DNA-Pb based 2+ Homogeneous electrochemical analysis method and application of functional nanohydrogel for exosome detection

Abstract

The present invention provides a DNA-Pb 2+ The present invention relates to a homogeneous electrochemical analysis method and application of functional nano-hydrogel for exosome detection, which relates to the technical field of biomedical diagnostic analysis methods. The present invention is based on the DNA nano-hydrogel prepared by the rolling circle amplification (RCA) method, combining the G-quadruplex structure with Pb 2+ The specific recognition mechanism was constructed, and the epithelial cell adhesion molecule (EpCAM) on the surface of exosomes was selected as the detection target. The specific binding between EpCAM and its aptamer triggered the disintegration of DNA nanohydrogel, thereby exposing a large number of G-quadruplex structures generated by RCA, which could bind to Pb 2+ By selectively binding and regulating the electrochemical signal output, this method realizes a one-pot, rapid and efficient detection process, and the entire process is completed within 45 minutes.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical diagnostic analysis methods, in particular to a method based on DNA-Pb 2+ Homogeneous electrochemical analysis method and application of functional nanohydrogel for exosome detection. Background Art

[0002] Accurate diagnosis and staging of breast cancer are crucial for guiding clinical treatment strategies and predicting patient survival outcomes. However, existing diagnostic technologies still have certain limitations: imaging methods lack sensitivity in detecting microtumors and micrometastases, while tissue biopsy, the current "gold standard," is invasive and unsuitable for repeated testing, limiting its clinical application in real-time dynamic monitoring. In recent years, liquid biopsy using exosomes as tumor markers has become a promising technology for cancer screening and early diagnosis. Studies have shown that exosome levels are significantly correlated with tumor stage, with exosome levels typically higher in the late stages of cancer. Exosomes are considered promising biomarkers for monitoring tumor progression.

[0003] However, existing methods for quantitative exosome detection, such as nanoparticle tracking analysis (NTA) and immunology-based flow cytometry, have limitations. They often fail to effectively distinguish between different exosome subtypes or require complex labeling and separation steps, resulting in cumbersome operations and low detection efficiency. They also require high laboratory equipment and technical expertise, and their sensitivity is insufficient, limiting their widespread clinical application. Therefore, there is an urgent need for new exosome detection methods that can achieve precise identification, high sensitivity, and simple operation, thereby overcoming the shortcomings of existing methods and improving the accuracy and practicality of exosome detection.

[0004] In recent years, a variety of novel sensing technologies have been developed for exosome analysis, including surface-enhanced Raman spectroscopy, fluorescence detection, electrochemical detection, electrochemiluminescence, and colorimetry. While these methods demonstrate excellent sensitivity, they generally rely on magnetic bead enrichment or complex material modification and synthesis processes, resulting in cumbersome detection workflows. Compared to antibodies, nucleic acid aptamers have been widely used in biosensing due to their ease of synthesis and strong target affinity. Aptamers can be combined with various nucleic acid amplification strategies, such as rolling circle amplification (RCA), polymerase chain reaction (PCR), and hybridization chain reaction (HCR), to achieve highly sensitive target detection. However, while cascade amplification methods can significantly enhance signal response, their operational complexity compared to single-signal amplification strategies limits their practical applicability. Currently, various DNA-based functional nanostructures, including DNA nanospheres, DNA tetrahedrons, and DNA nanohydrogels, have garnered significant attention due to their dual advantages of enhanced detection sensitivity and simplified operation. DNA nanohydrogels, primarily assembled from complementary RCA products, exhibit excellent programmability, high loading capacity, and responsiveness to specific analytes. Therefore, functionally integrated DNA nanohydrogels could serve as promising high-sensitivity sensing platforms integrating target recognition, signal amplification, and output.

[0005] Taking advantage of the advantages of DNA nanohydrogels in structural programmability and molecular responsiveness, more and more research is devoted to using them to encapsulate signal molecules to achieve rapid signal response. However, traditional physical encapsulation has problems such as low signal molecule loading, which leads to limited detection sensitivity and difficulty in ensuring stability. In order to overcome the above-mentioned defects, a strategy of introducing repeating functional DNA structural units (such as G-quadruplex or hairpin structure) into DNA nanohydrogels has been proposed in recent years to enhance signal amplification and output specificity. Among them, the G-quadruplex structure can specifically bind to a variety of signal molecules (such as methylene blue (MB), doxorubicin (DOX), lead ions (Pb 2+ For example, studies have shown that G-quadruplexes can selectively bind to MB, and the precise quantification of target molecules can be achieved by detecting the difference in electrochemical signals between free MB and G-quadruplex-MB complexes. However, compared with MB and DOX, Pb 2+ It has a stronger affinity for G-quadruplex, which provides a basis for the construction of G-quadruplex-Pb 2+ The DNA nanohydrogel platform for the recognition mechanism provides a new technical path. Electrochemical sensors have attracted attention due to their miniaturization, ease of operation, and high sensitivity, but their reliance on electrode modification often leads to reduced reproducibility.

