Bionic electrode interface for capturing and analyzing tumor extracellular vesicles as well as preparation method and application of bionic electrode interface

Abstract

The invention discloses a bionic electrode interface for capturing and analyzing tumor extracellular vesicles (EVs) as well as a preparation method and application of the bionic electrode interface, and belongs to the technical field of biosensing. The method comprises the following steps: extracting tumor cell membrane vesicles and modifying the vesicles on the surface of an electrode to construct a bionic electrode interface with homologous recognition capability; after the interface is used for specifically capturing target tumor EVs, an electroactive molecular probe is used for labeling, and finally quantitative analysis is realized through electrochemical detection. According to the method disclosed by the invention, multivalent recognition is carried out by utilizing an integral antigen spectrum of a natural cell membrane, so that the limitation of traditional point-to-point single ligand recognition is overcome, and the capture comprehensiveness and firmness of homologous EVs are remarkably improved; meanwhile, due to inherent biocompatibility and anti-fouling characteristics of the bionic interface, non-specific adsorption in a complex sample can be effectively inhibited, so that high-sensitivity detection of the target EVs is realized on the premise of ensuring high selectivity.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically a biomimetic electrode interface for capturing and analyzing extracellular vesicles of tumor cells, its preparation method, and its application. Background Technology

[0002] Tumor extracellular vesicles (EVs) have shown great potential as important biomarkers in early cancer diagnosis, treatment monitoring, and prognostic assessment. They carry molecular information such as proteins and nucleic acids specific to the maternal cell, making it possible to obtain non-invasive tumor molecular profiles. However, achieving highly selective capture and accurate quantification of tumor EVs from complex clinical samples (such as blood) remains a key technological challenge.

[0003] Currently, the separation and enrichment of EVs mainly relies on physical principles. Ultracentrifugation, as the "gold standard," is cumbersome, time-consuming, and has low recovery rates, and it cannot distinguish between EVs of tumor origin and those of non-tumor origin. While methods such as size exclusion chromatography, ultrafiltration, and polymer precipitation have improved throughput, they are still limited by separation purity and struggle to remove interference from high-abundance proteins (such as albumin and lipoproteins) and other vesicles with overlapping sizes and densities. These methods are essentially coarse separations based on physical properties and lack the ability to specifically identify tumor EVs. To improve selectivity, affinity-based capture techniques have been developed. These methods typically rely on immobilizing one or a few antibodies or nucleic acid aptamers targeting specific surface markers (such as EpCAM and CD63) on a solid-phase support, achieving capture through "point-to-point" binding with the target EVs. However, such strategies have inherent limitations: First, the expression of tumor EV surface markers is highly heterogeneous, and a single marker may coexist on multiple EVs, leading to incomplete capture (false negatives) or non-specific binding (false positives); second, these artificially modified solid-liquid interfaces are prone to non-specific protein adsorption in complex biological samples, severely interfering with the target signal and affecting detection accuracy; third, most affinity capture methods only perform separation and enrichment functions, requiring combination with independent detection technologies such as nucleic acid detection, Western blotting, or nanoparticle tracking analysis, which is complex, costly, and difficult to achieve integrated and convenient quantitative analysis.

[0004] Therefore, there is an urgent need for an integrated detection platform that can simulate the recognition characteristics of biological systems, possesses high selectivity for capture, excellent anti-interference capabilities, and direct quantification functions. This is of great significance for promoting the clinical application of tumor EV detection technology. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method for preparing a biomimetic electrode interface, wherein the biomimetic electrode interface prepared by the method can specifically capture EVs secreted by homologous tumor cells and effectively resist non-specific protein adsorption by virtue of the inherent biocompatibility of the cell membrane.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a biomimetic electrode interface, the method comprising the following steps: Cell membrane vesicles are extracted from target tumor cells; the cell membrane vesicles are modified onto the surface of an electrode substrate to form a biomimetic electrode interface for capturing and analyzing extracellular vesicles of homologous tumor cells.

[0007] Preferably, the method for extracting cell membrane vesicles includes the following steps: subjecting target tumor cells to hypotonic lysis and repeated freeze-thaw treatment, followed by differential centrifugation to obtain purified cell membrane vesicles.

[0008] More preferably, the conditions for the repeated freeze-thaw treatment are: freezing at -80°C to -75°C for 1-2 minutes, followed by thawing at 22°C to 25°C for 1-2 minutes; repeating this cycle 3-5 times.

[0009] Preferably, the modification method includes the following steps: adding cell membrane vesicle resuspension to the pretreated electrode surface and incubating at 50°C to 60°C for 30-35 minutes.

[0010] The present invention also provides a method for preparing a biomimetic electrode interface as described above.

