Preparation method and application of conductive nanofiber membrane

Abstract

This invention relates to the field of sensing technology, providing a method for preparing and applying a conductive nanofiber membrane. The preparation method includes: preparing a polycaprolactone / β-cyclodextrin / graphene composite nanofiber membrane by electrospinning; after amination treatment, sequentially constructing a conductive catalytic interface on its surface through gold seed adsorption and in-situ growth of gold nanowires; integrating the resulting functionalized membrane into the working electrode region of a three-electrode chip, and further bonding it with PDMS microfluidic channels to construct a biomimetic chamber. This invention combines a cell culture interface with an electrochemical sensing interface, enabling in-situ, real-time, and dynamic electrochemical monitoring of nitric oxide released by vascular endothelial cells in a microfluidic system simulating a physiological environment, providing an innovative technical means for real-time assessment of vascular endothelial function and related pathological research.

Description

Technical Field

[0001] This invention relates to the field of sensing technology, and more specifically, to a method for preparing and applying a conductive nanofiber membrane. Background Technology

[0002] Cardiovascular diseases (CVDs) are one of the leading causes of death and disability worldwide, seriously endangering public health. Studies have shown that although the etiology of cardiovascular diseases is complex, their occurrence and development are often accompanied by continuous changes in vascular structure and function, among which arteriosclerosis is the most core and common pathological feature.

[0003] As organs that dynamically regulate blood flow and substance exchange, blood vessels possess excellent elasticity and compliance in a healthy state, maintaining stable blood delivery. However, under the long-term influence of factors such as aging, adverse metabolic environment, and inflammatory stimulation, the elasticity of the blood vessel walls gradually decreases, the lumen narrows, and structural remodeling occurs. Arteriosclerosis, especially the pathological process represented by atherosclerosis, is the common pathological basis for various cardiovascular and cerebrovascular events such as coronary heart disease, stroke, and peripheral artery disease.

[0004] Numerous studies have confirmed that endothelial dysfunction is a key early event in the occurrence and progression of arteriosclerosis. Under the stimulation of multiple factors such as abnormal blood flow shear stress, metabolic stress, oxidative stress, and immune inflammation, endothelial cells shift from a homeostatic state to an activated or damaged state, which in turn triggers a series of chain reactions, including decreased endothelial diastolic function, enhanced inflammatory response, migration and proliferation of vascular smooth muscle cells, and extracellular matrix remodeling, ultimately leading to plaque formation, vascular wall stiffening, and functional impairment.

[0005] It is noteworthy that arteriosclerosis progresses slowly and is highly reversible in its early stages. Therefore, identifying and intervening in early endothelial function changes in arteriosclerosis is of great significance in reducing the risk of cardiovascular events. However, current clinical and experimental research still lacks sensitive detection methods and functional indicators that can reflect the state of endothelial function in real time.

[0006] Nitric oxide (NO) is a key signaling molecule secreted by vascular endothelial cells, playing a central role in maintaining vasodilation, inhibiting platelet aggregation, and regulating vascular homeostasis. Changes in its release level are considered important indicators for assessing endothelial cell functional status and related vascular pathological changes (such as arteriosclerosis and endothelial dysfunction). Therefore, developing a technique for real-time, in-situ detection of NO release from cells under simulated physiological conditions has significant research value and application prospects.

[0007] Currently, research on the electrochemical detection of NO mainly focuses on the construction and performance optimization of electrode materials. For example, Shi Wang et al. proposed a thin-film electrode for NO detection and its preparation method. By constructing an electrochemically active functional thin film on the surface of a conductive substrate, NO undergoes electrochemical oxidation on the electrode surface, thereby achieving detection. This technology has certain advantages in terms of material design and electrochemical response, but its detection is mainly for solution systems. It does not combine the electrode with the interface of live cell culture, nor does it consider the simulation of the real microenvironment during the NO release process from cells. Therefore, it is difficult to achieve in-situ, dynamic detection of cell-derived NO.

[0008] Furthermore, Hui Liu et al. proposed a porous membrane electrode structure with catalytic and separation functions. By constructing a porous conductive membrane to improve reactant mass transfer efficiency and enhance electrochemical signal output, it exhibited good performance in gas or small molecule detection. However, this technology focuses on the optimization of electrode structure and material function, and its system is not integrated with microfluidic or cell culture systems. It cannot simulate the vascular endothelial microenvironment in a controlled fluid environment, nor can it achieve real-time monitoring of the NO release process after cell stimulation.