[0006] Therefore, if the Pb 2+ With G-quadruplex-Pb 2+The effective differentiation of complexes makes it feasible to establish a homogeneous (one-pot) electrochemical strategy. Summary of the Invention

[0007] In view of this, one of the objects of the present invention is to provide a DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is proposed to solve technical problems such as low signal molecule loading in traditional physical encapsulation, which leads to limited detection sensitivity and difficulty in ensuring stability.

[0008] The second object of the present invention is to provide a DNA-Pb 2+ Application of homogeneous electrochemical analysis method for exosome detection based on functional nanohydrogels.

[0009] In order to achieve one of the above objects, the present invention provides a DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels, the method comprising constructing a DNA nanohydrogel based on an RCA product, wherein the DNA nanohydrogel comprises a nucleic acid aptamer for recognition, a complementary base hybridization region, and a G-quadruplex for signal output;

[0010] Based on whether the nucleic acid aptamer specifically binds to the target to cause the DNA nanohydrogel to disintegrate and expose the G-quadruplex, and based on whether the G-quadruplex binds to Pb 2+ Formation of G-quadruplex-Pb 2+ Complex, through Pb 2+ The target is quantified by an electrochemical signal, and the target is an exosome.

[0011] Optionally, the exosomes are positive for expression of epithelial cell adhesion molecules.

[0012] Optionally, the Pb 2+ With the G-quadruplex-Pb 2+ The complexes have obvious electrochemical signal differences. 2+ The electron transport performance is much greater than that of the G-quadruplex-Pb 2+ Complex.

[0013] Optionally, when epithelial cell adhesion molecules are present, they bind to the corresponding nucleic acid aptamers to trigger the disintegration of the DNA nanohydrogel, exposing the G-quadruplex and binding to free Pb 2+ , reducing free Pb 2+ The concentration of , resulting in a decrease in the electrochemical signal intensity;

[0014] When epithelial cell adhesion molecules were absent, the DNA nanohydrogel remained intact, the G-quadruplex was not exposed, and Pb 2+ It can freely diffuse to the surface of the ITO electrode and significantly enhance the electrochemical signal output.

[0015] Optionally, the G-quadruplex is GT GGG TA GGG C GGG TT GG.

[0016] Optionally, the primers of the DNA nanohydrogel include Primer-1 and Primer-2;

[0017] The Primer-1 is GTG GGT AGG GCG GGT TGG TGA AGG TTC GTT GTT T;

[0018] The Primer-2 is ACA GAT TTT GGG AAT GGT GGG TAG GGC GGG TTG G.

[0019] Optionally, the padlocks of the DNA nanohydrogel include Padlock-1 and Padlock-2;

[0020] The Padlock-1 is Phosphate-CCA ACC CGC CCT ACC CAC GTG TGA TGA GAC TAAATA CTA AAG CAT TCC CAA AAT CTG TAT CTC TTC TAA AGA GTC TAC ACC CAC CGA AACAAC GAA CCT TCA;

[0021] The Padlock-2 is Phosphate-CAT TCC CAA AAT CTG TAT CTC TTC TAA AGA GTCTAC ACC CAC CGA AAC AAC GAA CCT TCA CTT TAG TAT TTA GTC TCA TCA CAC CCA ACCCGC CCT ACC CAC.

[0022] Optionally, the Pb 2+ The linear regression equation of the peak current and the logarithmic concentration of the epithelial cell adhesion molecule is Y = -100LogC + 1242, and the correlation coefficient is R 2 The concentration of epithelial cell adhesion molecule ranged from 100 ag / mL to 10 pg / mL.

[0023] Optionally, the Pb 2+Methylene blue or doxorubicin can be used as an alternative.

[0024] In order to achieve the second of the above purposes, a DNA-Pb 2+ Application of a homogeneous electrochemical analysis method for exosome detection using functional nano-hydrogels, the application comprising the steps of: 2+ A homogeneous electrochemical analysis method for exosome detection based on functional nanohydrogels was used for the quantitative analysis of epithelial cell adhesion molecules.

[0025] The present invention provides a DNA-Pb 2+ The homogeneous electrochemical analysis method and application of functional nano-hydrogel for exosome detection have the following technical effects:

[0026] The DNA nanohydrogel prepared by the rolling circle amplification (RCA) method combines the G-quadruplex structure with Pb 2+ The specific recognition mechanism was constructed, and the epithelial cell adhesion molecule (EpCAM) on the surface of exosomes was selected as the detection target. The specific binding between EpCAM and its aptamer triggered the disintegration of DNA nanohydrogel, thereby exposing a large number of G-quadruplex structures generated by RCA, which could bind to Pb 2+ By selectively binding and regulating the electrochemical signal output, this method realizes a one-pot, rapid and efficient detection process, and the entire process is completed within 45 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0028] Figure 1 Schematic diagram of the strategy for homogeneous electrochemical detection of breast cancer-derived exosomes using DNA nanohydrogels for targeted protein regulation;

