Homogeneous electrochemical analysis method and application for exosome detection based on programmable G4@DNA nanohighway network

Abstract

The present invention provides a homogeneous electrochemical analysis method and application for exosome detection based on a programmable G4@DNA nanohighway network, which relates to the technical field of biomedical diagnostic analysis methods. The present invention constructs a universal homogeneous electrochemical detection platform. By integrating ligand-triggered hybridization chain reaction and streptavidin-biotin cross-linking technology, the platform constructs a DNA nanohighway network embedded with a large number of split G4 structures. The network can be rapidly synthesized in one step at room temperature. It relies on the specific binding of aptamers to vimentin in G4@DNA-NHWN, which leads to the disintegration of the network and the destruction of the embedded split G4 structure, thereby preventing the split G4 from interacting with free Pb. 2+ Formation of G4-Pb 2+ The complex ultimately achieves selective identification and precise quantification of vimentin-positive exosomes. This "one-pot" reaction system can rapidly quantify tumor-derived exosomes within 60 minutes.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical diagnostic analysis methods, and in particular to a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network and its application. Background Art

[0002] Breast cancer is the leading cause of cancer-related death among women worldwide. Early assessment of axillary lymph node metastasis (ALNM) status is crucial for optimizing prognosis and developing personalized treatment strategies, directly impacting patient survival. While tissue biopsy remains the gold standard for ALNM diagnosis, its invasive nature limits the possibility of repeated testing. Imaging techniques such as magnetic resonance imaging (MRI) are unable to accurately detect micrometastases. Liquid biopsy provides a non-invasive diagnostic solution for the early detection and dynamic assessment of ALNM by analyzing circulating tumor cells (CTCs), tumor-derived exosomes (TDEs), or circulating tumor DNA (ctDNA). TDEs are the most ideal liquid biopsy marker, entering the bloodstream earlier than CTCs and offering advantages over ctDNA such as greater stability and carrying more biological information. Existing TDE detection technologies fall into two main categories: methods based on protein-specific recognition (e.g., ELISA, immunohistochemistry, and flow cytometry) are highly specific but complex and time-consuming. Physical property-based analytical techniques (e.g., nanoparticle tracking analysis and transmission electron microscopy) can measure exosome size distribution and ultrastructure, but they are significantly limited in sensitivity, accuracy, and equipment cost. Therefore, there is an urgent need to develop highly sensitive, rapid, and user-friendly detection methods to enhance the clinical value of TDE in early cancer diagnosis and metastasis assessment.

[0003] Numerous studies have exploited the high affinity of antibodies to improve the sensitivity and accuracy of TDE detection, but these methods are costly and unstable. Aptamers, short, single-stranded oligonucleotides, offer advantages such as excellent protein-binding specificity, chemical stability, and low cost, making them an attractive alternative to antibodies. Researchers often combine aptamers with techniques such as CRISPR or rolling circle amplification for signal amplification, demonstrating their suitability as recognition elements for high-throughput analysis of exosomal proteins. However, these techniques rely on enzymatic reactions, which are susceptible to enzyme purity and batch variability, limiting their detection capabilities. In contrast, non-enzymatic amplification techniques such as hybridization chain reaction (HCR) and catalytic hairpin assembly effectively circumvent this dependency, achieving highly sensitive detection through cascaded signal amplification. However, these techniques typically require sophisticated instrumentation such as mass spectrometers and Raman spectrometers for signal readout. Therefore, combining non-enzymatic amplification techniques with ease of use and integrated detection elements equipped with simple detectors could help achieve highly sensitive target identification and immediate detection.

[0004] In recent years, DNA functionalized nanomaterials (nanoflowers, hydrogels, nanomeshes, etc.) have become the preferred choice for integrated detection elements due to their excellent programmability and multivalent binding properties. Among them, DNA nanomeshes can be rapidly self-assembled at room temperature, and their three-dimensional network structure can provide rich binding sites, which has unique advantages in target capture efficiency. However, traditional DNA nanomeshes rely on chemically labeled fluorescence detection, which is cumbersome to operate and has limited sensitivity improvement. To solve these problems, the programmable properties of DNA nanomeshes are used to precisely integrate functional nucleic acid units such as G-quadruplexes (G4) and hairpin structures into the nanoframe. These endogenous structures can spontaneously bind to signal molecules without the need for additional modification. Among them, G4 can specifically bind to methylene blue (MB), doxorubicin and Pb 2+ For example, the specific binding of G4 to MB was used to compare the signal difference between G4-MB complex and free MB to achieve accurate quantification of the target. 2+ The binding affinity of Pb is significantly higher than that of MB or DOX, making it an ideal medium for direct target quantification and can be used with appropriate detectors to achieve rapid and sensitive detection. 2+ Detection methods include electrochemistry (EC), fluorescence, colorimetry and inductively coupled plasma mass spectrometry. Among them, EC analysis can significantly improve the robustness and ease of operation of the method due to its higher sensitivity, label-free operation and cost-effectiveness. Therefore, if the EC system can reliably distinguish Pb 2+ With G4-Pb 2+ complex, it is feasible to develop a homogeneous EC detection strategy. Summary of the Invention