[0009] In summary, current technologies for the electrochemical detection of NO still have the following shortcomings: firstly, they lack a working electrode interface that combines cell culture functionality with electrochemical detection capabilities; secondly, they lack an integrated detection system capable of simulating the vascular endothelial microenvironment and achieving controllable fluid conditions. Therefore, how to achieve in-situ, real-time monitoring of NO release from cells while ensuring detection sensitivity is of significant theoretical and practical value for revealing the mechanisms of early endothelial dysfunction in arteriosclerosis, identifying functional biomarkers of disease progression, and evaluating the effectiveness of interventions. Summary of the Invention

[0010] To overcome the shortcomings of the prior art, the first objective of this invention is to provide a method for preparing a conductive nanofiber membrane, comprising the following steps: Step S1: Preparation of composite spinning solution: A composite spinning solution is prepared using polycaprolactone, β-cyclodextrin and graphene as raw materials; Step S2: Preparation of conductive nanofiber membrane: Conductive nanofiber membrane is prepared by electrospinning using the composite spinning solution obtained in step S1 as raw material.

[0011] Compared with existing technologies, the preparation method employed in this invention involves combining polycaprolactone, β-cyclodextrin, and graphene, and then using electrospinning technology to prepare a conductive nanofiber membrane with a three-dimensional porous structure. The surface of the fiber membrane constructed by this method facilitates endothelial cell adhesion and growth, while its conductive network provides an effective reaction interface for the electrochemical oxidation of nitric oxide, thus forming an integrated substrate material that combines cell culture support and electrochemical sensing functions.

[0012] In one possible implementation, step S1 is performed as follows: Step S11: Dissolve polycaprolactone in an organic solvent to obtain a polymer solution; Step S12: Add β-cyclodextrin to the polymer solution and stir until homogeneous to obtain a composite polymer solution; Step S13: Graphene is uniformly dispersed in the composite polymer solution by mechanical stirring and ultrasonic dispersion to obtain a composite spinning solution.

[0013] Compared with existing technologies, in this invention, polycaprolactone is used as the film-forming matrix in step S11, providing structural support and biocompatibility for the fibers; in step S12, β-cyclodextrin is introduced as a hydrophilic modifying component, effectively improving the wettability of the fiber membrane surface and facilitating subsequent cell culture; in step S13, graphene is uniformly dispersed through a combination of mechanical stirring and ultrasonic dispersion, forming a continuous conductive network within the fibers, laying the foundation for constructing an electrochemically active sensing interface. This composite solution system is a key prerequisite for achieving subsequent spinning and obtaining nanofiber membranes with both cell compatibility and conductive functions.

[0014] In one possible implementation, step S2 is specifically operated as follows: the composite spinning solution is injected into a syringe, a metal spinning needle is connected and installed on an electrospinning device; a conductive plate is used as a receiving device, and electrospinning is performed under the action of an external high voltage electric field, so that the composite spinning solution forms continuous nanofibers under the traction of the electric field and is deposited on the surface of the receiving device. After spinning is completed, the nanofibers are removed and dried to obtain a conductive nanofiber membrane.

[0015] Compared with existing technologies, the above-mentioned technical solution employs high-voltage electrospinning technology to stretch, refine, and solidify the composite spinning solution under a strong electric field, ultimately depositing a nanofiber membrane with a three-dimensional interconnected network structure onto a conductive plate receiving device. This method is direct, and the resulting fiber membrane possesses excellent porosity and structural stability. Its unique biomimetic topology provides an ideal physical support interface for the adhesion and growth of endothelial cells, forming the basis for subsequent cell culture and in-situ electrochemical detection.

[0016] The second objective of this invention is to provide a method for preparing a conductive nanofiber film with a surface modified with gold nanowires, specifically comprising: Preparation of conductive nanofiber membranes; Surface-modified nanowires: First, the pretreated conductive nanofiber membrane is immersed in a gold seed suspension. After a first immersion treatment, a gold seed layer is formed on the surface. Then, it is immersed in a gold nanowire growth solution for a second immersion treatment to obtain a conductive nanofiber membrane with gold nanowires on its surface.