[0029] Figure 2 Figure 2 shows the preparation and characterization of DNA nanohydrogels containing G-quadruplexes;

[0030] Figure 2 a is a schematic diagram of DNA nanohydrogel synthesis based on RCA;

[0031] Figure 2 b is the agarose gel electrophoresis image of RCA;

[0032] Figure 2c1-c3 are representative AFM images of primers and padlock probes before and after ligation and RCA products;

[0033] Figure 2 d is the image of RCA product 1, RCA product 2 and DNA nanohydrogel under sunlight and ultraviolet light;

[0034] Figure 2 e is the rheological properties diagram of DNA nanohydrogel;

[0035] Figure 2 f is a fluorescence microscopy image of DNA nanohydrogel stained with SYBR green I;

[0036] Figure 2 g is a scanning electron microscope image of DNA nanohydrogel;

[0037] Figure 3 For electrochemical analysis of Pb 2+ and G-quadruplex-Pb 2+ Specific recognition map of the complex;

[0038] Figure 3 a is Pb 2+ Schematic diagram of electrochemical analysis as a G-quadruplex binding probe;

[0039] Figure 3 b shows different G-quadruplexes and Pb 2+ Electrochemical signal diagram after binding;

[0040] Figure 3 c is the relationship between the electrochemical peak current and the logarithmic concentration of PS2.M;

[0041] Figure 3 d is the RCA product and Pb in the presence of different padlock probe concentrations. 2+ Electrochemical signal diagram between

[0042] Figure 3 e is the CD spectrum of G-quadruplex;

[0043] Figure 4 Optimization diagram for EpCAM analysis conditions;

[0044] Figure 4 a is the volume diagram of T4 ligase;

[0045] Figure 4 b is the connection time graph;

[0046] Figure 4 c is the volume diagram of dNTPs;

[0047] Figure 4d is the volume diagram of Phi29 DNA polymerase;

[0048] Figure 4 e is the amplification time diagram;

[0049] Figure 5 is the analytical performance diagram of EpCAM;

[0050] Figure 5 a is the detection of free Cu after EpCAM was added to DNA nanohydrogel using ICP-MS 2+ and Pb 2+ concentration map;

[0051] Figure 5 bc are DLS and Zeta potential images of RCA products 1 and 2 and DNA hydrogel;

[0052] Figure 5 d is the DPV measurement value diagram;

[0053] Figure 5 e The corresponding peak current graphs at different EpCAM concentrations;

[0054] Figure 5 f is the linear correlation graph between the peak current and the logarithm of EpCAM concentration;

[0055] Figure 5 g is the protein selectivity of EpCAM, and the error bars represent the data obtained from three repeated measurements;

[0056] Figure 6 for the analytical performance of exosomes;

[0057] Figure 6 a Schematic diagram of the workflow for exosome isolation, characterization, and analytical performance evaluation;

[0058] Figure 6 bc are NTA assay images of exosomes isolated from MCF-7 cell culture supernatant or plasma samples;

[0059] Figure 6 de are TEM images of exosomes derived from MCF-7 cells and plasma;

[0060] Figure 6 f is a Western blot analysis of exosome markers derived from MCF-7;

[0061] Figure 6 gh is the DPV response and Pb at different exosome concentrations 2+ The corresponding peak current diagram;

[0062] Figure 6i is the linear correlation diagram between peak current and logarithmic concentration of exosomes;

[0063] Figure 7 Diagram of clinical diagnosis and results analysis of breast cancer patients;

[0064] Figure 7 a is a flow chart of clinical sample collection and analysis;

[0065] Figure 7 b is a bar chart of diagnosis results;

[0066] Figure 7 c is a box plot of the diagnosis results;

[0067] Figure 7 d is the heat map of diagnosis results;

[0068] Figure 7 e is the ROC analysis graph;

[0069] Figure 7 f is the patient's clinical MRI result image;

[0070] Figure 7 g is the pathological result diagram;

[0071] Figure 8 To analyze clinical breast cancer staging and outcomes;

[0072] Figure 8 a is the analysis flow chart of clinical sample staging;

[0073] Figure 8 b is a bar chart of staging results;

[0074] Figure 8 c is a box plot of the staging results;

[0075] Figure 8 d is the heat map of staging results;

[0076] Figure 8 e is the ROC analysis graph;

[0077] Figure 8 f is the corresponding CT and Figure 8 g is the pathological result diagram. DETAILED DESCRIPTION

[0078] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0079] The principle of the analytical method of the present invention is as follows:

[0080] like Figure 1 The figure shows a homogeneous electrochemical analysis method based on DNA nanohydrogel for detecting exosomes from breast cancer. This analysis method achieves the detection of Pb by introducing G-quadruplex structure into DNA hydrogel system. 2+ The specific identification of glioma has potential application value in early diagnosis and staging of breast cancer.

