Homogeneous electrochemical analysis method for exosome detection based on DNA-Pb < 2 + > functional nano hydrogel and application

Abstract

The invention provides a homogeneous electrochemical analysis method for exosome detection based on DNA-Pb < 2 + > functional nano-hydrogel and application, and relates to the technical field of biomedical diagnostic analysis method.The homogeneous electrochemical analysis method is constructed based on DNA nano-hydrogel prepared through a rolling circle amplification (RCA) method by combining a G-quadruplex structure and a Pb < 2 + > specific recognition mechanism; an epithelial cell adhesion molecule (EpCAM) on the surface of the exosome is selected as a detection target, the DNA nano-hydrogel is triggered to disintegrate by utilizing specific binding between the EpCAM and an aptamer thereof, then a large number of G-quadruplex structures generated by RCA are exposed, the G-quadruplex structures can be selectively bound with Pb < 2 + > and regulate and control electrochemical signal output, and the detection sensitivity is high. According to the method, a one-pot method, a rapid and efficient detection process is realized, and the whole process is completed within 45 minutes.

Description

Technical Field

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

[0002] Accurate breast cancer diagnosis and staging are crucial for guiding clinical treatment strategies and predicting patient survival prognosis. However, existing diagnostic techniques still have certain limitations: imaging methods lack sufficient sensitivity in detecting small tumors and micrometastases, while tissue biopsy, as the current "gold standard", is invasive and not suitable for repeated testing, limiting its clinical application in real-time dynamic monitoring. In recent years, exosomes as tumor markers for liquid biopsy have become a promising cancer screening and early diagnosis technique. Studies have shown that there is a significant correlation between exosome levels and tumor staging. Usually, at the advanced stage of cancer, the exosome level is relatively high, and exosomes are considered promising biomarkers for monitoring tumor progression.

[0003] However, existing exosome quantitative detection methods, such as nanoparticle tracking analysis (NTA) and immunology-based flow cytometry, have certain limitations. They often cannot effectively distinguish different exosome subtypes, or require complex labeling and separation steps, resulting in cumbersome operations and low detection efficiency. They have high requirements for experimental equipment and technicians and lack sufficient sensitivity, limiting their popularization and application in clinics. Therefore, there is an urgent need for a new exosome detection method that can achieve precise identification, high sensitivity, and simple operation, so as to overcome the deficiencies of existing methods and improve the accuracy and practicality of exosome detection.

[0004] In recent years, a variety of novel sensing technologies have been developed, including surface-enhanced Raman spectroscopy, fluorescence detection, electrochemical detection, electrochemiluminescence, and colorimetry for exosome analysis. Although the above methods exhibit excellent detection sensitivity, they generally rely on magnetic bead enrichment or complex material modification and synthesis processes, resulting in cumbersome detection procedures. Compared with antibodies, aptamers have been widely used in the field of biosensing due to their advantages such as simple synthesis and strong target affinity. Aptamers can be combined with various nucleic acid amplification strategies (such as rolling circle amplification (RCA), polymerase chain reaction, and hybridization chain reaction) to achieve highly sensitive target detection. However, although cascade amplification methods can significantly enhance the signal response, their operation processes are more complex than single signal amplification strategies, limiting the convenience of practical applications. Currently, various DNA-based functional nanostructures (including DNA nanospheres, DNA tetrahedrons, and DNA nanohydrogels) have received extensive attention due to their dual advantages in enhancing detection sensitivity and simplifying operations. Among them, DNA nanohydrogels are mainly composed of complementary assembly of RCA products, with good programmability, high loading capacity, and the ability to respond to specific analytes. Therefore, functional integrated DNA nanohydrogels can serve as a promising high-sensitivity sensing platform 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 studies have been 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, resulting in limited detection sensitivity and difficult-to-guarantee stability. To overcome the above defects, a strategy of introducing repetitive functional DNA structural units (such as G-quadruplexes or hairpin structures) into DNA nanohydrogels has been proposed in recent years to enhance signal amplification and output specificity. Among them, the G-quadruplex structure has been widely used because it can specifically bind to a variety of signal molecules (such as methylene blue (MB), doxorubicin (DOX), lead ions (Pb 2+ ), etc.). For example, some studies have shown that G-quadruplexes can selectively bind to MB, and accurate quantification of target molecules can be achieved by detecting the electrochemical signal difference between free MB and the G-quadruplex-MB complex. However, compared with MB and DOX, Pb 2+ has a stronger affinity for G-quadruplexes, which provides a new technical path for constructing a DNA nanohydrogel platform based on the G-quadruplex-Pb 2+ recognition mechanism. Electrochemical sensors have received attention due to their characteristics such as miniaturization, simple operation, and high sensitivity, but their reliance on electrode modification often leads to a decrease in repeatability.