[0005] In view of this, one of the objectives of the present invention is to provide a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network, so as to solve the technical problems that traditional DNA nanonetworks rely on fluorescent detection of chemical labels, which is cumbersome to operate and has limited sensitivity improvement.

[0006] A second object of the present invention is to provide an application of a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network.

[0007] In order to achieve one of the above objectives, the present invention provides a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network, wherein the method comprises forming a linear HCR product (H1-bio-H3) based on an aptamer, H1, H2, bio-H3 and H4-bio. n -aptamer-(H2-bio-H4) n, bio-H3 and bio-H4 were cross-linked with streptavidin, and the streptavidin-biotin binding sites were used as regulatory nodes to achieve the transformation of the topological structure into a three-dimensional network to construct G4@DNA-NHWN;

[0008] Based on whether the aptamer of G4@DNA-NHWN specifically binds to the target, resulting in the disintegration of G4@DNA-NHWN and the destruction of the embedded split G4 structure, and based on whether the split G4 binds to the free Pb 2+ Whether G4-Pb is formed 2+ Complex, through Pb 2+ The target is quantified by an electrochemical signal, and the target is tumor-derived exosomes.

[0009] Optionally, the split G4 sequence

[0010] Comprises G1 and G1', wherein G1 is GTGGGT, and G1' is AGGGCGGGTTGG;

[0011] or includes G2 and G2', wherein G2 is GTGGGTA, and G2' is GGGCGGGTTGG;

[0012] or includes G3 and G3', wherein G3 is GTGGGTAGGG, and G3' is CGGGTTGG;

[0013] Or include G4 and G4', wherein G4 is GTGGGTAGGGC, and G4' is GGGTTGG.

[0014] Optionally, the Pb 2+ With the G4-Pb 2+ The complexes have obvious electrochemical signal differences. 2+ The electron transport performance is much greater than that of G4-Pb 2+ Electron transport properties of the composites.

[0015] Alternatively, when the tumor-derived exosomes are absent, the G4@DNA-NHWN remains intact, and the split G4 sequences of H1 and H2 are aligned with the Pb 2+ Forming the G4-Pb 2+ The complex does not generate electrochemical signals;

[0016] When the tumor-derived exosomes are present, the tumor-derived exosomes bind to the aptamer of the G4@DNA-NHWN, causing the G4@DNA-NHWN to disintegrate, thereby hindering the G4-Pb 2+ Complex formation, free Pb 2+ Produces a strong electrochemical signal.

[0017] Optionally, H1 is: CGG GTT GGG TTC CAG ACG AGG AGC AAA GCT AAG AAT ACTTTG CTC CTC GGT GGG TAGG G;

[0018] The H2 is: CGG GTT GGT TTA GTT CTG GGA TAC TTA CGC CCA GAA CTA AAG CTGCGA TGT GGG TAG GG;

[0019] The H3 is: CTT TGC TCC TCG TCT GGA ACC GAG GAG CAA AGT ATT CTT AGT TTTTTT TTT TTT TTT-biotin;

[0020] The H4 is: Biotin-TTT TTT TTT TTT TTT TCG TAA GTA TCC CAG AAC TAA AATCGC AGC TTT AGT TCT GGG.

[0021] Optionally, the tumor-derived exosomes are vimentin.

[0022] Optionally, the aptamer of vimentin is: CAC GCA TAG CCT TTG CTC CTC GTC TGG AACGTC GCA GCT TTA GTT CTG GGC CTA TGC GTG.

[0023] Optionally, the linear regression equation of the electrochemical signal and the logarithmic value of the concentration of vimentin is Y = 200.9LogC + 580.5, and the correlation coefficient R 2 The concentration of vimentin ranged from 10 ag / mL to 100 fg / mL.