[0081] Specifically, DNA nanohydrogels are constructed based on RCA products. The hydrogel structure integrates nucleic acid aptamers for target identification, complementary base hybridization regions, and G-quadruplex sequences for signal output. When the target molecule specifically binds to the aptamer in the nanohydrogel, the complementary base pairing structure in the adjacent region is destroyed, leading to the disintegration of the DNA nanohydrogel structure. This disintegration process exposes a large number of G-quadruplex structures, which quickly react with free Pb in the system. 2+ Selective binding occurs to form G-quadruplex-Pb 2+ complex to achieve signal amplification.

[0082] Due to free Pb 2+ It has excellent electron transport properties and can be directly detected by electrochemical instruments; while G-quadruplex-Pb 2+ The complex is inert on the ITO electrode, and there is a clearly distinguishable electrochemical signal difference between the two, thus enabling homogeneous electrochemical quantitative detection without modification.

[0083] During the detection process, when EpCAM is present, it binds to the aptamer and triggers the disintegration of the nanohydrogel, exposing the G-quadruplex and binding to free Pb 2+ , thereby reducing free Pb 2+ In contrast, in the absence of EpCAM, the hydrogel remained intact, the G-quadruplex was not exposed, and Pb 2+ It can freely diffuse to the surface of the ITO electrode and significantly enhance the electrochemical signal output.

[0084] The technical solution of the present invention is specifically verified below with reference to the embodiments.

[0085] 1 Materials and Methods

[0086] 1.1 Materials and Reagents

[0087] All oligonucleotides with different sequences were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences are shown in Table 1 .

[0088] T4 DNA ligase, phi29 DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), bovine serum albumin (BSA), and agarose were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0089] Recombinant human epidermal cell adhesion molecule (EpCAM) protein was purchased from Shanghai Novozyme Biotechnology Co., Ltd.

[0090] Lead nitrate (Pb(NO3)2) and sodium chloride (NaCl) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

[0091] DNA molecular weight markers (25–500 bp) and 4S Gel-Red dye were purchased from BBI Biotechnology Co., Ltd. (Shanghai, China); 6× DNA loading buffer was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0092] Human serum albumin (HSA), trypsin, glucose oxidase (GOD), immunoglobulin G (IgG), pepsin, streptavidin (SA), prostate-specific antigen (PSA), transferrin, interferon-γ (IFN-γ), papain, nuclear matrix protein 22 (NMP-22), glycocortin 3 (GPC3), lysozyme, thrombin, human immunodeficiency virus (HIV) p24 protein, and tetanus toxoid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0093] Dulbecco's modified Eagle's medium (DMEM) and human breast cancer MCF-7 cell line were purchased from Zhanjiang Tongbo Medical Technology Co., Ltd.

[0094] Fetal bovine serum (FBS), penicillin / streptomycin, phosphate-buffered saline (PBS), and 0.22 μm syringe filters were purchased from Gibco Invitrogen Corporation (California, USA).

[0095] Anti-CD9, anti-CD63, and anti-CD81 antibodies were purchased from Proteintech (Rosemont, IL, USA), and EpCAM antibody was purchased from Thermo Fisher.

[0096] Ultra-clean centrifuge tubes (25 × 89 mm, 38.5 mL, sterile, thin-walled opening) were purchased from Beckman Coulter (Indiana, USA).

[0097] All water used in experiments was purified by an ultrapure water system from Chengdu Ultrapure Technology Co., Ltd., with a resistivity of 18.25 MΩ·cm. All working solutions were prepared in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS and 100 mM NaNO3, pH 7.4).

[0098] All reagents used in this invention were of analytical grade or higher and used directly without further purification. All solutions were stored at 4°C until use. Healthy human plasma samples and breast cancer patient whole blood samples were provided by West China Hospital, Sichuan University. This study was approved by the Biomedical Ethics Committee of West China Hospital, Sichuan University (Chengdu, China, Approval No. 20221489).

[0099] Table 1. Sequences of oligonucleotides used in this study

[0100]

[0101] 1.2 Cell culture and exosome isolation

[0102] Cell Culture: MCF-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v / v) FBS at 37°C in a 5% CO2 incubator. When cell confluency reached 70%-80%, cells were washed twice with DMEM without any additives and then replaced with serum-free DMEM for an additional 48 hours. After culture, all cell supernatants were collected in sterile 50 mL conical centrifuge tubes and subjected to gradient centrifugation to extract exosomes.