[0006] Therefore, if the detection of Pb 2+ and G-quadruplex-Pb 2+For the effective discrimination of the complex, it is feasible to establish a homogeneous (one-pot) electrochemical strategy. Summary of the Invention

[0007] In view of this, one of the purposes of the present invention is to provide a homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogels, so as to solve the technical problems such as low loading amount of signal molecules in traditional physical encapsulation, resulting in limited detection sensitivity and difficult to guarantee stability.

[0008] Another purpose of the present invention is to provide an application of a homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogels.

[0009] To achieve the above purpose, the present invention provides a homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogels. The method includes constructing DNA nanohydrogels based on RCA products. The DNA nanohydrogels include nucleic acid aptamers for recognition, complementary base hybridization regions, and G-quadruplexes for signal output; Based on whether the nucleic acid aptamer specifically binds to the target to cause the disintegration of the DNA nanohydrogel to expose the G-quadruplex, and based on whether the G-quadruplex binds to Pb 2+ to form a G-quadruplex-Pb 2+ complex, the target is quantified by the electrochemical signal of Pb 2+ and the target is exosomes.

[0010] Optionally, the exosomes are positive for epithelial cell adhesion molecule expression.

[0011] Optionally, there is a significant difference in the electrochemical signals between Pb 2+ and the G-quadruplex-Pb 2+ complex, and the electron transport performance of Pb 2+ is much greater than that of the G-quadruplex-Pb 2+ complex.

[0012] Optionally, when epithelial cell adhesion molecule is present, it binds to the corresponding nucleic acid aptamer to trigger the disintegration of the DNA nanohydrogel, exposing the G-quadruplex and binding free Pb 2+ , reducing the concentration of free Pb 2+ , resulting in a decrease in the intensity of the electrochemical signal; When epithelial cell adhesion molecule is absent, the DNA nanohydrogel remains intact, the G-quadruplex is not exposed, and Pb 2+It can freely diffuse to the surface of the ITO electrode, significantly enhancing the electrochemical signal output.

[0013] Optionally, the G-quadruplex is GT GGG TA GGG C GGG TT GG.

[0014] Optionally, the primers of the 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.

[0015] Optionally, the padlocks of the DNA nanohydrogel include Padlock-1 and Padlock-2; 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; 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.

[0016] Optionally, the Pb 2+ The linear regression equation of the current peak value of and the logarithm of the concentration of the epithelial cell adhesion molecule is Y = -100LogC + 1242, and the correlation coefficient R 2 is 0.988, and the concentration range of the epithelial cell adhesion molecule is 100 ag / mL to 10 pg / mL.

[0017] Optionally, the Pb 2+ can be replaced with methylene blue or doxorubicin.

[0018] To achieve the second above-mentioned purpose, a DNA-Pb-based 2+Application of a homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels, said application comprising using any one of the above DNA-Pb-based 2+ The homogeneous electrochemical analysis method for exosome detection using functional nanohydrogels is used for quantitative analysis of epithelial cell adhesion molecule.

[0019] The DNA-Pb-based 2+ Homogeneous electrochemical analysis method and application for exosome detection using functional nanohydrogels have the following technical effects: The DNA nanohydrogel prepared by the present invention based on the rolling circle amplification (RCA) method is constructed by combining the G-quadruplex structure with the Pb 2+ specific recognition mechanism. The epithelial cell adhesion molecule (EpCAM) on the surface of exosomes is selected as the detection target. The specific binding between EpCAM and its aptamer triggers the disintegration of the DNA nanohydrogel, thereby exposing a large number of G-quadruplex structures generated by RCA. The latter can selectively bind to Pb 2+ and regulate the electrochemical signal output. This method realizes a one-pot, rapid and efficient detection process, and the whole process is completed within 45 minutes. Brief Description of the Drawings