[0011] The present invention also provides a method for capturing and analyzing extracellular vesicles of tumor cells using the aforementioned biomimetic electrode interface, the method comprising the following steps: The sample containing extracellular vesicles of the target tumor cells is brought into contact with the biomimetic electrode interface, so that the extracellular vesicles of the target tumor cells are captured. Electroactive molecular probes are used to label captured tumor extracellular vesicles; signals are obtained through electrochemical detection technology to achieve qualitative or quantitative analysis of target tumor extracellular vesicles.

[0012] Preferably, the electroactive molecular probe includes a recognition unit and a signal reporting unit; the recognition unit is a nucleic acid aptamer, antibody, or affinity polypeptide that specifically binds to markers on the surface of extracellular vesicles of tumor cells.

[0013] Preferably, the electrochemical detection technology includes AC impedance voltammetry, square wave voltammetry, or differential pulse voltammetry.

[0014] Preferably, the target tumor cells are selected from breast cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, or colorectal cancer cells.

[0015] The present invention also provides the application of the aforementioned biomimetic electrode interface, or the method thereof, in the preparation of products for tumor diagnosis or prognostic assessment.

[0016] Compared with the prior art, the present invention has the following advantages: (1) The biomimetic electrode interface constructed in this invention uses tumor cell membranes rich in native tumor antigens as recognition elements, enabling the overall recognition and capture of EVs secreted by homologous cancer cells. This method not only effectively eliminates interference from non-target vesicles but also overcomes the problem of poor capture stability and accuracy caused by the reliance on a single antigen in traditional "point-to-point" recognition strategies. Experimental data clearly show that, at the same concentration, this interface only produces a strong specific signal for its homologous EVs, while the signal produced for heterologous EVs is no different from that of the blank control. This fully demonstrates that this invention achieves efficient and precise capture of homologous EV populations.

[0017] (2) The biomimetic electrode interface constructed in this invention benefits from the inherent fluidity and antifouling properties of the cell membrane, and can significantly inhibit the non-specific adsorption of high-abundance proteins (such as albumin and apolipoproteins) and cell debris in complex samples such as blood. Electrochemical impedance spectroscopy characterization results show that as the cell membrane and EVs are orderly modified at the interface, the charge transfer resistance changes regularly, indicating that the interface modification is successful and the binding event is effective. In principle, this biocompatible interface can effectively reduce the non-specific adsorption of high-abundance proteins in complex samples, laying a solid foundation for reducing detection background noise and improving the signal-to-noise ratio.

[0018] (3) The biomimetic electrode interface constructed in this invention facilitates the integration of specific electrochemical probes, thereby achieving highly sensitive and wide-linear-range electrochemical quantification of target vesicles. Data from this invention show that the detection limit for MCF-7 EVs is as low as 269 particles / mL, and the linear range spans 1×10⁻⁶. 3 Up to 1×10 8 The detection limit is on the order of five orders of magnitude (particles / mL). The biomimetic electrode interface constructed in this invention has a significantly lower detection limit than many previously reported cutting-edge electrochemical methods based on DNA machines and glycomolecular imprinting, demonstrating its outstanding advantages in trace detection.

[0019] (4) This invention has verified the successful capture and analysis of EVs from three different tumor cell sources: MCF-7 (breast cancer), SK-BR-3 (breast cancer), and A549 (lung cancer). This demonstrates that the biomimetic interface constructed using homologous cell membranes in this invention is universal and not limited to specific tumor types or specific surface markers. By simply changing the cell membranes of different tumors, corresponding specific interfaces can be constructed, providing a universal technical platform for the detection of EVs for multiple cancers. Attached Figure Description

[0020] Figure 1 This is a technical schematic diagram of a method for capturing and analyzing extracellular vesicles of tumor cells based on a biomimetic electrode interface. Figure 2 The preparation of a functionalized biomimetic electrode interface for MCF-7 cell membranes and the electrochemical impedance spectroscopy characterization results of its use in MCF-7 EVs capture; Figure 3 The figures show the electrochemical analysis results of MCF-7 EVs based on the functionalized biomimetic electrode interface of the MCF-7 cell membrane; in the figure, A is the square wave voltammetry obtained when detecting different concentrations of MCF-7 EVs, and B is the detection of 1×10 9 Square wave voltammetric peak current values ​​obtained for particles / mL MCF-7 EVs, SK-BR-3 EVs, A549 EVs and blank control; Figure 4 The preparation of the functionalized biomimetic electrode interface for SK-BR-3 cell membrane and the electrochemical impedance characterization results of its use in SK-BR-3 EVs capture; Figure 5 The figures show the electrochemical analysis results of A549 EVs based on the A549 cell membrane functionalized biomimetic electrode interface; A in the figure represents the detection depth of 1×10⁻⁶. 9 Square wave voltammetry spectra obtained for particles / mL A549 EVs, MCF-7 EVs, SK-BR-3 EVs and blank control; B is the square wave voltammetry peak current value obtained when detecting different concentrations of A549 EVs; Figure 6 This is the electrochemical analysis result of a clinical plasma sample based on an A549 cell membrane-functionalized biomimetic electrode interface. Detailed Implementation

[0021] The present invention provides a method for preparing a biomimetic electrode interface, wherein the preparation method preferably includes the following steps: extracting cell membrane vesicles from target tumor cells; modifying the cell membrane vesicles onto the surface of an electrode substrate to form a biomimetic electrode interface for capturing and analyzing extracellular vesicles of homologous tumor cells.