[0103] Exosome extraction: Cells and debris were pelleted by centrifugation at 500 × g and 4°C for 10 minutes. The resulting supernatant was then centrifuged at 2,000 × g and 4°C for 20 minutes to further remove cell debris and apoptotic bodies. The supernatant was then centrifuged at 10,000 × g and 4°C for 30 minutes to remove larger extracellular vesicles. The supernatant was then filtered through a 0.22 μm filter to minimize particulate contamination. The filtered medium was ultracentrifuged at 150,000 × g and 4°C for 2 hours. The pelleted exosomes were washed with PBS and centrifuged again under the same conditions. Finally, the extracted exosomes were resuspended in 200 μL of PBS and stored at -80°C until further use. The extracted exosomes were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting.

[0104] 1.3 Analysis steps of EpCAM / MCF-7 cells

[0105] First, 5′-phosphorylated padlock-1 (100 μM, 1 μL) and primer-1 (100 μM, 1 μL) were mixed with NaCl solution (800 mM, 2 μL) and 16 μL of ultrapure water. 5′-phosphorylated padlock-2 (100 μM, 1 μL) and primer-2 (100 μM, 1 μL) were treated in the same manner. The mixtures were heated at 95°C for 5 minutes and then slowly cooled to room temperature.

[0106] T4 DNA ligase (5 U / μL, 1 μL), T4 DNA ligase reaction buffer (10×, 5 μL), and 24 μL of ultrapure water were then added to the mixture. The mixture was incubated at 37°C for 75 minutes to complete the ligation reaction. The reaction mixture was then heated at 65°C for 10 minutes. A deoxyribonucleoside triphosphate mix (dNTPs, 25 mM, 4 μL), phi29 DNA polymerase (10 U / μL, 1.25 μL), phi29 reaction buffer (10×, 10 μL), bovine serum albumin (25 mg / mL, 1 μL), NaCl solution (800 mM, 5 μL), and 29.5 μL of ultrapure water were added to the ligation reaction product and incubated at 37°C for 2.5 hours. The reaction was then heated at 65°C for 10 minutes to inactivate the phi29 DNA polymerase.

[0107] Finally, the reaction solution 1 (30 μL) and reaction solution 2 (30 μL) obtained above were mixed to form a DNA hydrogel.

[0108] To this mixture, 60 μL of MOPS buffer (10 mM MOPS, 100 mM NaNO₃, pH 7.3) was added, followed by 60 μL of EpCAM protein solutions at varying concentrations. The mixture was incubated at room temperature for 30 minutes. Pb(NO₃)₂ (10 μM, 20 μL) was then added, and the reaction continued for 15 minutes. Finally, 200 μL of 10 mM MOPS buffer was added to the solution.

[0109] All tests were performed on a CHI660E electrochemical workstation (Shanghai, China) equipped with a three-electrode system consisting of a pretreated indium tin oxide (ITO) electrode as the working electrode, a saturated Ag / AgCl reference electrode, and a platinum wire electrode as the auxiliary electrode. Differential pulse voltammetry (DPV) was used for measurements in the range of −0.7 to 0.1 V. The pulse amplitude was set to 0.05 V, the pulse width to 0.05 s, the potential increment to 0.004 V, and the cycle to 0.5 s.

[0110] 1.4 Pretreatment of clinical samples

[0111] Peripheral blood samples were collected from patients at West China Hospital, Sichuan University (Chengdu, China). All procedures were performed under ethical approval (approval number: 20221489). Four milliliters of venous whole blood was collected from each subject in sterile, EDTA-coated vacuum anticoagulant tubes. To ensure efficient plasma separation, the collected blood samples were processed within 2 hours. The upper plasma layer was obtained by centrifugation at 2500 g for 15 minutes at 25°C. The separated plasma layer was then carefully transferred and centrifuged again under the same conditions to remove residual platelets. The resulting plasma samples were stored at −80°C until further analysis.

[0112] 2 Synthesis of DNA Nanohydrogels Containing G-quadruplexes

[0113] The present invention successfully synthesized DNA nanohydrogel based on RCA. Figure 2 shown.

[0114] To verify the effective formation of RCA circular template and its amplification product, 2.5% agarose gel electrophoresis analysis was performed (e.g. Figure 2 b). The results showed that the circular RCA template (lanes 7 and 8) migrated significantly slower than the linear single-stranded DNA (lanes 1 to 6), while RCA products 1 and 2 (lanes 9 and 10) remained within the gel pores, indicating that their molecular weights were much larger. At the same time, atomic force microscopy (AFM) was used to image and analyze the changes in DNA structure during the RCA process ( Figure 2 c). Before connection, the AFM image shows uniformly distributed nanodots ( Figure 2 c1), corresponding to a single primer and padlock probe, with a size consistent with a short DNA fragment. After ligation, a ring-shaped nanostructure with a diameter of approximately 40 nm was observed ( Figure 2 c2), confirming the successful formation of the RCA template. Subsequently, the RCA reaction formed a large number of entangled DNA nanofilaments, indicating the generation of ultra-long RCA products ( Figure 2 c3).