[0024] Optionally, the research model of vimentin is the MDA-MB-231 cell line, and the linear regression equation of the electrochemical signal and the logarithmic value of the vimentin concentration is Y = 184.2LogC + 565, with a correlation coefficient R 2 is 0.986, and the concentration range of vimentin is 10 2 - 10 7 particles / mL.

[0025] In order to achieve the second of the above objectives, the present invention provides an application of a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network, wherein the application comprises applying any of the above-mentioned homogeneous electrochemical analysis methods for exosome detection based on a programmable G4@DNA nanohighway network to the quantitative analysis of vimentin.

[0026] The homogeneous electrochemical analysis method and application of exosome detection based on the programmable G4@DNA nanohighway network provided by the present invention have the following technical effects:

[0027] The present invention constructs a universal homogeneous electrochemical detection platform. By integrating ligand-triggered hybridization chain reaction and streptavidin-biotin cross-linking technology, the platform constructs a DNA nanohighway network (G4@DNA-NHWN) embedded with a large number of split G4 structures, which can be rapidly synthesized in one step at room temperature.

[0028] This study selected vimentin as a proof-of-concept marker—it is overexpressed during the epithelial-mesenchymal transition and tumor metastasis of cancer and is an ideal target for exosome-based assessment of axillary lymph node metastasis in breast cancer. The core detection mechanism relies on the specific binding of the aptamer in G4@DNA-NHWN to vimentin, which leads to network disassembly and destruction of the embedded split G4 structure, thereby preventing the split G4 from interacting with free Pb. 2+ Formation of G4-Pb 2+ The complex ultimately achieves selective identification and precise quantification of vimentin-positive exosomes. This "one-pot" reaction system can rapidly quantify tumor-derived exosomes within 60 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 Schematic diagram of one-pot electrochemical detection of breast cancer exosomes based on the G4@DNA nanonetwork platform;

[0031] Figure 2 Figure 2 is the characterization result of G4@DNA-NHWN;

[0032] Figure 2 a is a schematic diagram of the synthesis of G4@DNA-NHWN;

[0033] Figure 2 b is the effect of different G4 structures on Pb²+ Histogram of the impact of electrochemical signals;

[0034] Figure 2 c is the linear regression analysis diagram of peak current and logarithm of PS2.M concentration;

[0035] Figure 2 d is a bar graph showing the regulatory effects of different splitting configurations of PS2.M on the electrochemical signal of Pb2+;

[0036] Figure 2 e1-3 are atomic force microscopy (AFM) images of H1-H4, hybridization chain reaction products, and G4@DNA-NHWN, respectively;

[0037] Figure 2 f1-3 are transmission electron microscope images of the corresponding samples;

[0038] Figure 2 g is the H1-H4, hybridization chain reaction and G4@DNA-NHWN to Pb 2+ Line graph of the regulation effect of current;

[0039] Figure 2 h is the circular dichroism spectrum characteristic diagram of G4 structure;

[0040] Figure 3 A specific vimentin biosensor pattern was achieved for functionalized G4@DNA-NHWN; wherein:

[0041] Figure 3 ab are the Zeta potential and dynamic light scattering analysis results of the system under different conditions;

[0042] Figure 3 c is the effect of vimentin on Pb 2+ Histogram of the regulatory effects of signals;

[0043] Figure 3 d is agarose gel electrophoresis analysis of G4@DNA-NHWN under different conditions;

[0044] Figure 3 e: Electrochemical evaluation of G4@DNA-NHWN under different conditions before centrifugation;

[0045] Figure 4 Optimize the analytical conditions for current intensity;

[0046] Figure 4 ab are the corresponding plots of the concentration of hairpin structures H1-H4 and current intensity;

[0047] Figure 4 cd is the corresponding plot of aptamer concentration and current intensity;

[0048] Figure 4 ef is the corresponding graph of streptavidin concentration and current intensity;

[0049] Figure 4 gh is the corresponding diagram of the assembly time of DNA nanomachines and current intensity;

[0050] Figure 5 is the analytical performance of vimentin; wherein:

[0051] Figure 5 a is based on Pb 2+ Schematic diagram of electrochemical analysis as a G-quadruplex binding probe;

[0052] Figure 5 b EC response curves of different concentrations of vimentin and (5c) peak current values;

[0053] Figure 5 c is the peak current value;

