Fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nano material

Abstract

The invention provides a fluorescence analysis method for performing one-step tumor liquid biopsy on the basis of DNA functional hydrogel of an embedded nano-material, and relates to the technical field of biomedical diagnos.The fluorescence analysis method comprises the steps that the DNA hydrogel of the embedded nano-material is prepared through multiple rolling circle amplification reactions, and the one-step tumor liquid biopsy is performed on the basis of combination of part of aptamers and a programmed death ligand 1; the method comprises the following steps: taking DNA (Deoxyribose Nucleic Acid) hydrogel as a template, adding an aptamer into the template, adding the template into the template, adding the aptamer, destroying the structure of the DNA hydrogel by the residual aptamer, triggering the release of the encapsulated nano-material, and quantifying a single target object based on a fluorescence signal of the nano-material, the target object being a tumor marker including circulating tumor cells (CTCs) and tumor-derived exosomes (TDE). According to the invention, tumor markers including CTCs and TDE are tested, and the universality and robustness of the method in proteins, exosomes and cells are verified. The sensitivity of fg / mL, the treatment time of about one hour, verification of 20 clinical samples and the like show that the method is expected to promote the development of the precision medical field.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical diagnosis, and in particular to a fluorescence analysis method for one-step tumor liquid biopsy using a DNA-functional hydrogel embedded with nanomaterials. Background Art

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates globally. In China, both the incidence and mortality rates of lung cancer rank first, bringing a huge disease and economic burden. Due to its non-obvious early symptoms, most patients are in the middle and late stages at the time of diagnosis, resulting in poor treatment effects.

[0003] Traditional diagnostic methods (such as imaging and tissue biopsy) have limitations such as low sensitivity and high invasiveness. Therefore, there is an urgent need to develop non-invasive and highly efficient early diagnosis and dynamic monitoring technologies. In recent years, the main core tools of liquid biopsy technology include circulating tumor cells (CTCs), tumor-derived exosomes (TDE), and circulating tumor DNA (ctDNA), providing a new direction for the diagnosis and treatment of lung cancer. Compared with ctDNA, CTCs and TDE have significant advantages in providing complete cell information, functional research, tumor heterogeneity analysis, and dynamic monitoring, making up for the deficiencies of ctDNA in terms of cell function and protein expression information, and providing a more comprehensive tool for precise tumor diagnosis and treatment. CTCs are tumor cells that shed from primary or metastatic tumors into the blood circulation and can reflect the real-time state of the tumor, while exosomes are nanoscale vesicles secreted by tumor cells and carry abundant tumor-specific biomarkers. Both can be non-invasively detected through body fluids such as blood, avoiding the invasiveness and limitations of traditional tissue biopsy. Their concentration changes are closely related to the tumor condition and have high clinical application potential.

[0004] Existing CTCs and TDE quantification techniques include relatively mature systems, flow cytometry, immunofluorescence, nanoparticle tracking analyzers, and nanoflow cytometry, etc. However, these techniques have disadvantages such as high cost, precise instruments, and long detection cycles. In addition, sensing techniques developed in recent years, including Raman spectroscopy, colorimetry, electrochemistry, microfluidic chips, etc., still cannot meet the sensitivity and specificity requirements of clinical liquid biopsy while being easy to operate. However, the detection of CTCs and TDE still faces problems such as insufficient technical sensitivity and specificity, tumor heterogeneity, lack of standardization, high cost, and technical thresholds. Therefore, developing a rapid, sensitive, and simple method for the precise detection strategy of CTCs and TDE will help promote their clinical application.

[0005] Although significant progress has been made in the quantitative detection techniques of CTCs and exosomes in terms of sensitivity, specificity, and high-throughput detection in recent years, there are still certain challenges. First, existing detection methods are often affected by sample complexity and low-concentration markers, resulting in weak signal intensity and limited sensitivity. Second, some techniques (such as electrochemical sensing and Raman spectroscopy) face high costs and operational complexity in practical applications, are not suitable for use in scenarios with limited conditions, and are difficult to popularize. Nucleic acid aptamers with high affinity and good stability have been widely used in the development of biosensors compared with other recognition probes. Moreover, single-stranded DNA aptamers are easy to modify without affecting target specificity and can integrate various nucleic acid-based amplification reactions for signal amplification to improve detection sensitivity. DNA nanostructures can autonomously regulate their functions according to external stimuli (such as pH, light, temperature, and target aptamers). At the same time, their hyperbranched structure provides many binding sites for target recognition probes and signal molecules. These characteristics enable DNA nanomaterials to contribute to achieving high efficiency and sensitivity in detection, providing new possibilities for the field of biosensing. Therefore, specifically responsive DNA nanomaterials can be pre-designed and prepared based on DNA aptamer sequences, which will help to achieve a rapid, accurate, and sensitive homogeneous method for one-step detection. Summary of the Invention