[0022] In this invention, the preferred method for extracting the cell membrane vesicles includes the following steps: taking 1 mL of a concentration of 0.5-1×10 7 Tumor cells per milliliter were placed in a microtube and centrifuged at 1500-2000 rpm for 3-5 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. The centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (15-20 mM Tris-HCl buffer, pH 7.0-7.5, containing 10-15 mM KCl and 1.5-2.5 mM...). The precipitate was resuspended in MgCl2 and 1%-1.2% protease / phosphatase inhibitor. The resulting solution was frozen at -75 to -80°C for 1-2 minutes and then rapidly transferred to 22 to 25°C to thaw for 1-2 minutes. The freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10-15 minutes and centrifuged at 700-800g for 10-12 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000-21000g for 25-30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate was the tumor cell membrane vesicle, which was resuspended in 1 mL of 1× phosphate buffer for later use.

[0023] The specific steps for modifying the cell membrane vesicles onto the electrode substrate surface in this invention are as follows: First, the surface of the indium tin oxide (ITO) electrode is ultrasonically cleaned sequentially with acetone, ethanol, and double-distilled water. Then, the electrode is placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide, and double-distilled water (volume ratio 1:1:6) and treated at 80-90°C for 25-30 minutes. After treatment, the electrode surface is thoroughly rinsed with double-distilled water, and 50 μL of the obtained tumor cell membrane vesicle resuspension is added dropwise to the electrode surface and incubated at 50-60°C for 30-35 minutes. Finally, the resulting tumor cell membrane-functionalized biomimetic electrode interface is rinsed with 1× phosphate buffer and stored for later use.

[0024] This invention also provides a method for preparing a biomimetic electrode interface as described above. This invention utilizes a repeated freeze-thaw cycle and centrifugation under hypotonic conditions to extract stable tumor cell membrane vesicles from cultured tumor cells; subsequently, the obtained membrane vesicles are modified onto the surface of an indium tin oxide (ITO) electrode using a vesicle fusion strategy, thereby constructing a biomimetic electrode interface. The biomimetic electrode interface prepared by this invention, on the one hand, utilizes the specific recognition and binding ability between the tumor cell membrane and homologous EVs to achieve efficient capture of target EVs; on the other hand, it significantly improves capture efficiency by leveraging the inherent fluidity and antifouling properties of the cell membrane, and effectively suppresses interference from non-target components such as free proteins and cell debris in blood samples.

[0025] The present invention also provides a method for capturing and analyzing tumor extracellular vesicles using the aforementioned biomimetic electrode interface. The method preferably includes the following steps: contacting a sample containing target tumor extracellular vesicles with the biomimetic electrode interface to capture the target tumor extracellular vesicles; labeling the captured tumor extracellular vesicles using an electroactive molecular probe; and acquiring signals through electrochemical detection technology to achieve qualitative or quantitative analysis of the target tumor extracellular vesicles.

[0026] In a specific implementation of this invention, the specific steps of the method are as follows: 100 μL of a solution containing a certain concentration of tumor EVs is added dropwise to the prepared biomimetic electrode interface, and the reaction is incubated to capture the target EVs; the reaction temperature is 35-40℃, and the reaction time is 1-2 hours; after the reaction, the electrode surface is thoroughly rinsed with 1× phosphate buffer. Subsequently, 100 μL of a 1-2 μM electroactive molecular probe is added dropwise to the prepared biomimetic electrode interface to react, so that the probe is labeled onto the EVs surface through specific binding with a specific marker; the reaction temperature is 20-25℃, and the reaction time is 1-2 hours; after the reaction, the electrode surface is thoroughly rinsed with 1× phosphate buffer, and electrochemical signals are collected using appropriate electrochemical testing techniques.