[0017] Compared with existing technologies, this step, based on the already prepared conductive nanofiber membrane, introduces gold seeds and gold nanowire growth solution sequentially via a stepwise impregnation method, successfully growing gold nanowire structures in situ on the surface of the fiber membrane. The composite interface constructed by this method retains the three-dimensional porous morphology and biocompatibility of the nanofiber membrane, while significantly enhancing the electrochemical response capability of the interface through the highly conductive and catalytically active gold nanowires. This achieves integrated functionality of the sensing material in both cell culture support and nitric oxide electrochemical detection.

[0018] In one possible implementation, the gold seed suspension is prepared by the following method: chloroauric acid is used as the gold source and sodium citrate is used as the stabilizer, both dissolved in a solvent to form a mixed solution; then sodium borohydride is added to the mixed solution as a reducing agent, and the mixture is subjected to rapid reaction at low temperature while being stirred to obtain the gold seed suspension.

[0019] Compared with existing technologies, this invention uses chloroauric acid as the gold source and sodium citrate as a stabilizer, and prepares a gold seed suspension through a rapid reduction reaction with sodium borohydride, a strong reducing agent, at low temperatures. The addition of sodium citrate effectively prevents the aggregation of nanoparticles, while the low temperature and stirring operation help control the reaction rate and particle size uniformity, thereby obtaining a gold seed suspension with good dispersibility and high stability, providing a reliable guarantee for the subsequent formation of a uniform and dense gold seed adsorption layer on the fiber membrane surface.

[0020] In one possible implementation, the gold nanowire growth solution is prepared by the following method: chloroauric acid as a gold precursor, mercaptobenzoic acid as a linking / stabilizing molecule, and L-ascorbic acid as a reducing agent are dissolved together in a solvent and stirred evenly to obtain the gold nanowire growth solution.

[0021] Compared with existing technologies, this invention prepares a gold nanowire growth solution by dissolving chloroauric acid, mercaptobenzoic acid, and L-ascorbic acid in a mixed solvent. Mercaptobenzoic acid, as a linker molecule, guides the directional growth and assembly of gold atoms, while L-ascorbic acid, as a mild reducing agent, controls the reduction rate to achieve controllable growth of gold nanowires. This formulation provides the necessary chemical environment and reaction driving force for the in-situ, directional growth of gold nanowires on gold seeds on the surface of a fiber membrane.

[0022] In one possible implementation, the pretreatment step is as follows: after oxygen plasma treatment, the conductive nanofiber membrane is immersed in an organic solution containing 3-aminopropyltriethoxysilane, amino functional groups are introduced on the surface, and then the membrane surface is cleaned and dried.

[0023] Compared to existing technologies, this pretreatment step first cleans and activates the nanofiber membrane surface through oxygen plasma treatment, increasing its surface energy and hydrophilicity, and introducing active groups. Subsequently, immersion in an APTES solution chemically bonds amino functional groups to the activated fiber membrane surface. The introduction of amino groups makes the fiber membrane surface positively charged, enabling efficient and uniform adsorption of negatively charged gold seeds via electrostatic interactions, laying a crucial surface chemical foundation for the subsequent uniform growth of gold nanowires.

[0024] The second objective of this invention is to provide a three-electrode detection chip, comprising an insulating substrate and a working electrode, a reference electrode, and a counter electrode disposed on the insulating substrate, wherein the surface of the working electrode is loaded with a conductive nanofiber film with gold nanowires modified on the surface obtained by the above preparation method.

[0025] Compared to existing technologies, this three-electrode detection chip uses a conductive nanofiber membrane with gold nanowires modified on its surface as a functional interface, directly loading it onto the working electrode region of a traditional three-electrode system. This design integrates the originally separate cell culture substrate and electrochemical sensing electrode into one, creating an integrated working electrode that can directly support endothelial cell growth and perform in-situ electrochemical capture of its metabolite nitric oxide, thus forming an integrated detection core component for biological systems.

[0026] The third objective of this invention is to provide an electrochemical detection method for nitric oxide, the electrochemical detection method specifically comprising the following steps: Step S1: Prepare a saturated nitric oxide solution; Step S2: Use phosphate buffer solution with a pH of 7.4 to perform gradient dilution of the saturated solution prepared in S1 to obtain working solutions of nitric oxide with different concentrations. Step S3: Introduce the nitric oxide working solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal to obtain a standard curve between the oxidation current response and the nitric oxide concentration. Step S4: Introduce the nitric oxide test solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal. Obtain the concentration of the nitric oxide test solution through the standard curve obtained in step S3. In steps S3 and S4, the working electrode surface is loaded with a conductive nanofiber film with gold nanowires modified on the surface, prepared by the above method.