[0006] In view of this, the present invention provides a fluorescence analysis method for one-step tumor liquid biopsy based on a DNA-functional hydrogel embedding nanomaterials, so as to solve the technical problem of weak signal intensity and limited sensitivity in the quantitative detection techniques of CTCs and exosomes in terms of sensitivity, specificity, and high-throughput detection in the prior art.

[0007] To achieve the above object, the present invention provides a fluorescence analysis method for one-step tumor liquid biopsy based on a DNA-functional hydrogel embedding nanomaterials. The method includes preparing a DNA hydrogel embedding nanomaterials through multiple rolling circle amplification reactions, binding some aptamers to programmed death ligand 1, and the remaining aptamers destroying the structure of the DNA hydrogel to trigger the release of the encapsulated nanomaterials, and quantitatively detecting a single target based on the fluorescence signal of the nanomaterials, where the target is a tumor marker including circulating tumor cells (CTCs) and tumor-derived exosomes.

[0008] The biosensor of the present invention based on a DNA hydrogel converts a specific molecular recognition event into an acoustic, optical, electrical, or other detectable signal by means of a gel-solution conversion, making it highly versatile in biological applications.

[0009] Rolling circle amplification (RCA) is a mature isothermal amplification strategy that is currently often used to prepare DNA hydrogels, but there is still a problem of a long preparation time. Using multiple RCA may solve this problem.

[0010] In addition, although single-component DNA hydrogels are relatively easy to prepare, their applications in the biological field are still limited. To address these constraints, the three-dimensional structure of DNA hydrogels can be used to encapsulate nanomaterials (such as quantum dots, gold nanoparticles, carbon nanotubes, etc.), and their sequences can be carefully designed to achieve specific target recognition, which can promote the development of intelligent stimulus-responsive DNA hydrogels. These advanced hydrogels can serve as efficient biosensing elements to achieve a simplified and homogeneous "sample in, signal out" one-step detection mechanism.

[0011] Optionally, the target is inversely proportional to the intensity of the fluorescence signal. The higher the concentration of the target, the weaker the intensity of the fluorescence signal.

[0012] Optionally, the nanomaterial embedded in the DNA hydrogel is a quantum dot.

[0013] Optionally, the emission wavelength of the quantum dot is 530 nm.

[0014] Optionally, the repeating fragments of the RCA product forming the DNA hydrogel are evenly divided into three parts, serving as the three sides of an equilateral triangle. The waists of the three equilateral triangles are pairwise complementary, and the base is a palindromic sequence.

[0015] Optionally, the partial sequences on the waists of the equilateral triangle are complementary to the aptamer of programmed death ligand 1.

[0016] Optionally, the target is lung cancer A549 cells and their derived exosomes.

[0017] Optionally, the linear relationship between the fluorescence intensity and the concentration of exosomes of lung cancer A549 cells is: Y = -239LogC + 2541 (R 2 = 0.99).

[0018] The fluorescence analysis method for one-step tumor liquid biopsy using the DNA-functional hydrogel based on encapsulated nanomaterials provided by the present invention has the following technical effects:

[0019] This fluorescence analysis method is based on the sensing technology of DNA hydrogels encapsulated with quantum dots and is used for lung cancer liquid biopsy. The DNA hydrogel is prepared by triple RCA, and cadmium telluride quantum dots (CdTe QDs) are encapsulated during the gelation process. The DNA hydrogel serves as a structural scaffold, while the QDs are encapsulated in the hydrogel matrix to provide fluorescence signal output. After the aptamer binds to the target, the remaining aptamers will disrupt the stability of the DNA hydrogel structure, resulting in the release of QDs, and a detectable fluorescence signal, which is inversely proportional to the target concentration.