[0020] 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 use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 Schematic diagram of a homogeneous electrochemical detection strategy for breast cancer-derived exosomes by a DNA nanohydrogel regulated by a target protein; Figure 2 Preparation and characterization of DNA nanohydrogels containing G-quadruplex; Figure 2 a is a schematic diagram of DNA nanohydrogel synthesis based on RCA; Figure 2 b is the agarose gel electrophoresis pattern of RCA; Figure 2 c1-c3 are representative AFM images of primers and padlock probes before and after ligation and RCA products; Figure 2 d is the photographed image of RCA product 1, RCA product 2 and DNA nanohydrogel under daylight and ultraviolet light; Figure 2 e is the rheological property diagram of DNA nanohydrogel; Figure 2 f is the fluorescence microscopy image of the DNA nanohydrogel stained with SYBR green I; Figure 2 g is the scanning electron microscopy image of the DNA nanohydrogel; Figure 3 For electrochemical analysis to achieve specific recognition of Pb 2+ and the G-quadruplex-Pb 2+ complex; Figure 3 a is the schematic diagram of Pb 2+ serving as a G-quadruplex binding probe for electrochemical analysis; Figure 3 b is the diagram of the electrochemical signal situation after different G-quadruplexes bind to Pb 2+ ; Figure 3 c is the relationship diagram between the electrochemical peak current and the logarithmic concentration of PS2.M; Figure 3 d is the diagram of the electrochemical signal situation between the RCA product and Pb 2+ in the presence of different concentrations of padlock probes; Figure 3 e is the CD spectrum of the G-quadruplex; Figure 4 For the optimization of the EpCAM analysis conditions; Figure 4 a is the volume diagram of T4 ligase; Figure 4 b is the ligation time diagram; Figure 4 c is the volume diagram of dNTPs; Figure 4 d is the volume diagram of Phi29 DNA polymerase; Figure 4 e is the amplification time diagram; Figure 5 For the analysis performance of EpCAM; Figure 5 a is the concentration diagram of free Cu 2+ and Pb 2+ after adding EpCAM to the DNA nanohydrogel detected by ICP-MS; Figure 5 b-c are the DLS and Zeta potential diagrams of the RCA products 1 and 2 and the DNA hydrogel; Figure 5 d is the DPV measurement value diagram; Figure 5 e is the corresponding peak current diagram at different EpCAM concentrations; Figure 5f is a linear correlation diagram of the peak current versus the logarithm of the EpCAM concentration; Figure 5 g is the protein selectivity of EpCAM, and the error bars represent the data diagram obtained from three repeated measurements; Figure 6 is the analytical performance of exosomes; Figure 6 a is a schematic workflow diagram for the evaluation of exosome isolation, characterization, and analytical performance; Figure 6 b - c are NTA measurement diagrams of exosomes isolated from MCF - 7 cell culture supernatants or plasma samples; Figure 6 d - e are TEM images of exosomes derived from MCF - 7 cells and plasma; Figure 6 f is a Western blot analysis diagram of exosome markers from MCF - 7; Figure 6 g - h are DPV responses and the corresponding peak current diagrams of Pb 2+ at different exosome concentrations; Figure 6 i is a linear correlation diagram of the peak current versus the logarithm of the exosome concentration; Figure 7 is a diagram for the diagnosis and result analysis of clinical breast cancer patients; Figure 7 a is a flowchart for the collection and analysis of clinical samples; Figure 7 b is a bar chart of the diagnosis results; Figure 7 c is a box plot of the diagnosis results; Figure 7 d is a heat map of the diagnosis results; Figure 7 e is an ROC analysis diagram; Figure 7 f is a diagram of the clinical MRI results of the patients; Figure 7 g is a diagram of the pathological results; Figure 8 is for the staging and result analysis of clinical breast cancer patients; Figure 8 a is a flowchart for the analysis of the staging of clinical samples; Figure 8 b is a bar chart of the staging results; Figure 8 c is a box plot of the staging results; Figure 8 d is a heat map of the staging results; Figure 8 e is an ROC analysis diagram; Figure 8 f is the corresponding CT for early and late breast cancer patients and Figure 8 g is the pathological result diagram. Specific implementation manner

[0022] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other implementation manners obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope protected by the present invention.

[0023] The principle of the analysis method of the present invention is as follows: As Figure 1 shown, it is a homogeneous electrochemical analysis method based on DNA nanohydrogel for detecting exosomes derived from breast cancer. This analysis method introduces G-quadruplex structure into the DNA hydrogel system, realizing specific recognition of Pb 2+ and having potential application value for early diagnosis and staging of breast cancer.

[0024] Specifically, a DNA nanohydrogel is constructed based on RCA products. The hydrogel structure integrates nucleic acid aptamers for target recognition, 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 selectively bind to the free Pb 2+ in the system, forming a G-quadruplex-Pb 2+ complex and achieving signal amplification.