[0054] Figure 5 d is the linear regression analysis of peak current and logarithmic concentration of vimentin;

[0055] Figure 5 e is the specificity evaluation of vimentin detection;

[0056] Figure 6 is the analytical performance of exosomes; where:

[0057] Figure 6 a is a schematic diagram of the exosome extraction and analysis process;

[0058] Figure 6 bc are the results of nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells and blood samples;

[0059] Figure 6 de is the transmission electron microscopy image of exosomes from MDA-MB-231 cells and blood samples;

[0060] Figure 6 f, Western immunoblot analysis of exosomes from MDA-MB-231 cells;

[0061] Figure 6 gi is the electrochemical response change of exosomes at different concentrations;

[0062] Figure 7 This is a schematic diagram of the clinical diagnosis and analysis of breast cancer patients;

[0063] Figure 7 a is a schematic diagram of the blood sample testing process;

[0064] Figure 7b is the electrochemical signal histogram of 42 clinical samples;

[0065] Figure 7 c is a heat map;

[0066] Figure 7 d is a violin plot showing the significant differences between healthy controls (n=10) and patients (n=32);

[0067] Figure 7 e is the receiver operating characteristic curve of exosome detection;

[0068] Figure 7 f is the confusion matrix of the EC detection method;

[0069] Figure 7 g is magnetic resonance imaging of a breast cancer patient;

[0070] Figure 7 h is a schematic diagram of pathological examination results;

[0071] Figure 8 This is a clinical breast cancer diagnosis and results analysis chart; among them:

[0072] Figure 8 a is a schematic diagram of the blood sample testing process;

[0073] Figure 8 b is a histogram of clinical samples;

[0074] Figure 8 c is a heat map;

[0075] Figure 8 d is a violin plot showing the distribution characteristics of electrochemical signals in breast cancer samples;

[0076] Figure 8 e is the receiver operating characteristic curve analysis of the EC detection method;

[0077] Figure 8 f is the confusion matrix of the EC detection method;

[0078] Figure 8 g is the pathological examination results of breast cancer patients. DETAILED DESCRIPTION

[0079] 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.

[0080] The principle of the analytical method of the present invention is as follows:

[0081] like Figure 1 As shown, the present invention provides an integrated aptamer-mediated target recognition, G4-Pb 2+ A universal G4@DNA-NHWN platform for signal transduction and nanonetwork signal amplification for accurate quantitative detection of biomarkers.

[0082] Four hairpin DNA structures were designed based on the aptamer sequences (Table 1), in which H1 and H2 carried split G4 sequences at both ends, and the 5' end of bio-H3 and the 3' end of H4-bio were modified with biotin.

[0083] Specifically, the aptamer initiates the HCR reaction by unlocking the H1 and H2 hairpin structures. The exposed sticky ends guide the biotinylated bio-H3 and H4-bio hairpins to unfold sequentially, releasing the same sequence as the aptamer to drive bidirectional HCR. This process forms a linear HCR product (H1-bio-H3). n -aptamer-(H2-bio-H4) n Streptavidin (SA) cross-links biotinylated bio-H3 and bio-H4 at a 1:4 ratio. The SA-biotin binding sites serve as regulatory nodes (toll booths) to achieve a topological transition to a three-dimensional network, creating a robust structure similar to a highway network, thereby improving target capture efficiency.

[0084] The split G4 sequences carried by H1 and H2 can be combined with Pb 2+ Formation of electrochemically inert G4-Pb 2+ complex, as a signal reporting unit (service area) ( Figure 1 AB). Electrochemical detection can directly distinguish free Pb 2+ With G4-Pb 2+ The signal difference of the complexes enables true homogeneous detection.

[0085] To validate the platform, we selected vimentin, which is overexpressed in breast cancer-derived exosomes, as a target. In the absence of exosomes, the nanomesh maintains its intact structure, with the split G4 sequence of H1 / H2 forming G4-Pb. 2+ complex, no electrochemical signal is generated; on the contrary, when exosomes are present, the binding of trace amounts of vimentin to the aptamer will cause the nanonet to disintegrate, hindering the large amount of G4-Pb 2+ Complex formation, released free Pb 2+ The enhanced electron transfer generates a strong electrochemical signal, enabling highly sensitive TDE detection, which allows breast cancer patients to be distinguished from healthy controls.