[0027] In this invention, the electroactive molecular probe comprises a recognition unit and a signal reporting unit. The recognition unit can bind with high affinity and high specificity to specific marker molecules on the surface of target EVs, thereby anchoring the entire probe to the target vesicle. In a preferred embodiment, the recognition unit is a nucleic acid aptamer, antibody, or affinity polypeptide that specifically binds to markers on the surface of tumor extracellular vesicles. Nucleic acid aptamers are single-stranded DNA or RNA molecules obtained through in vitro screening, capable of folding into specific three-dimensional structures to bind to the target, similar to antibodies. Their advantages include small molecular weight, ease of chemical synthesis and modification, good stability, and minimal batch-to-batch variation. In specific implementations of this invention, DNA aptamers targeting mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are preferred. Antibodies refer to monoclonal antibodies, polyclonal antibodies, or fragments thereof (such as Fab, scFv) capable of specifically recognizing EV surface antigens (such as CD63, CD81, EpCAM, HER2, etc.). Affinity peptides refer to short peptide sequences that specifically bind to target proteins, obtained through techniques such as phage display. These peptides have smaller molecular weights and are easier to synthesize and penetrate. The signal reporter unit generates or modulates a measurable electrical signal during electrochemical detection, and its signal intensity is related to the number of probes bound to the interface. In specific embodiments of this invention, the signal reporter unit preferably includes organic electroactive molecules such as methylene blue, ferrocene, and their derivatives, which can undergo reversible redox reactions at specific electrode potentials to generate Faraday currents; and inorganic nanomaterials such as gold nanoparticles, silver nanoparticles, and quantum dots, which can generate strong electrochemical signals themselves or during their dissolution and catalysis. Through the above design, the conversion of antibody-antigen and aptamer-target binding biorecognition events into quantitatively readable electrical signals is key to achieving quantitative analysis. In this invention, the recognition unit and the signal reporter unit are typically covalently coupled via chemical bonds or flexible connecting arms. For example, in nucleic acid aptamer probes, the signal molecule can be directly modified chemically at specific ends or internal bases of the aptamer chain.

[0028] In this invention, the electrochemical detection technology preferably includes AC impedance voltammetry, square wave voltammetry, or differential pulse voltammetry.

[0029] The core of the biomimetic electrode interface of this invention lies in utilizing the principle of homology recognition. Those skilled in the art should understand that as long as the cell membrane of the target tumor cells can be cultured and obtained, the corresponding specific biomimetic interface can be constructed using the same technical approach of this invention, thereby achieving the capture and analysis of extracellular vesicles (EVs) originating from that specific tumor. The method for capturing and analyzing extracellular vesicles of tumor cells in this invention is universal, and its application is not limited to the specific cell lines listed in the examples, but can be extended to other epithelial-derived malignant tumors, such as, but not limited to, breast cancer, lung cancer, liver cancer, gastric cancer, colorectal cancer, etc.

[0030] This invention also provides the application of the aforementioned biomimetic electrode interface, or the method described herein, in the preparation of products for tumor diagnosis or prognostic assessment. In this invention, the products include, but are not limited to, reagent kits, detectors, etc.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0033] Example 1 The steps for capturing estrogen receptor-positive breast cancer EVs based on a biomimetic electrode interface are as follows: (1) Extraction of tumor cell membrane vesicles, the specific process is as follows: take 1 mL of vesicles with a concentration of 1×10 7 MCF-7 estrogen receptor-positive breast cancer cells (cells / mL) were placed in a microtube and centrifuged at 1500 rpm for 3 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. This centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (20 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, 2 mM MgCl2, and 1% protease / phosphatase inhibitor). The resulting solution was frozen at -80°C for 2 minutes and then rapidly transferred to 25°C to thaw for 2 minutes. This freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10 minutes and centrifuged at 700 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000 g for 30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate, which is the tumor cell membrane vesicle, was resuspended in 1 mL of... Store in 1× phosphate buffer for later use.

[0034] (2) Preparation of the biomimetic electrode interface: First, the ITO electrode was ultrasonically cleaned sequentially with acetone, ethanol, and double-distilled water. Then, the electrode was placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide, and double-distilled water in a volume ratio of 1:1:6 and treated at 85°C for 30 minutes. After treatment, the electrode surface was thoroughly rinsed with double-distilled water, and 50 μL of the tumor cell membrane vesicle resuspension obtained in step (1) was added to the electrode surface and incubated at 50°C for 30 minutes. Finally, the obtained tumor cell membrane functionalized biomimetic electrode interface was rinsed with 1× phosphate buffer and stored for later use.

[0035] (3) Take 100 μL containing 1×109 A solution of particles / mL MCF-7 cell-derived EVs was added dropwise to the interface of the biomimetic electrode prepared in step (2), and the incubation reaction was carried out to capture the target EVs; the reaction temperature was 37℃ and the reaction time was 1.5 hours.