[0025] Since free Pb 2+ has excellent electron transport performance and can be directly detected by an electrochemical instrument; while the G-quadruplex-Pb 2+ complex is inert on the ITO electrode and there is an obvious distinguishable electrochemical signal difference between the two, therefore, homogeneous electrochemical quantitative detection without modification can be achieved.

[0026] During the detection process, when EpCAM is present, its binding to the aptamer triggers the disintegration of the nanohydrogel, exposing G-quadruplex and binding free Pb 2+ , thus reducing the concentration of free Pb 2+ and resulting in a decrease in the electrochemical signal intensity. On the contrary, when EpCAM is absent, the hydrogel remains intact, G-quadruplex is not exposed, and Pb 2+It can freely diffuse to the surface of the ITO electrode, significantly enhancing the electrochemical signal output.

[0027] The technical solution of the present invention will be specifically verified below in conjunction with embodiments.

[0028] 1 Materials and Methods 1.1 Materials and Reagents Oligonucleotides of all different sequences were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences are shown in Table 1.

[0029] T4 DNA ligase, phi29 DNA polymerase, deoxynucleoside triphosphates (dNTPs), bovine serum albumin (BSA), and agarose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

[0030] Recombinant human epidermal cell adhesion molecule (EpCAM) protein was purchased from Novoprotein Scientific Inc. (Shanghai, China).

[0031] Lead nitrate (Pb(NO3)2) and sodium chloride (NaCl) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

[0032] DNA molecular weight standard (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, Massachusetts, USA).

[0033] 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), glypican 3 (GPC3), lysozyme, thrombin, human immunodeficiency virus (HIV) p24 protein, and tetanus toxoid were all purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0034] Dulbecco's modified Eagle's medium (DMEM) and human breast cancer MCF-7 cell line were purchased from Zhanjiang Tongbo Medical Technology Co., Ltd.

[0035] Fetal bovine serum (FBS), penicillin / streptomycin, phosphate buffer solution (PBS), and 0.22 μm syringe filter were purchased from Gibco Invitrogen Corporation (California, USA).

[0036] Anti-CD9, anti-CD63, and anti-CD81 antibodies were purchased from Proteintech (Rosemont, Illinois, USA), and EpCAM antibody was purchased from Thermo Fisher.

[0037] Ultra-clear centrifuge tubes (25 × 89 mm, 38.5 mL, sterile, open thin-walled) were purchased from Beckman Coulter (Indiana, USA).

[0038] All water used in the experiments was purified by the ultrapure water system of Chengdu Ultra-pure Technology Co., Ltd., with a resistivity of 18.25 MΩ·cm. All working solutions were prepared with 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (10 mM MOPS and 100 mM NaNO3, pH 7.4).

[0039] All reagents used in this invention were of analytical grade or higher purity and were used directly without further purification. All solutions were stored at 4 °C in the refrigerator until use. Healthy human plasma samples and whole blood samples from breast cancer patients were provided by West China Hospital of Sichuan University, and the relevant research has been approved by the Biomedical Ethics Committee of West China Hospital of Sichuan University (Chengdu, China, approval number: 20221489).

[0040] Table 1. Sequences of oligonucleotides used in the study

[0041] 1.2 Cell culture and exosome isolation Cell culture: MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) in a cell culture incubator at 37 °C and 5% CO2. When the cell confluence reached 70%-80%, the cells were washed twice with pure DMEM without any additives, and then replaced with serum-free DMEM and cultured for another 48 hours. After the culture, all cell supernatants were collected in 50 mL sterile conical centrifuge tubes and subjected to gradient centrifugation to extract exosomes.

[0042] Exosome extraction: First, centrifuge at 500 ×g and 4 °C for 10 minutes to precipitate cells and their debris; then centrifuge the obtained supernatant at 2,000 ×g and 4 °C for 20 minutes to further remove cell debris and apoptotic bodies; subsequently, centrifuge the supernatant at 10,000 ×g and 4 °C for 30 minutes to remove larger extracellular vesicles. After that, filter the supernatant through a filter membrane with a pore size of 0.22 μm to minimize particulate contamination as much as possible. The filtered medium is ultracentrifuged at 150,000 ×g and 4 °C for 2 hours, and the precipitated exosomes are washed with PBS and centrifuged again under the same conditions. Finally, the extracted exosomes are resuspended in 200 μL of PBS and stored at -80 °C for later use. The extracted exosomes are characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot.