[0086] Similarly, the expression of vimentin on the surface of exosomes differed between ALNM-positive and -negative patients - metastatic patients showed higher vimentin expression, resulting in a stronger electrochemical signal, thus accurately distinguishing ALNM status ( Figure 1 C). Validated by exosome analysis of blood samples, the platform demonstrated high accuracy in early diagnosis of breast cancer and assessment of ALNM status, highlighting its potential for clinical translation.

[0087] The technical solution of the present invention is specifically verified below with reference to the embodiments.

[0088] 1. Materials and Methods

[0089] All oligonucleotides with different sequences were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd. The oligonucleotide sequences used in this study are detailed in Tables 1-3.

[0090] Recombinant human vimentin was purchased from Novozymes Biotech (Shanghai) Co., Ltd., and lead nitrate (Pb(NO3)2) was purchased from Aladdin Reagent Co., Ltd. (Shanghai).

[0091] DNA Marker (25–500 bp) and 4S Gel-Red were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai), and 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), glypican 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 MDA-MB-231 cell line were purchased from Zhanjiang Tongbo Medical Technology Co., Ltd.

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

[0095] Anti-CD9, anti-CD63, anti-CD81, anti-MUC1, and anti-PD-L1 antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

[0096] Ultra-clear centrifuge tubes (25 × 89 mm, 38.5 mL, sterile thin-walled open-end) were purchased from Beckman Coulter (Indianapolis, IN, USA).

[0097] The experimental water was prepared using an ultrapure water system (Chengdu Ultrapure Technology Co., Ltd.) with a resistivity of 18.25 MΩ·cm.

[0098] All working solutions were prepared in 3-(N-morpholino)propanesulfonic acid buffer (10 mM, containing 100 mM NaNO3, pH 7.4).

[0099] All reagents used in this study were of analytical grade or higher and were used without further purification. All solutions were stored at 4°C until use. Plasma from healthy individuals and whole blood from breast cancer patients were donated 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).

[0100] Table 1. Sequences of oligonucleotides used in this study

[0101]

[0102] Table 2. Sequences of oligonucleotides used in the study

[0103]

[0104] Table 3. Sequences of oligonucleotides used in the study

[0105]

[0106] 2. Pretreatment of Clinical Samples

[0107] Peripheral blood samples were collected with the approval of West China Hospital, Sichuan University (Approval No. 20221489). Four milliliters of whole blood were collected into disposable, sealed vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant. Within 2 hours of collection, plasma was separated by centrifugation at 2500 g for 15 minutes at 25°C. Platelet-poor plasma was obtained by a second centrifugation under the same conditions and stored at −80°C for subsequent analysis.

[0108] 3. Synthesis of target regulatory DNA functional network

[0109] By integrating hybridization chain reaction and streptavidin-biotin system, the G4@DNA-NHWN platform was developed for target protein detection. Figure 2 As shown in a, the synthesis process of G4@DNA-NHWN was systematically designed.

[0110] First, G4-Pb2+ Combined with experimental verification of binding efficiency, by systematically evaluating the EC signals and signal-to-noise ratios (SNRs) of different G4 sequences (Table 2), it was found that the concentration of the PS2.M G4 sequence was significantly negatively correlated with the decrease in EC signal (SNR = 1.6). This steady downward trend laid a solid foundation for the subsequent development of hairpin sequences ( Figure 2 bc).

[0111] By comparing the EC signals of four base splitting modes (Table 3), the G3-G3' (10+8) base splitting mode was finally selected for subsequent research because of its 2+ Minimal signal interference ( Figure 2 d). Nanostructure characterization shows that atomic force microscopy and transmission electron microscopy observations show that the individual H1-H4 hairpin structures are uniformly distributed in small sizes ( Figure 2 e1 / f1), and after hybridization chain reaction, it combines with the aptamer to form a linear structure with an average length of 200 nm ( Figure 2 e2 / f2). G4@DNA-NHWN showed a multi-directional growth network structure, confirming the successful construction of DNA nanonetwork ( Figure 2 e3 / f3).

[0112] By analyzing the different components of Pb 2+ It was found that with the increase of hairpin concentration, the current signal of the hairpin group alone was stable, while the Pb 2+ The signals showed a downward trend, with the latter decreasing more significantly, confirming the formation of the G4 structure and its signal inhibition advantage ( Figure 2 g). Circular dichroism analysis further verified the structural characteristics of G4, and positive / negative characteristic peaks were observed at 310 nm and 285 nm, respectively, indicating that Pb 2+ In the presence of Figure 2 h).