[0020] Using programmed death ligand 1 (PD-L1) as a biomarker, tumor markers including CTCs and TDEs were tested, and the generality and robustness of this method in proteins, exosomes, and cells were verified. The sensitivity of fg / mL, the processing time of about one hour, and the verification of 20 clinical samples, etc. indicate that this method is expected to promote the development of the field of precision medicine. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 Is a one-step strategy for liquid biopsy based on DNA hydrogels embedded with quantum dots: (a) Synthesis of DNA hydrogels embedded with quantum dots; (b) Detection process of the target; (c) Interaction between the aptamer and the DNA hydrogel embedded with quantum dots;

[0023] Figure 2 Is the exosome extraction process;

[0024] Figure 3 Is the feasibility verification;

[0025] Figure 4 Is the condition optimization of the RCA reaction process;

[0026] Figure 5 Is the condition optimization of the PD-L1 analysis process;

[0027] Figure 6 Is the analytical performance of PD-L1 and TDE;

[0028] Figure 7 Is a schematic diagram of the one-step method for lung cancer A549 based on DNA hydrogels embedded with quantum dots;

[0029] Figure 8 Is the extraction process of A549 cells in clinical samples. Detailed Embodiments

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will describe the technical solutions of the present invention in detail. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0031] The principle of the analysis method of the present invention is as follows:

[0032] As Figure 1 shown, the detection process can be completed in only one step.

[0033] First, DNA hydrogel / quantum dots are prepared through a triple rolling circle amplification (RCA) reaction ( Figure 1 a). DNA acts as a cross-linking unit, recognition element, and medium for encapsulating quantum dots in the hydrogel, thus forming a biosensor with integrated signal recognition and output functions. Programmed death ligand 1 (PD-L1) highly expressed in lung cancer tumor cells is selected as the detection target, and three groups of parallel RCAs are designed according to the PD-L1 aptamer sequence. The repeating fragments of each RCA product can be evenly divided into three parts, serving as the three sides of an equilateral triangle. The waists of the three triangles are pairwise complementary, and the base is a palindromic sequence. Such a design not only shortens the preparation time but also ensures the stability of the hydrogel.

[0034] Part of the base of each waist of the triangle is complementary to the aptamer of a specific target. After introducing the PD-L1 aptamer and its corresponding targets (PD-L1 / A549 cells / A549 cell-derived exosomes), part of the aptamer binds to the target, while the other part will destroy the stability of the DNA hydrogel, trigger the release of encapsulated quantum dots, and generate a detectable fluorescence signal ( Figure 1 b and 1c).

[0035] Simply put, a higher target concentration will result in a lower fluorescence intensity, and the target can be quantitatively analyzed through the change in fluorescence intensity.

[0036] The technical solution of the present invention will be specifically verified below in combination with embodiments.

[0037] Example 1 Materials and Methods

[0038] 1. Materials and Reagents

[0039] Oligonucleotides with different sequences were synthesized and purified by Shanghai Sangon Biotech Co., Ltd.;

[0040] Table 1 lists the sequences of the oligonucleotides used in the present invention;

[0041] T4 DNA ligase, phi 29 DNA polymerase, deoxynucleotide (dNTPs) mixed solution, bovine serum albumin, agarose, recombinant human programmed cell death ligand 1 (PD-L1) protein were purchased from Shanghai Sangon Biotech Co., Ltd.;

[0042] SYBR Green I and 6×DNA loading Dye were purchased from Thermo Fisher Scientific (Massachusetts, USA);

[0043] Human serum albumin (HSA), trypsin, glucose oxidase (GOD), immunoglobulin G (IgG), pepsin, streptavidin (SA), prostate specific antigen (PSA), transferrin, interferon-γ (IFN-γ), papain, cluster of differentiation 3 (CD3), lysozyme, thrombin and histidine were all ordered from Sigma-Aldrich (St. Louis, MO, USA);

[0044] Recombinant human mucin 1 (MUC1) protein was ordered from Sino Biological (Beijing, China);

[0045] All working solutions were prepared with MOPS buffer solution (10 mM MOPS, 150 mM NaCl, 50 mM MgCl 2 , pH 7.4). Phosphate buffered saline (PBS) was ordered from Corning (New York, USA);