[0043] 1.3 Analysis steps of EpCAM / MCF-7 cells First, mix 5'-phosphorylated padlock-1 (100 μM, 1 μL), primer-1 (100 μM, 1 μL) with NaCl solution (800 mM, 2 μL) and 16 μL of ultrapure water. In the same way, treat 5'-phosphorylated padlock-2 (100 μM, 1 μL) with primer-2 (100 μM, 1 μL). The mixed solutions are heated at 95 °C for 5 minutes and then slowly cooled to room temperature.

[0044] Subsequently, add T4 DNA ligase (5 U / μL, 1 μL), T4 DNA ligase reaction buffer (10×, 5 μL), and 24 μL of ultrapure water to the above mixed solutions, and incubate at 37 °C for 75 minutes to complete the ligation reaction. Then heat the reaction mixture at 65 °C for 10 minutes. Add deoxynucleoside triphosphate mixture (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 to the ligation reaction product, and react at 37 °C for 2.5 hours. Then heat at 65 °C for 10 minutes to inactivate phi29 DNA polymerase.

[0045] Finally, mix the obtained reaction solution 1 (30 μL) with reaction solution 2 (30 μL) to form a DNA hydrogel.

[0046] Add 60 μL of MOPS buffer (10 mM MOPS, 100 mM NaNO3, pH 7.3) to the mixture, then add 60 μL of EpCAM protein solution at different concentrations, and incubate at room temperature for 30 minutes. Then add Pb(NO3)2 (10 μM, 20 μL) and continue the reaction for 15 minutes. Finally, add 200 μL of 10 mM MOPS buffer to the solution.

[0047] All tests were carried out on a CHI660E electrochemical workstation (Shanghai, China), which was equipped with a three-electrode system, including a pre-treated indium tin oxide (ITO) electrode as the working electrode, a saturated Ag / AgCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. Differential pulse voltammetry (DPV) was selected for measurements in the range of -0.7 - 0.1 V. The pulse amplitude was set to 0.05 V, the pulse width was 0.05 s, the potential increment was 0.004 V, and the period was 0.5 s.

[0048] 1.4 Pretreatment of clinical samples Peripheral blood samples of patients were collected from West China Hospital of Sichuan University (Chengdu, China), and all operations were carried out after ethical approval (approval number: 20221489). 4 mL of venous whole blood was collected from each subject, and the collection container was a sterile, EDTA-coated vacuum anticoagulant tube. To achieve effective separation of plasma, the collected blood samples needed to be processed within 2 hours. First, centrifuge at 2500 g for 15 minutes at 25°C to obtain the upper plasma layer. Subsequently, the separated plasma layer was carefully transferred and centrifuged again under the same conditions to remove residual platelets. The finally obtained plasma samples were stored at -80°C until subsequent analysis.

[0049] 2 Synthesis of DNA nanohydrogel containing G-quadruplex In this invention, DNA nanohydrogel was successfully synthesized based on RCA, as Figure 2 shown.

[0050] To verify the effective formation of the RCA circular template and its amplification products, 2.5% agarose gel electrophoresis analysis was carried out (as Figure 2 shown in b). The results showed that the migration speed of the circular RCA template (lanes 7 and 8) was significantly slower than that of the linear single-stranded DNA (lanes 1 to 6), while RCA product 1 and product 2 (lanes 9 and 10) remained in the gel wells, indicating that their molecular weights were much larger. At the same time, atomic force microscopy (AFM) was used to image and analyze the DNA structure changes during the RCA process ( Figure 2 c). Before ligation, the AFM images showed uniformly distributed nanodots ( Figure 2c1), corresponding to a single primer and a padlock probe, with a size consistent with that of the short DNA fragment. After the ligation was completed, a circular nanostructure with a diameter of about 40 nm was observed ( Figure 2 c2), confirming the successful formation of the RCA template. Subsequently, the RCA reaction formed a large number of intertwined DNA nanofibers, indicating the generation of ultra-long RCA products ( Figure 2 c3).

[0051] After mixing RCA product 1 and product 2, a translucent gel can be formed ( Figure 2 d), whose physical properties are significantly different from the initial solution state, further confirming the successful construction of the hydrogel. In the rheological property test, the storage modulus (G’, about 10 Pa) of the DNA nanohydrogel was significantly higher than the loss modulus (G’’, about 1 Pa), indicating its ultra-soft solid state characteristics. Through the time sweep test, the G’ value remained higher than G’’ throughout the test, further verifying the mechanical stability of the hydrogel ( Figure 2 e). Fluorescence microscopy and scanning electron microscopy (SEM) were used to analyze the structure of the hydrogel, revealing a clear porous three-dimensional structure inside. The fluorescence image after SYBR Green I staining showed uniformly distributed green fluorescence ( Figure 2 f). High-resolution SEM images further revealed a three-dimensional cross-linked network structure with fiber diameters ranging from dozens of nanometers to hundreds of nanometers ( Figure 2 g). This multi-scale pore structure endows the DNA nanohydrogel with great application potential in exosome detection and related biomedical fields.