[0027] Compared with existing technologies, this method first prepares a saturated nitric oxide stock solution under simulated physiological pH conditions and obtains a series of standard solutions through gradient dilution, establishing a standardized sample pretreatment procedure. Then, using the functionalized working electrode, the standard solutions are detected by amperometric method in a physiological buffer system, and a standard curve of concentration versus current response is constructed by recording the oxidation current signal. Finally, quantitative analysis of unknown samples is performed based on this curve. This method combines a stable material interface, a detection system simulating a physiological environment, and a standardized electrochemical analysis procedure, achieving sensitive, stable, and quantitative detection of nitric oxide.

[0028] The fourth objective of this invention is to provide an application of a conductive nanofiber membrane with gold nanowires modified on its surface, prepared by the above method, in evaluating the function of vascular endothelial cells. The invention is characterized by integrating a working electrode loaded with the conductive nanofiber membrane with a microfluidic structure to construct a biomimetic vascular chamber. During endothelial cell culture, the functional status or response to stimuli is evaluated by detecting the nitric oxide signal released by the cells.

[0029] Compared to existing technologies, this application integrates a working electrode loaded with a functionalized conductive nanofiber membrane with a PDMS microfluidic channel, constructing a closed, fluid-controlled biomimetic vascular chamber. Within this chamber, endothelial cells can be directly cultured on the sensing interface and subjected to various stimuli under simulated physiological or pathological shear stress environments induced by microfluidics. This method, through real-time, in-situ monitoring of changes in the electrochemical signal of nitric oxide release after cell stimulation, can dynamically and directly assess the functional state of endothelial cells and their stress response under simulated pathological conditions, providing an innovative real-time monitoring method for vascular function research.

[0030] Compared with the prior art, the present invention has the following advantages: (1) Achieve stable and controllable detection of nitric oxide under simulated physiological conditions. This invention uses phosphate-buffered saline (PBS) at pH 7.4 as the nitric oxide detection system, simulating the human physiological environment and effectively avoiding interference from strong acids, strong bases, or organic solvents on the stability of nitric oxide and its subsequent biological applications. Under these conditions, by applying a predetermined potential to the working electrode, nitric oxide undergoes a repeatable electrochemical oxidation reaction on the surface of the conductive nanofiber membrane, ensuring the physiological relevance of the detection environment and the reliability of the results.

[0031] Compared to non-physiological systems, the current signal obtained by this invention is closer to the behavioral characteristics of nitric oxide in real biological systems, and is suitable for subsequent cell or tissue-related research.

[0032] (2) Conductive nanofiber membranes significantly improve the electrochemical response sensitivity of nitric oxide. This invention selects a conductive nanofiber membrane with a high specific surface area and a continuous conductive network structure as the sensitive interface of the working electrode. On the one hand, this structure provides a large number of effective reaction sites for nitric oxide molecules; on the other hand, the nanoscale conductive pathway shortens the electron transport path and reduces the interfacial charge transfer impedance.

[0033] Under the same potential conditions, the present invention can achieve a higher response current and a better signal-to-noise ratio compared to planar electrodes, which is beneficial for the quantitative detection of low concentrations of nitric oxide.

[0034] (3) Rapid and real-time detection of nitric oxide based on electrochemical methods This invention employs the amperometric / differential pulse voltammetry method for nitric oxide detection. The current signal responds instantly to changes in nitric oxide concentration, eliminating the need for complex pretreatment or labeling steps. By recording steady-state or transient current changes and comparing them with a standard curve, quantitative analysis of nitric oxide concentration can be directly achieved.

[0035] Compared with traditional detection methods such as fluorescence and colorimetry, this invention has the advantages of fast response speed, strong real-time performance, and high instrument integration.

[0036] (4) The detection system is stable, with good repeatability and scalability. This invention ensures the consistency of initial nitric oxide concentration across different batches of experiments through a standardized process for preparing saturated nitric oxide solution, deoxygenation treatment, and working solution dilution. Furthermore, the conductive nanofiber membrane electrode fabrication process is stable and can be reused or produced in batches.

[0037] This is beneficial for establishing a reliable nitric oxide concentration-current response relationship, meeting the requirements for repeatability and consistency in scientific research and engineering applications.