[0115] Mixing RCA product 1 and product 2 can form a translucent gel ( Figure 2 d), its physical properties are significantly different from the initial solution state, further confirming the successful construction of the hydrogel. In the rheological performance test, the storage modulus (G', about 10 Pa) of the DNA nanohydrogel is significantly higher than the loss modulus (G'', about 1 Pa), indicating that it has ultra-soft solid properties. Through time sweep testing, the G' value continues to be higher than G'' throughout the test process, further verifying the mechanical stability of the hydrogel ( Figure 2e). Fluorescence microscopy and scanning electron microscopy (SEM) were used to analyze the structure of the hydrogel, revealing that it has a clear porous three-dimensional structure. The fluorescence image after SYBR Green I staining shows uniformly distributed green fluorescence ( Figure 2 f). High-resolution SEM images further reveal a three-dimensional cross-linked network structure with fiber diameters ranging from tens to hundreds of nanometers ( Figure 2 g). This multi-scale pore structure gives the DNA nanohydrogel great application potential in exosome detection and related biomedical fields.

[0116] 3 Electrochemical analysis of Pb 2+ and Pb 2+ Specific recognition of G-quadruplex complexes

[0117] The present invention provides a method for realizing Pb 2+ Specific identification method, in which Pb 2+ As an electrochemical signal indicator molecule, it specifically binds to the G-quadruplex structure to form a stable complex.

[0118] G-quadruplex is a unique nucleic acid secondary structure that is closely related to Pb 2+ It has specific binding ability and is a key medium in the signal conversion process. As the concentration of G-quadruplex increases, it reacts with Pb 2+ The electrochemical signal generated by the binding showed a gradually weakening trend, indicating that there was a negative correlation between the G-quadruplex concentration and the electrochemical signal intensity ( Figure 3 a).

[0119] A series of G-quadruplexes with different sequences were further subjected to Pb 2+ The binding capacity test was conducted and the signal-to-noise ratio (SNR) of the corresponding electrochemical signal was analyzed. Among them, the PS2.M sequence showed the most significant linear response relationship. The correlation between its G-quadruplex concentration and the degree of electrochemical signal attenuation was high, and the signal-to-noise ratio reached 1.7, making it suitable for constructing a high-sensitivity electrochemical detection system ( Figure 3 b and Figure 3 c).

[0120] Based on this finding, we further studied the effect of circular template concentration on G-quadruplex amplification. The results showed that as the RCA product accumulated, the electrochemical signal continued to decrease, proving that the amplified product could be combined with Pb 2+ Binding, promoting the formation of G-quadruplex structure, thereby enhancing the detection sensitivity ( Figure 3 d).

[0121] To further verify the formation of the G-quadruplex structure, circular dichroism (CD) spectroscopy was used to confirm the structure. Figure 3 As shown in e, the artificially synthesized G-quadruplex shows a positive band at 310 nm and a negative band at 265 nm. This spectral feature is consistent with the known G-quadruplex, confirming that the Pb 2+ Under the presence of , RCA successfully generated a G-quadruplex structure with an antiparallel conformation.

[0122] 4. Feasibility and analytical performance of EpCAM

[0123] To show the best results, optimize the experimental conditions, e.g. Figure 4 As shown, with particular attention to EpCAM and Pb 2+ incubation time and concentration.

[0124] Under the optimized conditions, the feasibility of EpCAM protein response in the system was verified in multiple dimensions, such as Figure 5 shown.

[0125] First, the free Cu in the system 2+ and Pb 2+ The content was quantitatively analyzed. After adding DNA nanohydrogel with different concentrations of EpCAM, the originally encapsulated Cu 2+ Release, resulting in an increase in signal intensity; contains G-quadruplex-Pb 2+ The DNA chains in the complex structure were also released into the supernatant, enhancing the electrochemical signal intensity. Dynamic light scattering (DLS) further verified the structural changes in the system: when EpCAM protein was added at concentrations of 100 ag / mL, 10 fg / mL, and 1 pg / mL, the particle sizes of RCA products 1, 2, and the DNA hydrogel were 106 nm, 122 nm, 955 nm, 825 nm, 712 nm, and 531 nm, respectively. Zeta potential measurements showed that during DNA hydrogel formation, the potential of the RCA product changed from -4.0 mV to -0.1 mV, indicating that the exposure of the negative charges carried by the phosphate backbone decreased due to structural densification and charge shielding. With increasing protein concentration, the hydrogel particle size decreased significantly and the absolute value of the zeta potential increased, reflecting the collapse of the network structure and the redispersion of the DNA chains, confirming the responsive properties of the hydrogel for biosensing.

[0126] Subsequently, the detection performance of the system was quantitatively evaluated. In the range of EpCAM concentration from 100 ag / mL to 10 pg / mL, Pb 2+ The electrochemical signal showed a significant downward trend, and the peak current showed a good linear relationship with the logarithm of EpCAM concentration. The linear regression equation was Y = -100LogC + 1242, and the correlation coefficient R 2The detection limit (LOD) calculated based on a 3-fold signal-to-noise ratio was 30 ag / mL.