[0052] 3 Electrochemical analysis for specific recognition of Pb 2+ and Pb 2+ -G-quadruplex complex The present invention provides a method for specific recognition of Pb 2+ based on electrochemical analysis technology, where Pb 2+ acts as an electrochemical signal indicator molecule and specifically binds to the G-quadruplex structure to form a stable complex.

[0053] G-quadruplex, as a unique nucleic acid secondary structure, has the specific binding ability with Pb 2+ and is a key medium in the signal conversion process. As the concentration of G-quadruplex increases, the electrochemical signal generated by binding to Pb 2+ shows a gradually decreasing trend, indicating an inverse correlation between the G-quadruplex concentration and the electrochemical signal intensity ( Figure 3 a).

[0054] Furthermore, a series of G-quadruplexes with different sequences were further subjected to Pb 2+Combined with the binding ability test, 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, with a relatively high correlation between the G-quadruplex concentration and the degree of attenuation of the electrochemical signal, and the signal-to-noise ratio reached 1.7, which was suitable for constructing a highly sensitive electrochemical detection system ( Figure 3 b and Figure 3 c).

[0055] Based on this discovery, the effect of the circular template concentration on the amplification effect of G-quadruplex was further studied. The results showed that as the RCA product accumulated, the electrochemical signal continued to decline, demonstrating that the amplification product could bind to Pb 2+ and promote the formation of the G-quadruplex structure, thereby enhancing the detection sensitivity ( Figure 3 d).

[0056] To further verify the formation of the G-quadruplex structure, circular dichroism spectroscopy (CD) was used for structure confirmation. As Figure 3 shown in e, the artificially synthesized G-quadruplex showed a positive band at 310 nm and a negative band at 265 nm, and this spectral feature was consistent with the known G-quadruplex, confirming that under the condition of the presence of Pb 2+ RCA successfully generated a G-quadruplex structure with an antiparallel conformation.

[0057] 4 Feasibility and analytical performance of EpCAM To show the best results, the experimental conditions were optimized, as Figure 4 shown, with particular attention paid to the incubation time and concentration of EpCAM and Pb 2+ .

[0058] Under the optimized conditions, the feasibility of the EpCAM protein response in the system was verified in multiple dimensions, as Figure 5 shown.

[0059] First, the contents of free Cu 2+ and Pb 2+ in the system were quantitatively analyzed. After adding different concentrations of EpCAM to the DNA nanohydrogel, as the hydrogel disintegrated, the originally encapsulated Cu 2+ was released, resulting in an increase in the signal intensity; the G-quadruplex-Pb 2+The DNA strands of the composite structure are also released into the supernatant, enhancing the electrochemical signal intensity. The structural changes of the system were further verified by dynamic light scattering (DLS) technology: when the concentrations of EpCAM protein were 100 ag / mL, 10 fg / mL, and 1 pg / mL, the particle sizes of RCA product 1, product 2, and the DNA hydrogel were 106 nm, 122 nm, 955 nm, 825 nm, 712 nm, and 531 nm, respectively. The results of Zeta potential measurement showed that during the formation of the DNA hydrogel, the potential of the RCA product changed from -4.0 mV to -0.1 mV, indicating that due to the structural densification and charge screening effect, the exposure of the negative charges carried by the phosphate backbone decreased. With the increase in protein concentration, the particle size of the hydrogel decreased significantly and the absolute value of the Zeta potential increased, reflecting the disintegration of the network structure and the re-dispersion of DNA strands, verifying the response characteristics of the hydrogel in biosensing.

[0060] Subsequently, a quantitative evaluation of the detection performance of the system was carried out. In the range of EpCAM concentration from 100 ag / mL to 10 pg / mL, the 2+ electrochemical signal showed a significant downward trend, and the peak current had a good linear relationship with the logarithm of the EpCAM concentration. The linear regression equation was Y = -100LogC + 1242, and the correlation coefficient R 2 was 0.988. The detection limit (LOD) calculated by 3 times the signal-to-noise ratio was 30 ag / mL.