[0046] Dulbecco's Modified Eagle Medium (DMEM / F12), fetal bovine serum (FBS), penicillin / streptomycin and syringe filters (0.22 μm) were purchased from Gibco Invitrogen (California, USA);

[0047] DNA marker (50 - 1031 bp) and 4SGelred were purchased from BBI Co., Ltd. (Shanghai, China);

[0048] Ultra-clear centrifuge tubes (25×89 mm, 38.5 mL, sterile, open thin-walled) were purchased from Beckman Coulter (Indianapolis, Indiana, USA);

[0049] All reagents used in this invention are analytical grade or higher grade reagents and do not require further purification when used. All experimental water was from the purified water system of Chengdu Ultra-pure Technology Co., Ltd. (Chengdu, China) with a resistivity of 18.25 MΩ·cm. Human lung cancer cells (A549), human umbilical vein endothelial cells (HUVEC), human embryonic kidney 293T cells (HEK-293T) and human normal lung epithelial cells (BEAS-2B) were provided by the scientific research base of West China Hospital. All solutions were stored in a 4°C refrigerator before use.

[0050] Table 1. Oligonucleotide sequences used in the study

[0051]

[0052]

[0053] *: Phosphorylation modification

[0054] 2. Synthesis of CdTe QDs

[0055] The common process for the synthesis of CdTe QDs is prior art.

[0056] Briefly, first prepare a 50 mL solution containing CdCl 2 (0.5 mM) and trisodium citrate (0.2 g). Then immediately add mercaptopropionic acid (MPA, 52 μL) to the above solution, and adjust the pH of the solution to 10.5 with NaOH. Subsequently, add Na 2 TeO 3 (0.1 mM) and KBH 4 (50 mg), and reflux in portions to obtain CdTe QDs. Subsequently, precipitate with n-propanol and centrifuge (11000 rpm) to obtain high-purity CdTe QDs. The purified red CdTe QDs are redispersed in ultrapure water before use.

[0057] 3. Cell resuscitation, culture and passage

[0058] Cell resuscitation: Take out the cryopreserved A549 cells from the liquid nitrogen tank, quickly thaw them at 37 °C, and then transfer them to a centrifuge tube. Add 5 mL of DMEM / F-12 medium containing 10% (v / v) FBS, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, add 5 mL of medium, and then transfer it to a culture dish. The cells are cultured at 37 °C under 5% CO 2 conditions.

[0059] Cell culture: A549 cells are cultured in 1640 medium containing 10% (v / v) FBS, and cultured at 37 °C and 5% CO 2 . When the cells are in the logarithmic growth phase, digest them into a single cell suspension and count. Finally, seed the cells in a culture dish.

[0060] Cell passage: When the cells are in the logarithmic growth phase, remove the old medium and wash with PBS buffer. Subsequently, digest with trypsin and centrifuge at 800 rpm for 3 minutes to collect the cells. Then add fresh medium. Finally, seed the cells in a new culture dish and continue culturing.

[0061] 4. Isolation of exosomes

[0062] After the cultured cells reach 70 - 80% confluence, wash them twice with DMEM medium.

[0063] As Figure 2 shown, all cell culture supernatants were collected into 50 mL sterile conical tubes for ultracentrifugation to isolate exosomes.

[0064] First, centrifuge at 500 g for 10 minutes at 4 °C to precipitate cells and cell debris.

[0065] Then, centrifuge the supernatant at 2000 g for 20 minutes at 4 °C to further remove cell debris and apoptotic bodies. The resulting supernatant was centrifuged at 10000 g for 30 minutes at 4 °C to remove large extracellular vesicles. The supernatant was then filtered through a 0.22 μm filter membrane to reduce microparticle contamination. The filtrate was ultracentrifuged at 150000 g for 2 hours at 4 °C. The resulting particles were resuspended in PBS and recentrifuged under the same conditions.

[0066] Finally, the isolated exosomes were resuspended in 200 μL PBS and stored at -80 °C.

[0067] Feasibility verification of Example 2

[0068] The preparation process of the DNA hydrogel material embedded with quantum dots is as Figure 3 shown in a, mainly relying on three groups of parallel RCA.

[0069] To characterize the RCA reaction process, agarose gel electrophoresis and atomic force microscopy (AFM) were first used. The successful synthesis of circular templates and ultra-long linear DNA is shown in lanes 10 - 12 and 13 - 15 respectively ( Figure 3 b). The AFM images further illustrate the products at different stages of the RCA reaction: before ligation, after ligation, and after amplification ( Figure 3 c).