[0036] (4) After thoroughly rinsing the electrode interface obtained in step (3) with 1× phosphate buffer, electrochemical signals were acquired using electrochemical impedance spectroscopy. The specific parameters were: electrochemical probe, 5 mM [Fe(CN)6]. 3- / 4- Initial potential: 0.224V; Frequency range: 0.1Hz to 10kHz. For the technical protocol of capturing and analyzing extracellular vesicles of tumor cells, see [link to technical documentation]. Figure 1 .

[0037] Figure 2 The Nyquist plots, characterized by electrochemical impedance spectroscopy (EIS) of the fabrication process of the MCF-7 cell membrane-functionalized biomimetic electrode interface and its performance in capturing MCF-7 EVs, are presented. As shown in the figure, with the sequential incubation of the ITO electrode surface with the MCF-7 cell membrane and MCF-7 EVs, the semicircle diameter in the high-frequency region of the plot gradually increases, indicating a corresponding increase in charge transfer resistance at the electrode interface. This result is in line with expectations, confirming the successful modification of the cell membrane and the effective capture of the target EVs, as the steric hindrance gradually formed at the interface hinders [Fe(CN)6]. 3- / 4- Electron transfer between electrodes.

[0038] Example 2 The quantitative analysis of estrogen receptor-positive breast cancer EVs based on a biomimetic electrode interface follows these steps: (1) Extraction of tumor cell membrane vesicles, the specific process is as follows: take 1 mL of vesicles with a concentration of 1×10 7MCF-7 estrogen receptor-positive breast cancer cells (cells / mL) were placed in a microtube and centrifuged at 1500 rpm for 3 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. This centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (20 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, 2 mM MgCl2, and 1% protease / phosphatase inhibitor). The resulting solution was frozen at -80°C for 2 minutes and then rapidly transferred to 25°C to thaw for 2 minutes. This freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10 minutes and centrifuged at 700 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000 g for 30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate, which is the tumor cell membrane vesicle, was resuspended in 1 mL of... Store in 1× phosphate buffer for later use.

[0039] (2) Preparation of the biomimetic electrode interface: First, the ITO electrode was ultrasonically cleaned sequentially with acetone, ethanol, and double-distilled water. Then, the electrode was placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide, and double-distilled water in a volume ratio of 1:1:6 and treated at 85°C for 30 minutes. After treatment, the electrode surface was thoroughly rinsed with double-distilled water, and 50 μL of the tumor cell membrane vesicle resuspension obtained in step (1) was added to the electrode surface and incubated at 50°C for 30 minutes. Finally, the obtained tumor cell membrane functionalized biomimetic electrode interface was rinsed with 1× phosphate buffer and stored for later use.

[0040] (3) Take 100 μL of solution containing different concentrations of MCF-7 cell-derived EVs and add it to the interface of the biomimetic electrode prepared in step (2). Incubate the reaction to achieve the capture of the target EVs. The reaction temperature is 37ºC and the reaction time is 1.5 hours. After the reaction is completed, use 1× phosphate buffer to thoroughly rinse the electrode surface.

[0041] (4) Take 100 μL of 1 μM methylene blue labeled mucin 1 aptamer probe (SEQ ID NO.1 is 5'-methylene blue-C6 spacer-GCAGTTGATCCTTTGGATACCCTGG-3') and drop it onto the biomimetic electrode interface prepared in step (3) to react, so that the probe is labeled onto the EVs surface by specific binding with a specific marker; the reaction temperature is 25℃ and the reaction time is 1.5 hours; after the reaction, use 1× phosphate buffer to thoroughly rinse the electrode surface, and use square wave voltammetry to collect electrochemical signals. The specific parameters are: scanning potential, -0.6V to -0.2V; amplitude, 25mV.

[0042] Figure 3 The experimental results obtained from electrochemical analysis of MCF-7 EVs based on the MCF-7 cell membrane functionalized biomimetic electrode interface are shown. Figure 3 As shown in A, in 1×10 3 granules / ml to 1×10 9 Within the particle / mL range, the square wave voltammetric peak increased with increasing MCF-7 EVs concentration. Furthermore, the square wave peak current value... I With MCF-7 EVs concentration C The logarithm of 1×10 3 granules / ml to 1×10 8 The correlation is linear within the particle / mL range, and the linear equation is: I =0.205 lg C -0.281 ( R 2 =0.997). Based on the linear equation, we calculated the detection limit of our method for detecting MCF-7 EVs to be 269 particles / mL, which is superior to most existing electrochemical methods (see Table 1 for comparative examples). Figure 3 B further demonstrates the application of this method to the same concentration (1×10⁻⁶). 9 The square-wave voltammetric peak current values ​​were obtained when analyzing MCF-7 EVs (particles / mL) and control EVs (including EVs derived from human epidermal growth factor receptor 2 positive breast cancer cells SK-BR-3 and non-small cell lung cancer cells A549). As shown in the figure, the square-wave voltammetric peak current values ​​generated by the control EVs were only comparable to those of the blank control group compared to MCF-7 EVs. These results fully demonstrate that this method can be used for the electrochemical quantitative analysis of target MCF-7 EVs, and thanks to the selective capture capability of the biomimetic electrode interface for target EVs, this method exhibits excellent analytical specificity.