[0127] To evaluate the specificity and anti-interference capabilities of this system in complex biological samples, a panel of common exogenous proteins (including human serum albumin and tumor markers such as glypican 3) were introduced as interfering substances for comparative analysis. At a concentration of 1 pg / mL, the electrochemical signals generated by these interfering substances were nearly identical to those of the blank control. However, even at low concentrations (100 ag / mL and 1 fg / mL) of EpCAM, a significant decrease in the electrochemical signal was observed. These results demonstrate the excellent sensitivity and high selectivity of this detection method, making it suitable for subsequent analysis of clinical samples.

[0128] 5. Analytical performance of exosomes

[0129] Before evaluating the analytical performance of the exosome detection method of the present invention, exosomes from MCF-7 cell supernatant and clinical plasma samples were first characterized, focusing on analyzing key parameters such as size, concentration, morphology, and surface protein expression (e.g. Figure 6 a).

[0130] The concentration and size distribution of exosomes were quantitatively analyzed using NTA. The results showed that the concentration of exosomes isolated from the supernatant of MCF-7 cells was approximately 1.6 × 10 10 particles / mL, with an average diameter of 160 nm (e.g. Figure 6 b).

[0131] In comparison, the concentration of exosomes isolated from clinical plasma samples was slightly lower, at 5.9 × 10 9 particles / mL, with a slightly larger average diameter of 172 nm (e.g. Figure 6 c). Real-time NTA images are shown Figure 6 b and Figure 6 As shown in c.

[0132] In order to further verify the morphology of exosomes, TEM observation was used. The results showed that exosomes have a typical cup-shaped structure with a double-layer membrane structure (such as Figure 6 d and Figure 6 The observed morphology is consistent with the size distribution data of NTA, further confirming the accuracy of the characterization.

[0133] In addition, Western blot was used to detect the expression of surface proteins of these exosomes, such as CD9, CD63, CD81 and EpCAM (e.g. Figure 6f). The presence of these proteins further indicates that the exosomes have been successfully isolated and are suitable for subsequent analysis, demonstrating their potential as reliable detection targets in clinical applications. A strong linear correlation was observed between the electrochemical signal and the logarithm of the exosome concentration. 3 to 10 7 When the concentration of exosomes was 0.1777 × 10.84 × 10.77 × 10.87 ... 2 is 0.993 (e.g. Figure 6 Compared with existing electrochemical methods, the method of the present invention has obvious advantages in quantifying exosomes by indirectly measuring the expression of surface protein EpCAM.

[0134] 6 Clinical Applicability of Exosome Analysis Methods

[0135] To further evaluate the practicality and accuracy of this electrochemical method, 39 clinical blood samples from healthy individuals (n = 12) and breast cancer patients (n = 27) were collected for validation. Electrochemical analysis of the clinical samples revealed significant differences in electrochemical peaks between healthy controls and breast cancer patients (see Figure 7 b and 7c).

[0136] Specifically, the electrochemical peak values measured in breast cancer patient samples were significantly reduced, which is closely related to the increased expression level of EpCAM in their exosomes. In addition, in order to more intuitively distinguish breast cancer patients from healthy individuals, the present invention generates a heat map, where lighter colors represent increased EpCAM concentrations ( Figure 7 d). To further verify the diagnostic performance of the system, the receiver operating characteristic curve (ROC curve) analysis method was used for evaluation. The diagnostic ability of the detection system of the present invention in clinical samples showed excellent specificity and sensitivity, specifically a specificity of 91.7% (11 / 12) and a sensitivity of 92.6% (25 / 27) ( Figure 7 e).

[0137] In addition, the area under the curve (AUC) was 0.944, further confirming the high accuracy of this method in breast cancer detection. Furthermore, the detection results of this exosome analysis method showed high consistency with the results of magnetic resonance imaging (MRI) and histopathology (see Figure 7 f and 7g), further verifying the reliability of this method. The high consistency with existing mainstream clinical diagnostic methods suggests that this method has potential clinical application value as a supplement or alternative to breast cancer screening and diagnosis.

[0138] To further evaluate the clinical application potential of the electrochemical detection system of the present invention in breast cancer staging ( Figure 8 a) The present invention systematically analyzed blood samples from 27 breast cancer patients of different stages.

[0139] The electrochemical detection results showed that as the tumor stage progressed, the electrochemical signal showed a gradual downward trend ( Figure 8 b). A quantitative comparison of electrochemical signals of early-stage (I-II) and late-stage (III-IV) breast cancer patients showed that the signal intensity of late-stage patients was significantly lower than that of early-stage patients ( Figure 8 cd).

[0140] To verify the diagnostic performance of the platform in breast cancer staging identification, ROC curve analysis was further performed. The results showed that the system had good diagnostic ability in distinguishing early from late breast cancer, with an area under the curve (AUC) of 0.864, a sensitivity of 86%, and a specificity of 70% ( Figure 8 e).

