillary immunoassay showed the expression of PD-L1 and exosomal markers CD63, CD81, and CD9 ( Figure 6 e), indicating that the target exosomes were successfully obtained for subsequent detection.

[0082] Cell-derived exosomes were used to evaluate the detection capability of the current method. The results showed a strong linear correlation between the fluorescence signal and the logarithm of the exosome concentration (Log C). In the exosome concentration range of 300-10,000 particles / mL, the calcein signal was negatively correlated, and R 2 =0.992( Figure 6 fh). In addition, based on a 3-fold signal-to-noise ratio, the LOD was 100 particles / mL. Compared with previous reports, the current method reduces the number of steps and does not require labeling and separation.

[0083] In addition, Cu 2+ The introduction of calcein promotes the generation of signal response, and the signal amplification effect of HCR improves the sensitivity.

[0084] 8. Clinical applicability of exosome analysis methods

[0085] Based on the previous test results, 37 clinical whole blood samples (including 25 cancer patients and 12 healthy volunteers) were collected for TDEs fluorescence detection. These samples were then compared with clinical diagnostic information to evaluate the clinical feasibility and accuracy of the method.

[0086] Clinical sample analysis process Figure 7 First, exosomes were isolated from whole blood of lung cancer patients according to the previous sample pretreatment procedures. The isolated exosomes were diluted 10 5 times to bring its concentration within the detection range and then measure it using a fluorescence instrument.

[0087] Figure 7 b and d show that the fluorescence values ​​of patient exosomes (sample numbers 1-25) are significantly lower than those of healthy donor exosomes (sample numbers 26-37), indicating that the former have higher PD-L1 levels (except for sample numbers 10 and 20). The heat map visually distinguishes negative and positive samples, and the darker the color, the lower the PD-L1 content ( Figure 7 c). The area under the receiver operating characteristic (ROC) curve (AUC) was 0.963, the specificity was 100% (12 / 12), and the sensitivity was 92% (23 / 25) ( Figure 7 e), indicating that this method has good clinical diagnostic value. In addition, the consistency between the fluorescence analysis results and the clinical diagnosis results is also high ( Figure 7 fg). In conclusion, the proposed strategy demonstrated its feasibility and reliability in a clinical setting.

[0088] 9. Conclusion

[0089] The present invention successfully developed a sensing platform that can achieve high sensitivity and quantitative detection of TDEs by targeting exosomal PD-L1. The use of aptamers as recognition probes ensures the specificity of exosome analysis, while tandem HCR provides detection sensitivity by amplifying nucleic acid signals without the use of enzymes. 2+ The selective ligand provides a convenient way for signal output. The current strategy showed good consistency with clinical diagnostic results through fluorescence analysis of 37 clinical whole blood samples, thus verifying its practicality.

[0090] Although the current study showed excellent clinical diagnostic performance, further evaluation on more clinical samples and practical applications is needed. It is worth noting that due to the ease of operation and mild experimental conditions of this technology, if combined with commercial automatic exosome isolation instruments and miniature fluorescence measurement devices, it will help to achieve bedside testing (POCT) and establish portable inspection stations. In summary, the sensing strategy proposed in this invention shows the potential for early diagnosis and real-time monitoring in the treatment and management of lung cancer.

[0091] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination, characterized in that: The method comprises a specific recognition reaction between the membrane protein PD-L1 overexpressed on tumor exosomes and the PD-L1 aptamer probe, and a double-stranded DNA product generated by a tandem hybridization chain reaction between the PD-L1 aptamer probe and a bidirectionally extendable hairpin DNA and a Cu 2+ The fluorescence signal of calcein is effectively changed, and a single target is quantified based on the fluorescence signal of calcein, wherein the target is a membrane protein PD-L1 or a tumor exosome.

2. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: When the Cu 2+ When the calcein coordinates with the base pairs and the phosphate backbone in the double-stranded DNA product to form a complex and the fluorescence signal of the calcein does not change, the target does not exist.

3. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: When the PD-L1 aptamer probe and the bidirectionally extendable hairpin DNA do not undergo a tandem hybridization chain reaction and do not generate a double-stranded DNA product, the Cu 2+ When recognized by calcein, the fluorescence signal of calcein decreases, indicating the presence of the target.

4. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: The bidirectionally extendable hairpin DNA includes H1, H2, H3 and H4; The sequence of H1 is: ATC AAT GAG TTG ATG TGG CCC GTC TAA GAA CGG GCC ACA TCA ACT; The sequence of H2 is: ACG GGC CAC ATC AAC TCA TTG ATA GTT GAT GTG GCC CGT TCT TAG; The H3 sequence is: ACG GGC AGT GGA CGC ATT GTC TTC ATA CAG ACA ATG CGT CCA CT; The H4 sequence is: AGA CAA TGC GTC CAC TGC CCG TAG TGG ACG CAT TGT CTG TAT GA.

5. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: The PD-L1 aptamer probe sequence is: ACG GGC CAC ATC AAC TCA TTG ATA GACAAT GCG TCC ACT GCC CGT.

6. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: The tumor exosomes are derived from lung cancer A549 cells using differential ultracentrifugation.

7. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 6, characterized in that: The lung cancer A549 cells can be extended to other circulating tumor cells expressing mucin 1.

8. The method for fluorescence detection of tumor exosomes based on tandem hybridization chain reaction and selective coordination according to claim 1, characterized in that: The calcein can be replaced by quantum dots, carbon dots, N-methylporphyrin dipropionate IX or other Cu-recognizable 2+ signaling molecules.

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