[0043] Example 3 The capture of human epidermal growth factor receptor 2-positive breast cancer EVs based on a biomimetic electrode interface is as follows: (1) Extraction of tumor cell membrane vesicles, the specific process is as follows: take 1 mL of vesicles with a concentration of 1×10 7Human epidermal growth factor receptor 2 positive breast cancer cells (SK-BR-3) were placed in a microtube and centrifuged at 1500 rpm for 3 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. This centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (20 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, 2 mM MgCl2, and 1% protease / phosphatase inhibitor). The resulting solution was frozen at -80°C for 2 minutes and then rapidly transferred to 25°C to thaw for 2 minutes. This freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10 minutes and centrifuged at 700 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000 g for 30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate, which was the tumor cell membrane vesicle, was resuspended in 1 mL of... Store in 1× phosphate buffer for later use.

[0044] (2) Preparation of the biomimetic electrode interface: First, the ITO electrode was ultrasonically cleaned sequentially with acetone, ethanol, and double-distilled water. Then, the electrode was placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide, and double-distilled water in a volume ratio of 1:1:6 and treated at 85°C for 30 minutes. After treatment, the electrode surface was thoroughly rinsed with double-distilled water, and 50 μL of the tumor cell membrane vesicle resuspension obtained in step (1) was added to the electrode surface and incubated at 50°C for 30 minutes. Finally, the obtained tumor cell membrane functionalized biomimetic electrode interface was rinsed with 1× phosphate buffer and stored for later use.

[0045] (3) Take 100 μL containing 1×10 9 A solution of SK-BR-3 cell-derived EVs per milliliter was added dropwise to the interface of the biomimetic electrode prepared in step (2), and the incubation reaction was carried out to capture the target EVs; the reaction temperature was 37°C and the reaction time was 1.5 hours.

[0046] (4) After thoroughly rinsing the electrode interface obtained in step (3) with 1× phosphate buffer, electrochemical signals were acquired using electrochemical impedance spectroscopy. The specific parameters were: electrochemical probe, 5 mM [Fe(CN)6]. 3- / 4- Initial potential, 0.224V; Frequency range, 0.1Hz to 10kHz.

[0047] Figure 4The Nyquist plot shows the results of characterizing the fabrication of the SK-BR-3 cell membrane-functionalized biomimetic electrode interface and its performance in capturing SK-BR-3 EVs using electrochemical impedance spectroscopy. As shown in the figure, after sequentially incubating the ITO electrode surface with SK-BR-3 cell membrane vesicles and SK-BR-3 EVs, the diameter of the semicircular portion in the high-frequency region of the Nyquist plot gradually increases, representing a gradual increase in charge transfer resistance at the electrode interface. This is consistent with our expectations, confirming the successful immobilization of the cell membrane and the successful capture of SK-BR-3 EVs, as both can form a gradually increasing steric hindrance effect at the interface, leading to [Fe(CN)6]... 3- / 4- The resistance to electron transfer between the electrodes gradually increases.

[0048] Example 4 The electrochemical analysis of non-small cell lung cancer EVs based on biomimetic electrode interfaces follows these steps: (1) Extraction of tumor cell membrane vesicles, the specific process is as follows: take 1 mL of vesicles with a concentration of 1×10 7 Non-small cell lung cancer (NSCLC) cells A549 per mL were placed in a microtube and centrifuged at 1500 rpm for 3 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. This centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (20 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, 2 mM MgCl2, and 1% protease / phosphatase inhibitor). The resulting solution was frozen at -80°C for 2 minutes and then rapidly transferred to 25°C to thaw for 2 minutes. This freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10 minutes and centrifuged at 700 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000 g for 30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate, which is the tumor cell membrane vesicle, was resuspended in 1 mL of 1× phosphate buffer for storage.

[0049] (2) Preparation of the biomimetic electrode interface: First, the ITO electrode was ultrasonically cleaned sequentially with acetone, ethanol, and double-distilled water. Then, the electrode was placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide, and double-distilled water in a volume ratio of 1:1:6 and treated at 85°C for 30 minutes. After treatment, the electrode surface was thoroughly rinsed with double-distilled water, and 50 μL of the tumor cell membrane vesicle resuspension obtained in step (1) was added to the electrode surface and incubated at 50°C for 30 minutes. Finally, the obtained tumor cell membrane functionalized biomimetic electrode interface was rinsed with 1× phosphate buffer and stored for later use.