[0113] 4. Functionalized G4@DNA-NHWN enables specific exosome detection

[0114] The feasibility of the G4@DNA-NHWN exosome detection platform was systematically evaluated using multi-dimensional analysis technology ( Figure 3 ). Zeta potential test shows: Pb 2+ The H1-H4 hairpin structure has a positive potential of +4.2 mV, while the H1-H4 hairpin structure has a negative potential of -23.3 mV due to the ionization of the phosphate backbone. 2+ When the G4@DNA-NHWN was assembled with the G4-containing hairpin through coordination, the negative charge was partially neutralized and the potential dropped to -13.5 mV, confirming that the complex was successfully constructed ( Figure 3 a).

[0115] After the specific binding of vimentin to the aptamer triggers structural dissociation, Pb 2+ Released from the G4 binding site, the internal negatively charged groups were exposed, resulting in a weakened charge neutralization effect and a potential increase to -10.1 mV, directly reflecting the disintegration of the nanonetwork. Dynamic light scattering analysis further confirmed that the average particle sizes of the H1-H4 hairpin, HCR product, and G4@DNA-NHWN were 40 nm, 95 nm, and 210 nm, respectively, while the particle size was reduced to 140 nm after protein interaction, indicating that the nanonetwork was broken ( Figure 3 b).

[0116] Control experiments confirmed that the EC signal change was due to the disassembly of G4@DNA-NHWN triggered by vimentin rather than its direct effect on Pb 2+ ( Figure 3 c). Agarose gel electrophoresis showed that the aptamer produced a high molecular weight product (lane 8) when it bound to the H1-H4 hairpin, while the products of vimentin-induced disassembly (lanes 10-12) had similar characteristics to the positive control (lane 9), confirming that the HCR reaction occurred ( Figure 3 d). The quantitative EC test results under different conditions were confirmed by electrophoresis analysis ( Figure 3 e), laying the foundation for subsequent research on vimentin and exosome detection performance.

[0117] 5. Vimentin Assay Performance

[0118] After the optimization of experimental conditions ( Figure 4 ), the analytical performance of vimentin was evaluated using EC sensor ( Figure 5 This detection strategy is based on the self-assembly properties of G4@DNA-NHWN and the signal amplification mechanism of vimentin-triggered nanonetwork dissociation, achieving high-sensitivity detection of exosomes ( Figure 5 a). Its excellent performance is attributed to three key factors: the specific binding of vimentin and aptamer, the nano-network signal amplification system and G4-Pb 2+ Electrochemical signal transduction.

[0119] When the concentration of vimentin increased from 10 ag / mL to 100 fg / mL, the EC signal showed a gradient increase ( Figure 5 bc). Within this concentration range, the EC signal showed a significant linear correlation with the logarithmic concentration of vimentin (R 2 = 0.996), the linear regression equation is Y = 200.9LogC + 580.5 ( Figure 5 d). The limit of detection (LOD) of this method is as low as 3 ag / mL, which is a million times more sensitive than existing detection technologies (generally at the μg / pg level).

[0120] Selectivity experiments showed that in the presence of non-target proteins such as interferon-γ, human serum albumin, and glypican-3 (10 pg / mL), the EC signal changed slightly; however, low concentrations of vimentin (100 ag / mL and 100 fg / mL) could induce significant signal enhancement ( Figure 5 e), demonstrating that this method has excellent specificity in complex biological samples.

[0121] The present invention uses the highly invasive and metastatic MDA-MB-231 cell line as an exosome research model. This cell line's high expression of vimentin accurately mimics the biological characteristics of metastatic breast cancer. This is to evaluate the analytical performance of exosome detection.

[0122] First, we systematically characterized the exosomes extracted from MDA-MB-231 cell supernatant and clinical plasma samples, focusing on key parameters such as particle size distribution, concentration, morphological characteristics, and surface protein expression ( Figure 6 a). The concentration of exosomes in the cell culture supernatant was measured by nanoparticle tracking analysis technology to be 1.1×10 11 particles / mL, and the concentration in clinical plasma samples was slightly lower at 4.2×10 10 particles / mL( Figure 6 bc). Transmission electron microscopy confirmed that exosomes exhibit a typical cup-shaped double membrane structure ( Figure 6 de), Western blot analysis showed that it expressed exosome marker proteins CD9, CD63, CD81 and tumor-associated vimentin, verifying the successful isolation of exosomes ( Figure 6 f).