[0061] To evaluate the specificity and anti-interference ability of this system in complex biological samples, a group of common exogenous proteins (including human serum albumin and tumor markers such as glypican 3) were further introduced as interferents for comparative analysis. When the concentration of the interferent was 1 pg / mL, the electrochemical signal generated by it was almost the same as that of the blank control, while EpCAM still caused a significant decrease in the electrical signal even at low concentrations (100 ag / mL and 1 fg / mL). These results together demonstrated that the detection method had excellent sensitivity and high selectivity, making it suitable for subsequent clinical sample analysis.

[0062] 5 Analytical performance of exosomes Before evaluating the analytical performance of the exosome detection method of the present invention, exosomes derived from MCF-7 cell supernatant and clinical plasma samples were first characterized, focusing on the analysis of key parameters such as their size, concentration, morphology, and surface protein expression (as Figure 6 shown in a).

[0063] NTA was used to quantitatively analyze the concentration and size distribution of exosomes. The results showed that the concentration of exosomes isolated from MCF-7 cell supernatant was approximately 1.6 × 1010 particles / mL, with an average diameter of 160 nm (as Figure 6 shown in b).

[0064] In contrast, 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 (as Figure 6 shown in c). Real-time NTA images are as Figure 6 shown in b and Figure 6 c.

[0065] To further verify the morphology of exosomes, TEM was used for observation, and the results showed that exosomes had a typical cup-shaped structure with a bilayer membrane structure (as Figure 6 shown in d and Figure 6 e). The observed morphology was consistent with the size distribution data of NTA, further confirming the accuracy of the characterization.

[0066] In addition, Western blot was used to detect the surface protein expression of these exosomes, such as CD9, CD63, CD81, and EpCAM (as Figure 6 shown in f). The presence of these proteins further indicated that the exosomes had been successfully isolated and were suitable for subsequent analysis, demonstrating their potential as reliable detection targets in clinical applications. There was a strong linear correlation between the electrochemical signal and the logarithm of the exosome concentration. In the exosome concentration range of 10 3 to 10 7 particles / mL, the electrochemical signal was negatively correlated with the exosome concentration. The regression equation for the exosome concentration was Y = -124.2LogC + 1769.4, and the correlation coefficient R 2 was 0.993 (as Figure 6 shown in g-i). Compared with existing electrochemical methods, the method of the present invention has obvious advantages in quantifying exosomes by indirectly measuring the expression of the surface protein EpCAM.

[0067] 6 Clinical applicability of the exosome analysis method 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 verification. After electrochemical analysis of the above clinical samples, it was found that there were significant differences in the electrochemical peaks between the healthy control group and breast cancer patients (see Figure 7 b and 7c).

[0068] Specifically, the electrochemical peak measured in breast cancer patient samples was significantly reduced, which was closely related to the increased expression level of EpCAM in their exosomes. In addition, to more intuitively distinguish breast cancer patients from healthy individuals, the present invention generated a heat map, in which lighter colors represent increased EpCAM concentration ( Figure 7 d). To further verify the diagnostic performance of the present system, the receiver operating characteristic curve (ROC curve) analysis method was used for evaluation. The detection system described in the present invention showed excellent specificity and sensitivity in the diagnosis of clinical samples, specifically 91.7% (11 / 12) for specificity and 92.6% (25 / 27) for sensitivity ( Figure 7 e).

[0069] In addition, the area under the curve (AUC) was 0.944, further confirming the high accuracy of the present method in breast cancer detection. Further, the detection results of this exosome analysis method showed high consistency with the results of magnetic resonance imaging (MRI) and histopathological detection (see Figure 7 f and 7g), further verifying the reliability of the present method. The high consistency with existing mainstream clinical diagnostic methods indicates that this method has potential clinical application value as a supplementary or alternative solution for breast cancer screening and diagnosis.

[0070] To further evaluate the clinical application potential of the electrochemical detection system described in the present invention in breast cancer staging ( Figure 8 a), the present invention conducted a systematic analysis of blood samples from 27 breast cancer patients at different stages.

[0071] The electrochemical detection results showed that as the tumor stage progressed, the electrochemical signal showed a gradually decreasing trend ( Figure 8 b). Quantitative comparison of the 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 c-d).

[0072] To verify the diagnostic performance of the platform in breast cancer staging recognition, further ROC curve analysis was performed, and the results showed that the system had good diagnostic ability in distinguishing early and 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).

[0073] In a specific experiment, the detection results of patient No. 1 and patient No. 7 were compared and analyzed. According to their computed tomography (CT) results and histopathological examinations, no distant organ metastasis was found in patient No. 1, while distant metastasis was present in patient No. 7, indicating that the breast cancer of patient No. 7 was in a more advanced stage ( Figure 8f-g). Correspondingly, there are significant differences in their electrochemical signals.