[0050] (3) Take 100 μL of solution containing different concentrations of A549 cell-derived EVs and add it to the interface of the biomimetic electrode prepared in step (2). Incubate the reaction to achieve the capture of the target EVs. The reaction temperature is 37℃ and the reaction time is 1.5 hours. After the reaction is completed, use 1× phosphate buffer to thoroughly rinse the electrode surface.

[0051] (4) Take 100 μL of 1 μM methylene blue labeled EGFR aptamer probe (sequence SEQ ID NO.2 is 5'-methylene blue-C6 spacer-TACCAGTGCGATGCTCAGTGCCGTTTCTTCTCTTTCGCTTTTTTTGCTTTTGAGCATGCTGACGCATTCGGTTGAC-3') and drop it onto the interface of the biomimetic electrode prepared in step (3) to react, so that the probe is labeled onto the EVs surface by specific binding with a specific marker; the reaction temperature is 25℃ and the reaction time is 1.5 hours; after the reaction, the electrode surface is thoroughly rinsed with 1× phosphate buffer, and the electrochemical signal is collected by square wave voltammetry. The specific parameters are: scanning potential, -0.6V to -0.2V; amplitude, 25mV.

[0052] Figure 5 The results of using a biomimetic electrode interface based on A549 cell membrane functionalization for electrochemical analysis of A549 EVs are presented. Figure 5 A shows the effect of this method on the same concentration (1×10⁻⁶). 9 Square wave voltammetric responses of A549 EVs (particles / mL) and control EVs (SK-BR-3 EVs and MCF-7 EVs) were measured. The results showed that the signals generated by the control EVs (including SK-BR-3 EVs and MCF-7 EVs) were similar to those of the blank control group, indicating that the method has high specificity for A549 EVs. Figure 5 B further indicates that at 1×10 3 granules / ml to 1×10 9 Within the particle / mL concentration range, the square wave voltammetric peak current increases with increasing A549 EVs concentration, exhibiting a good concentration dependence. In summary, this method enables specific electrochemical quantitative analysis of A549 EVs.

[0053] Example 5 The steps for capturing and analyzing tumor EVs in clinical plasma samples based on the A549 cell membrane functionalized biomimetic electrode interface are as follows: (1) Extraction of A549 cell membrane vesicles, the specific process is as follows: take 1 mL of vesicles with a concentration of 1×10 7A549 cells / mL were placed in a microtube and centrifuged at 1500 rpm for 3 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 1 mL of ice-cold 1× phosphate buffer. The centrifugation and resuspension process was repeated 3 times. After the last centrifugation, the supernatant was discarded, and the precipitate was resuspended in 1 mL of hypotonic lysis buffer (20 mM Tris-HCl buffer, pH 7.5, containing 10 mM KCl, 2 mM MgCl2, and 1% protease / phosphatase inhibitor). The resulting solution was frozen at -80°C for 2 minutes and then rapidly transferred to 25°C to thaw for 2 minutes. The freeze-thaw process was repeated 5 times. The solution was then sonicated on ice for 10 minutes and centrifuged at 700 g for 10 minutes at 4°C. After centrifugation, the supernatant was collected and centrifuged at 20000 g for 30 minutes at 4°C. Finally, the supernatant was discarded, and the resulting precipitate was the A549 cell membrane vesicle, which was resuspended in 1 mL of 1× phosphate buffer for later use.

[0054] (2) Preparation of the biomimetic electrode interface: First, the ITO electrode was ultrasonically cleaned sequentially with acetone, ethanol and double-distilled water; then, the electrode was placed in 500 μL of a mixed solution of 28% ammonia, 30% hydrogen peroxide and double-distilled water in a volume ratio of 1:1:6 and treated at 85°C for 30 minutes; after treatment, the electrode surface was thoroughly rinsed with double-distilled water, and 50 μL of the A549 cell membrane vesicle resuspension obtained in step (1) was added to the electrode surface and incubated at 50°C for 30 minutes. Finally, the obtained A549 cell membrane functionalized biomimetic electrode interface was rinsed with 1× phosphate buffer and stored for later use.

[0055] (3) Obtaining clinical plasma samples: After obtaining approval from the Ethics Committee of Shanghai University, peripheral blood samples (3 mL each, EDTA anticoagulated) were collected from 15 patients with non-small cell lung cancer, 15 patients with breast cancer and 10 healthy volunteers. All peripheral blood samples were centrifuged at 2500g for 15 minutes at room temperature within 2 hours after collection. The uppermost plasma layer was carefully aspirated and transferred to a new sterile centrifuge tube. Subsequently, the samples were centrifuged again for 15 minutes under the same conditions (room temperature, 2500g) to obtain high-purity plasma samples.