[0123] Based on the high vimentin expression characteristics of MDA-MB-231-derived exosomes, electrochemical detection signal ( Figure 6 g) At an exosome concentration of 10 2 - 10 7 particles / mL range and the logarithmic concentration showed a good linear relationship ( Figure 6 hi), the regression equation is Y = 184.2LogC + 565 (R 2 = 0.986), with LOD as low as 30 particles / mL.

[0124] The homogeneous electrochemical detection method of this invention significantly avoids the complex electrode modification required by traditional techniques. It requires no enzymes or labels and can complete detection within 60 minutes, significantly simplifying the process and improving detection efficiency. The method's high sensitivity, excellent reproducibility, and scalability make it a powerful tool for exosome analysis, with significant application value in clinical diagnosis and biomarker discovery. Its rapid detection capabilities are particularly well-suited for exosome-related diagnostic needs.

[0125] 6. Clinical Applicability of the G4@DNA-NHWN Detection Platform

[0126] Blood samples from 42 subjects (32 breast cancer patients and 10 healthy controls) were collected to evaluate the accuracy and diagnostic efficacy of the EC detection method. Figure 7 EC analysis of clinical samples showed that the peak signal of breast cancer patients was significantly higher than that of healthy controls, indicating that the expression of exosomal vimentin was increased ( Figure 7 b). The heat map can be used to visually distinguish breast cancer patients from healthy people. The darker the color, the higher the expression level of exosomal vimentin ( Figure 7 c). The violin plot showed that there was a significant difference in peak current between the two groups (P<0.05) ( Figure 7 d). The diagnostic performance of the EC system was evaluated using receiver operating characteristic (ROC) curves and confusion matrix. The results showed that the specificity reached 100% (10 / 10), the sensitivity was 87.5% (28 / 32), the accuracy was 90.5% (38 / 42), the precision was 100% (28 / 28), and the area under the curve (AUC) was 0.916, confirming that the method has high diagnostic accuracy ( Figure 7 ef). It is worth noting that the exosome detection results of the EC method are highly consistent with MRI and pathological diagnosis, further verifying the reliability of this method ( Figure 7 gh).

[0127] The method was further evaluated for its ability to determine ALNM status, a metric that is crucial for developing breast cancer treatment strategies ( Figure 8 a). The study found that EC signals in ALNM-positive patients were significantly higher than those in negative patients ( Figure 8 b), indicating that the expression level of exosomal vimentin in the metastatic group was higher. Heat map analysis showed that the color of the ALNM-positive group was darker than that of the negative group, showing good discrimination ability ( Figure 8 c). The violin plot confirmed that there was a significant difference in peak current between the two groups (P<0.05) ( Figure 8 d).

[0128] The receiver operating characteristic curve and confusion matrix evaluation showed that the method had excellent diagnostic performance: the area under the curve was 0.901, the specificity was 100% (24 / 24), the accuracy was 96.9% (31 / 32), the precision was 100% (7 / 7), and the sensitivity was 87.5% (7 / 8) ( Figure 8 ef). This result is highly consistent with the pathological evaluation ( Figure 8 g). For example, although both patients 3 and 10 were diagnosed with breast cancer, pathological examination revealed no lymph node metastasis in patient 10, while metastatic lesions were present in patient 3. This is consistent with the significant difference in EC signaling between the two. These results confirm the reliability and effectiveness of exosome detection in determining ALNM status and provide important evidence for clinical decision-making.

[0129] 7. Conclusion

[0130] This study successfully developed a highly sensitive target recognition system based on the G4@DNA-NHWN nanomaterial, which can be rapidly assembled through a one-pot reaction at room temperature. This innovative material integrates three core functional modules: (1) a target recognition unit; (2) a G4-Pb2+ signal transduction system; and (3) a nanonetwork signal amplification system. Clinical validation studies have shown that this platform exhibits excellent performance in distinguishing breast cancer patients from healthy controls, with a sensitivity and specificity exceeding 85% for ALNM status assessment, demonstrating significant clinical value.

[0131] The modular design of the platform allows for precise modification of nucleic acid recognition elements, thereby expanding its detection range to other biomarkers, providing an innovative technical solution for early tumor screening and metastasis monitoring. However, this study still has the following limitations: First, the current system only supports single-target detection, and multiple analysis can be achieved in the future by introducing multi-channel signal encoding; second, TDE detection requires additional sample pretreatment steps. Subsequent research will focus on the development of a pretreatment-free detection platform to simplify the operation process and improve detection efficiency. It is worth noting that this DNA-functionalized nanomaterial platform can not only serve as a multifunctional biosensor, but also shows significant development potential in biomedical applications such as targeted drug delivery.