[0074] In summary, the non-invasive electrochemical detection platform described in the present invention can achieve accurate identification of breast cancer staging, has important value as a clinical auxiliary tool, is particularly suitable for clinical scenarios where traditional imaging methods are limited, and has potential application prospects in formulating individualized treatment strategies.

[0075] 7 Conclusion The present invention successfully developed a one-pot electrochemical analysis method for detecting tumor-derived exosomes.

[0076] This method combines aptamer recognition with a target-responsive DNA hydrogel to induce the formation of a G-quadruplex-Pb 2+ complex, and utilizes the electrochemical differences in the Pb 2+ state for signal output. This one-pot detection process enables the detection to be completed quickly and efficiently in only 45 minutes. This 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 individualized treatment strategies and management. Although EpCAM was used as a representative biomarker in this study, it cannot fully capture the heterogeneity of breast cancer. Future research should focus on integrating more signal molecules to develop a multi-target system, thereby optimizing the sensitivity and specificity of tumor diagnosis and staging. In addition, this strategy can also be adapted to integrate aptamers targeting different biomarkers to achieve multiplex detection.

[0077] 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 can easily think of changes or substitutions within the technical scope disclosed by the present invention, and all should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogel, characterized in that The method includes constructing a DNA nano-hydrogel based on RCA products. The DNA nano-hydrogel includes nucleic acid aptamers for recognition, complementary base hybridization regions, and G-quadruplexes for signal output; Based on whether the nucleic acid aptamer specifically binds to the target, resulting in the disintegration of the DNA nanohydrogel to expose G-quadruplex, and based on whether the G-quadruplex binds to Pb 2+ to form a G-quadruplex-Pb 2+ complex, the target is quantified by the electrochemical signal of Pb 2+ and the target is exosome.

2. Homogeneous electrochemical analysis method for exosome detection based on the DNA-Pb 2+ functional nanohydrogel, characterized in that The said Pb 2+ has an obvious electrochemical signal difference from the said G-quadruplex-Pb 2+ complex, and the electron transport performance of the said Pb 2+ is much greater than that of the said G-quadruplex-Pb 2+ complex.

3. According to claim 2, the homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogel, characterized in that When the epithelial cell adhesion molecule is present, its binding to the corresponding nucleic acid aptamer triggers the disintegration of the DNA nanohydrogel, exposing the G-quadruplex and binding free Pb 2+ , reducing the concentration of free Pb 2+ , resulting in a decrease in the electrochemical signal intensity; In the absence of epithelial cell adhesion molecule, the DNA nanohydrogel remains intact, the G-quadruplex is not exposed, and Pb 2+ can freely diffuse to the surface of the ITO electrode, significantly enhancing the electrochemical signal output.

4. The homogeneous electrochemical analysis method for exosome detection based on the DNA-Pb 2+ functional nanohydrogel, characterized in that The G-quadruplex is GT GGG TA GGG C GGG TT GG.

5. The homogeneous electrochemical analysis method for exosome detection based on the DNA-Pb 2+ functional nanohydrogel, characterized in that The primers of the DNA nano-hydrogel 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.

6. According to claim 3, the homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogel, characterized in that The padlocks of the DNA nano-hydrogel 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.

7. The homogeneous electrochemical analysis method for exosome detection based on the DNA-Pb 2+ functional nanohydrogel, characterized in that The current peak value of the Pb 2+ and the logarithmic value of the concentration of the epithelial cell adhesion molecule has a linear regression equation of Y = -100LogC + 1242, and the correlation coefficient R 2 is 0.988, and the concentration range of the epithelial cell adhesion molecule is from 100 ag / mL to 10 pg / mL.

8. The homogeneous electrochemical analysis method for exosome detection based on the DNA-Pb 2+ functional nanohydrogel, characterized in that The Pb 2+ can be replaced with methylene blue or doxorubicin.

9. According to any one of claims 1-8, the homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogel, characterized in that The exosomes are positive for epithelial cell adhesion molecule expression.

10. An application of a homogeneous electrochemical analysis method for exosome detection based on DNA-Pb 2+ functional nanohydrogels, characterized in that The application includes using the homogeneous electrochemical analysis method for exosome detection with the DNA-Pb-based functional nano-hydrogel according to any one of claims 1-8 for quantitative analysis of epithelial cell adhesion molecule. 2+ ​

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