[0056] (4) Take 10 μL of the clinical plasma sample obtained in step (3) and mix it with 90 μL of 1× phosphate buffer. Then, drop the mixed solution onto the interface of the biomimetic electrode prepared in step (2) and incubate the reaction to capture the target tumor EVs in the sample. The reaction temperature is 37℃ and the reaction time is 1.5 hours. After the reaction is completed, use 1× phosphate buffer to thoroughly rinse the electrode surface.

[0057] (5) Take 100 μL of 1 μM methylene blue labeled EGFR aptamer probe (sequence SEQ ID NO.2 is 5'-methylene blue-C6 spacer-TACCAGTGCGATGCTCAGTGCCGTTTCTTCTCTTTCGCTTTTTTTGCTTTTGAGCATGCTGACGCATTCGGTTGAC-3') and drop it onto the interface of the biomimetic electrode prepared in step (4) to react, so that the probe is labeled onto the EVs surface by specific binding with a specific marker; the reaction temperature is 25℃ and the reaction time is 1.5 hours; after the reaction, the electrode surface is thoroughly rinsed with 1× phosphate buffer, and the electrochemical signal is collected by square wave voltammetry, with the following parameters: scan potential, -0.6V to -0.2V; amplitude, 25mV.

[0058] Figure 6 The results of using a biomimetic electrode interface based on A549 cell membrane functionalization in clinical plasma sample analysis are presented. The results show that the square wave voltammetric peak current value of non-small cell lung cancer patients is significantly higher than that of breast cancer patients and healthy controls. These results not only demonstrate that the A549 cell membrane functionalized biomimetic electrode interface can effectively distinguish non-small cell lung cancer patients from other groups, but also confirm the practical application value of the biomimetic electrode interface of this invention in the analysis of complex clinical samples.

[0059] Comparative Example 1 To objectively evaluate the analytical sensitivity of the method of the present invention, its detection limit was compared with that of various high-sensitivity detection methods for tumor EVs reported in recent literature. The results are shown in Table 1.

[0060] Table 1 Comparison of detection limits for electrochemical / electrochemiluminescence analysis methods for tumor extracellular vesicles

[0061] As shown in Table 1, the method established in this invention achieves a detection limit of 269 particles / mL without using complex enzymatic or nucleic acid cascade amplification strategies, which is lower than all the comparative methods listed in the table. This directly proves that the present invention can achieve efficient capture of tumor extracellular vesicles (EVs) through a biomimetic electrode interface, and has significant advantages in improving the sensitivity of tumor EV detection.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic electrode interface, characterized in that, The preparation method includes the following steps: Cell membrane vesicles are extracted from target tumor cells; the cell membrane vesicles are modified onto the surface of an electrode substrate to form a biomimetic electrode interface for capturing and analyzing extracellular vesicles of homologous tumor cells.

2. The preparation method according to claim 1, characterized in that, The method for extracting cell membrane vesicles includes the following steps: subjecting target tumor cells to hypotonic lysis and repeated freeze-thaw cycles, followed by differential centrifugation to obtain purified cell membrane vesicles.

3. The preparation method according to claim 2, characterized in that, The conditions for the repeated freeze-thaw treatment are: freezing at -80°C to -75°C for 1-2 minutes, followed by thawing at 22°C to 25°C for 1-2 minutes; repeat this cycle 3-5 times.

4. The preparation method according to claim 1, characterized in that, The modification method includes the following steps: adding cell membrane vesicle resuspension to the pretreated electrode surface and incubating at 50°C to 60°C for 30-35 minutes.

5. The biomimetic electrode interface is prepared by the preparation method according to any one of claims 1-4.

6. A method for capturing and analyzing extracellular vesicles of tumor cells using the biomimetic electrode interface described in claim 5, characterized in that, The method includes the following steps: The sample containing extracellular vesicles of the target tumor cells is brought into contact with the biomimetic electrode interface, so that the extracellular vesicles of the target tumor cells are captured. Electroactive molecular probes are used to label captured tumor extracellular vesicles; signals are obtained through electrochemical detection technology to achieve qualitative or quantitative analysis of target tumor extracellular vesicles.

7. The method according to claim 6, characterized in that, The electroactive molecular probe includes a recognition unit and a signal reporting unit; the recognition unit is a nucleic acid aptamer, antibody, or affinity polypeptide that specifically binds to markers on the surface of extracellular vesicles of tumor cells.

8. The method according to claim 6, characterized in that, The electrochemical detection techniques include AC impedance voltammetry, square wave voltammetry, or differential pulse voltammetry.

9. The method according to claim 6, characterized in that, The target tumor cells are selected from breast cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, or colorectal cancer cells.

10. The use of the biomimetic electrode interface of claim 5, or the method of any one of claims 6-9, in the preparation of products for tumor diagnosis or prognostic assessment.

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