[0141] In the specific experiment, the test results of patients No. 1 and No. 7 were compared and analyzed. According to their computed tomography (CT) results and histopathological examination, patient No. 1 had no distant organ metastasis, while patient No. 7 had distant metastasis, indicating that her breast cancer was in a more advanced stage ( Figure 8 fg). Correspondingly, there are significant differences in the electrochemical signals between the two.

[0142] In summary, the noninvasive electrochemical detection platform described in the present invention can accurately identify the stage of breast cancer and has important value as a clinical auxiliary tool. It is particularly suitable for clinical scenarios where traditional imaging methods are limited and has potential application prospects in the development of individualized treatment strategies.

[0143] 7 Conclusion

[0144] The present invention successfully developed a one-pot electrochemical analysis method for detecting tumor-derived exosomes.

[0145] This method combines aptamer recognition with target-responsive DNA hydrogels to induce the formation of G-quadruplex-Pb 2+ complex, and utilize Pb 2+The signal is output based on the electrochemical difference of the state. This one-pot detection process allows the detection to be completed quickly and efficiently in only 45 minutes. The method was further applied to the detection of exosomes in clinical samples, and the results were highly consistent with clinical diagnosis and staging, providing an auxiliary tool for personalized treatment strategies and management. Although EpCAM was used as a representative marker in this study, it cannot fully capture the heterogeneity of breast cancer. Future research should focus on integrating more signaling molecules to develop multi-target systems to optimize the sensitivity and specificity of tumor diagnosis and staging. In addition, this strategy can also be adapted to integrate multiple aptamers targeting different biomarkers to achieve multiple detection.

[0146] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A DNA-Pb-based 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The method comprises constructing a DNA nanohydrogel based on an RCA product, wherein the DNA nanohydrogel comprises a nucleic acid aptamer for recognition, a complementary base hybridization region, and a G-quadruplex for signal output; The specific binding of the nucleic acid aptamer to the target causes the DNA nanohydrogel to disintegrate and expose the G-quadruplex, and the G-quadruplex binds to the Pb 2+ Formation of G-quadruplex-Pb 2+ Complex, through Pb 2+ quantifying the target by an electrochemical signal, wherein the target is an exosome; The Pb 2+ With the G-quadruplex-Pb 2+ The complexes have obvious electrochemical signal differences. 2+ The electron transport performance is much greater than that of the G-quadruplex-Pb 2+ complex; When epithelial cell adhesion molecules are present, they bind to the corresponding nucleic acid aptamers to trigger the disintegration of the DNA nanohydrogel, exposing the G-quadruplex and binding to free Pb 2+ , reducing free Pb 2+ The concentration of , resulting in a decrease in the electrochemical signal intensity; When epithelial cell adhesion molecules were absent, the DNA nanohydrogel remained intact, the G-quadruplex was not exposed, and Pb 2+ It can freely diffuse to the surface of the ITO electrode and significantly enhance the electrochemical signal output.

2. according to claim 1 based on DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The G-quadruplex is GT GGG TA GGG C GGG TT GG.

3. according to claim 1 based on DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The primers of DNA nanohydrogel include Primer-1 and Primer-2; The Primer-1 is GTG GGT AGG GCG GGT TGG TGA AGG TTC GTT GTT T; The Primer-2 is ACA GAT TTT GGG AAT GGT GGG TAG GGC GGG TTG G.

4. according to claim 1 based on DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The padlocks of DNA nanohydrogel include Padlock-1 and Padlock-2; The Padlock-1 is Phosphate-CCA ACC CGC CCT ACC CAC GTG TGA TGA GAC TAA ATACTA AAG CAT TCC CAA AAT CTG TAT CTC TTC TAA AGA GTC TAC ACC CAC CGA AAC AACGAA CCT TCA; The Padlock-2 is Phosphate-CAT TCC CAA AAT CTG TAT CTC TTC TAA AGA GTC TACACC CAC CGA AAC AAC GAA CCT TCA CTT TAG TAT TTA GTC TCA TCA CAC CCA ACC CGCCCT ACC CAC.

5. according to claim 1 based on DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The Pb 2+ The linear regression equation of the peak current and the logarithmic concentration of the epithelial cell adhesion molecule is Y = -100LogC + 1242, and the correlation coefficient is R 2 The concentration of epithelial cell adhesion molecule ranged from 100 ag / mL to 10 pg / mL.

6. according to any one of claims 1-5 based on DNA-Pb 2+ A homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is characterized in that: The exosomes are positive for expression of epithelial cell adhesion molecules.

7. A DNA-Pb-based 2+ The application of the homogeneous electrochemical analysis method of functional nano-hydrogel for exosome detection is characterized in that: The application includes the DNA-Pb-based 2+ A homogeneous electrochemical analysis method for exosome detection based on functional nanohydrogels was used for the quantitative analysis of epithelial cell adhesion molecules.

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