[0070] Subsequently, the properties of the DNA hydrogel were verified. Figure 3 The elastic modulus (G', about 6 Pa) and viscous modulus (G", about 0.6 Pa) measured in d indicate that the hydrogel is soft and maintains a gel-like state. DNA hydrogels usually exhibit a three-dimensional, loose, and porous structure, which is confirmed by scanning electron microscopy (SEM) imaging ( Figure 3 e).

[0071] To achieve "sample in, result out" analysis, CdTe QDs were encapsulated in the DNA hydrogel. Transmission electron microscopy (TEM) images show that the diameter of the QDs is about 4 nm ( Figure 3 f). Ultraviolet (UV) absorption spectra ( Figure 3 g) show that a characteristic peak appears at 570 nm. Fluorescence microscopy (FM) was used to observe the DNA hydrogel stained with SYBR Green I, the encapsulated QDs, and their overlapping images.Figure 3 h 1 It shows that the morphology of the hydrogel is consistent with the SEM image.

[0072] To distinguish QDs from the stained dsDNA, QDs with an emission wavelength of 650 nm were selected. Figure 3 h 2-3 It was further confirmed that QDs had been successfully embedded in the hydrogel.

[0073] Subsequently, Zeta potential was used to monitor the formation of DNA hydrogel and DNA hydrogel embedded with quantum dots, as well as the interaction between the aptamer, PD-L1 and the hydrogel ( Figure 3 i). The observed increase in surface charge indicated that the introduced aptamer affected the DNA hydrogel and the DNA hydrogel embedded with quantum dots, resulting in the release of QDs, while the presence of PD-L1 inhibited this release.

[0074] Through the above experiments, the feasibility of the present invention being subsequently used for the quantitative analysis of the target was demonstrated.

[0075] Example 3 Optimization of RCA and PD-L1 Detection Conditions

[0076] To improve the analytical performance, the conditions of the entire analytical process were systematically optimized.

[0077] First, the RCA reaction process was optimized. When the concentrations of the linker strand and the padlock probe were selected to be 7.5 μM, the fluorescence difference induced between low concentration (10 fM) and high concentration (10 pM) of PD-L1 reached the highest ( Figure 4 a-c).

[0078] Subsequently, the ligation conditions of the linker strand and the padlock probe were evaluated, and the amount of T4 ligase used was selected to be 1.25 μL and the reaction time was 75 minutes ( Figure 4 d-f).

[0079] The optimal amounts of Phi 29 polymerase and dNTP were 1.75 μL and 3 μL respectively ( Figure 4 g-j). And the amplification time of the polymerase was optimized to 120 minutes ( Figure 4 k-l), achieving the best signal-to-noise ratio.

[0080] When the amount of the RCA product was 20 μL, the most obvious signal difference was presented ( Figure 5 a-b).

[0081] QDs with an emission wavelength of 530 nm had the best effect ( Figure 5 c-d). The optimal amount of QDs was 10 μL ( Figure 5 e-f).

[0082] The optimal concentration of the aptamer was set at 2.5 μM( Figure 5 g-h), and the interaction time between PD-L1, the aptamer, and the DNA hydrogel was adjusted to 60 minutes( Figure 5 i-j), further improving the signal-to-noise ratio.

[0083] Example 4 Analytical performance of PD-L1 and TDE

[0084] First, detection was performed on the protein itself and exosomes( Figure 6 a). Figure 6 b shows the negative correlation between the PD-L1 concentration and the fluorescence intensity. A linear relationship was observed between the fluorescence and the logarithm of the PD-L1 concentration( Figure 6 c), and the LOD was determined to be 0.6 fg / mL according to three times the standard deviation.

[0085] In addition, the property of the specific response of the system was tested. The influence of the interferent on the fluorescence of the system was negligible, equivalent to the blank signal or causing a slight decrease in fluorescence when the concentration was higher than the target protein( Figure 6 d). These findings laid a solid foundation for subsequent exosome analysis.