[0132] 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 homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network, characterized in that: The method comprises forming a linear HCR product (H1-bio-H3) based on aptamers, H1, H2, bio-H3 and H4-bio n -aptamer-(H2-bio-H4) n , n represents 1, 2, 3..., bio-H3 and bio-H4 were cross-linked with streptavidin, and the streptavidin-biotin binding sites were used as regulatory nodes to achieve the transformation of the topological structure into a three-dimensional network to construct G4@DNA-NHWN; Based on whether the aptamer of G4@DNA-NHWN specifically binds to the target, resulting in the disintegration of G4@DNA-NHWN and the destruction of the embedded split G4 sequence, and based on whether the split G4 sequence binds to the free Pb 2+ Whether G4-Pb is formed 2+ Complex, through Pb 2+ quantifying the target by an electrochemical signal, wherein the target is tumor-derived exosomes; When the tumor-derived exosomes were absent, the G4@DNA-NHWN remained intact, and the split G4 sequences of H1 and H2 were aligned with the Pb 2+ Forming the G4-Pb 2+ The complex does not generate electrochemical signals; When the tumor-derived exosomes are present, the tumor-derived exosomes bind to the aptamer of the G4@DNA-NHWN, causing the G4@DNA-NHWN to disintegrate, thereby hindering the G4-Pb 2+ Complex formation, free Pb 2+ Produces strong electrochemical signals; Four hairpin DNA structures were designed based on the aptamer sequences for H1-H4, where H1 and H2 carry split G4 sequences at both ends, and the 5' end of bio-H3 and the 3' end of H4-bio are modified with biotin; The split G4 sequence includes G1 and G1', wherein G1 is GTGGGT and G1' is AGGGCGGGTTGG; or includes G2 and G2', wherein G2 is GTGGGTA, and G2' is GGGCGGGTTGG; or includes G3 and G3', wherein G3 is GTGGGTAGGG, and G3' is CGGGTTGG; Or include G4 and G4', wherein G4 is GTGGGTAGGGC, and G4' is GGGTTGG.

2. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 1, characterized in that: The Pb 2+ With the G4-Pb 2+ The complexes have obvious electrochemical signal differences. 2+ The electron transport performance is much greater than that of G4-Pb 2+ Electron transport properties of the composites.

3. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 2, characterized in that: The H1 is: CGG GTT GGG TTC CAG ACG AGG AGC AAA GCT AAG AAT ACT TTG CTC CTCGGT GGG TAGG G; The H2 is: CGG GTT GGT TTA GTT CTG GGA TAC TTA CGC CCA GAA CTA AAG CTG CGATGT GGG TAG GG; The H3 is: CTT TGC TCC TCG TCT GGA ACC GAG GAG CAA AGT ATT CTT AGT TTT TTTTTT TTT TTT-biotin; The H4 is: Biotin-TTT TTT TTT TTT TTT TCG TAA GTA TCC CAG AAC TAA AAT CGCAGC TTT AGT TCT GGG.

4. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 2, characterized in that: The tumor-derived exosomes are vimentin.

5. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 4, characterized in that: The vimentin aptamer is: CAC GCA TAG CCT TTG CTCCTC GTC TGG AAC GTC GCA GCT TTA GTT CTG GGC CTA TGC GTG.

6. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 4, characterized in that: The linear regression equation of the electrochemical signal and the logarithmic value of the vimentin concentration is Y = 200.9LogC + 580.5, and the correlation coefficient R 2 The concentration of vimentin ranged from 10 ag / mL to 100 fg / mL.

7. The homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to claim 4, characterized in that: The research model of vimentin is MDA-MB-231 cell line, and the linear regression equation of the electrochemical signal and the logarithmic value of the vimentin concentration is Y = 184.2LogC + 565, with a correlation coefficient R 2 is 0.986, and the concentration range of vimentin is 10 2 - 10 7 particles / mL.

8. An application of a homogeneous electrochemical analysis method for exosome detection based on a programmable G4@DNA nanohighway network, characterized in that: The application includes using the homogeneous electrochemical analysis method for exosome detection based on the programmable G4@DNA nanohighway network according to any one of claims 4-7 for the quantitative analysis of vimentin.

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