[0086] After successfully enriching and purifying A549 cell-derived exosomes from the cell supernatant, the fluorescence intensity of the system in the presence of exosomes was measured, and it was found that the fluorescence intensity gradually decreased as the exosome level increased( Figure 6 e). A linear relationship was observed between the fluorescence intensity and the exosome concentration (10 4 to 10 7 particles / mL)( Figure 6 f), and the calculation formula was Y = -239LogC + 2541 (R 2 = 0.99). Figure 6 g shows that the fluorescence intensity of exosomes from other cell sources at 10 6 particles / mL did not decrease significantly, while the fluorescence intensity of A549 cell-derived exosomes at the same concentration decreased sharply.

[0087] The results indicate that this method not only ensures the simplicity of the system and shortens the detection time but also achieves high sensitivity, demonstrating the potential application value of this platform in clinical samples of lung cancer patients.

[0088] Example 5 Detection feasibility of lung cancer A549 cells

[0089] This example verified the ability of this platform in detecting A549 cells and its clinical applications( Figure 7 a). Consistent with expectations, the A549 cell concentration was negatively correlated with the fluorescence intensity within a certain range( Figure 7b), the fluorescence intensity showed a linear relationship with the logarithm of the A549 cell concentration ( Figure 7 c).

[0090] To evaluate its clinical utility, 4 mL of whole blood was collected from 15 non-small cell lung cancer (NSCLC) patients and 5 healthy volunteers. The A549 cells were isolated using the "12345" method ( Figure 8 ) for detection. The fluorescence data is shown in Figure 7 d, and to clearly distinguish between the healthy and diseased groups, the results were normalized and presented in the form of a heat map ( Figure 7 e).

[0091] Statistical analysis showed that there was a significant difference in the fluorescence signals between the healthy control group and the lung cancer patients (P < 0.001, Figure 7 f). Meanwhile, a receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic performance of the strategy, and the area under the curve was as high as 0.98.

[0092] To further verify the accuracy of the research results, the pathological and computed tomography (CT) images of the patients were collected synchronously, and the results showed a very good agreement with the fluorescence detection results ( Figure 7 h). This method showed comparable sensitivity to existing sensing methods while maintaining the simplicity of the system, demonstrating its potential as a reliable tool for assisting NSCLC diagnosis.

[0093] Conclusion of Example 6

[0094] In summary, based on the pre-prepared functional integrated DNA hydrogel material embedded with quantum dots, a rapid and simple one-step liquid biopsy method for lung cancer was obtained. While achieving specific response and sensitive quantitative detection of the target, this method simplifies the operation steps and reduces the reaction time required, is very suitable for clinical detection scenarios, and is conducive to promotion. Through verification from proteins to exosomes and then to tumor cells, it demonstrates the superiority of the method's performance and has good application prospects.

[0095] The above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials, characterized in that: The method includes preparing a DNA hydrogel encapsulating nanomaterials through a multiple rolling circle amplification reaction, based on the binding of some aptamers to programmed death ligand 1, the remaining aptamers destroy the structure of the DNA hydrogel, triggering the release of the encapsulated nanomaterials, and quantifying a single target based on the fluorescent signal of the nanomaterial, wherein the target is a tumor marker including circulating tumor cells and tumor-derived exosomes.

2. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 1, characterized in that: The intensity of the target object is inversely proportional to the intensity of the fluorescent signal. The higher the concentration of the target object, the weaker the intensity of the fluorescent signal.

3. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 1, characterized in that: The nanomaterial embedded in the DNA hydrogel is quantum dots.

4. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 3 is characterized in that: The emission wavelength of the quantum dots is 530 nm.

5. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 3, characterized in that: The repetitive fragments of the rolling circle amplification products that form the DNA hydrogel are evenly divided into three parts, which serve as the three sides of an equilateral triangle. The waists of the three equilateral triangles are complementary to each other, and the bases are palindromic sequences.

6. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 5, characterized in that: The partial sequence on the waist of the equilateral triangle is complementary to the aptamer of programmed death-ligand-1.

7. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 1, characterized in that: The target is lung cancer A549 cells and exosomes derived therefrom.

8. The fluorescence analysis method for one-step tumor liquid biopsy based on DNA functional hydrogel embedded with nanomaterials according to claim 7, characterized in that: The linear relationship between the fluorescence intensity and the exosome concentration derived from the lung cancer A549 cells is: Y = -239LogC + 2541 (R 2 =0.99).

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