[0038] (5) Provides a technical basis for the detection of nitric oxide release from cells and the study of vascular function. The electrochemical detection method for nitric oxide established in this invention can be directly extended to the in situ or quasi-in situ detection of nitric oxide release from cells, providing reliable technical support for assessing endothelial function, the degree of vascular sclerosis and related pathological changes.

[0039] It has good potential for application expansion and can be used in areas such as biosensors, organ-on-a-chip, and drug evaluation. Attached Figure Description

[0040] Figure 1 This is one of the schematic diagrams of a nitric oxide biosensor based on a nanofiber membrane; Figure 2 This is the second schematic diagram of the structure of a nitric oxide biosensor based on a nanofiber membrane; Figure 3This is the third schematic diagram of the structure of a nitric oxide biosensor based on a nanofiber membrane; Figure 4 This is one of the top views of the PDMS layer microchannel structure; Figure 5 This is the second top view of the PDMS layer microchannel structure; Figure 6 This is a top view of the electrode layer structure. Detailed Implementation

[0041] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0042] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0043] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0044] The first aspect of this invention provides a method for preparing a conductive nanofiber membrane, primarily addressing the problem of stable in-situ, real-time detection of nitric oxide released from endothelial cells. To solve this problem, this invention proposes directly constructing a conductive nanofiber membrane as an endothelial cell culture interface to maintain good cell compatibility, enabling electrochemical capture and detection of nitric oxide released from endothelial cells near its production site, thereby achieving in-situ, real-time response to nitric oxide. The conductive nanofiber membrane is prepared by the following method: Step S1: Preparation of spinning solution: Polycaprolactone (PCL) was selected as the matrix material and dissolved in an organic solvent to prepare a uniform and transparent polymer solution. β-cyclodextrin (β-CD) was added to the polymer solution as a hydrophilic modifier, and the solution was stirred thoroughly at room temperature to ensure uniform dispersion and form a stable composite polymer solution. Subsequently, graphene (Gr) was introduced into the composite polymer solution as a functional filler. Graphene was uniformly dispersed in the solution system through a combination of mechanical stirring and ultrasonic dispersion to obtain a composite spinning solution for electrospinning.

[0045] Step S2, Preparation of Conductive Nanofiber Membrane: The composite spinning solution is injected into a syringe, connected to a metal spinning needle, and mounted on an electrospinning device. Using a conductive plate as a receiving device, electrospinning is performed under an applied high-voltage electric field, causing the composite spinning solution to form continuous nanofibers under the electric field's pull, which are then deposited on the surface of the receiving device to prepare a conductive nanofiber membrane. After spinning, the obtained conductive nanofiber membrane is removed from the receiving device and dried under environmental conditions to remove residual solvent, obtaining a structurally stable conductive nanofiber membrane culture interface.

[0046] The inventors characterized the hydrophilicity of the conductive nanofiber membrane surface: The surface wetting properties of the conductive nanofiber membrane were tested using a contact angle meter. Water droplets were added to the surface of the fiber membrane, and the hydrophilicity level of the fiber membrane was evaluated by measuring the contact angle of the water droplets on the membrane surface. The hydrophilicity modification effect was analyzed by comparing the contact angle results of fiber membranes prepared from different material systems.

[0047] The inventors described the inoculation and culture of endothelial cells from the prepared conductive nanofiber membrane: The prepared conductive nanofiber membrane was used as the cell culture interface and placed in a culture container after routine sterilization. Human umbilical vein endothelial cells (HUVECs) were selected and inoculated onto the surface of the conductive nanofiber membrane, and cultured under cell culture conditions. The adhesion, spreading, and growth of cells on the fiber membrane surface were observed at different culture time points.

[0048] Example 1 This embodiment provides a specific method for preparing conductive nanofiber membranes, which includes the following steps: S1. Preparation of polymer solution before spinning: Weigh 0.5 g of polycaprolactone granules and dissolve them in 4.5 mL of hexafluoroisopropanol. Place the mixture on a magnetic stirrer and stir continuously at room temperature for 2 hours until the PCL is completely dissolved, forming a homogeneous and transparent solution.

[0049] Add 0.07 g of β-cyclodextrin powder to the above PCL solution. Continue stirring overnight (at least 12 hours) at room temperature to ensure that β-CD is fully dispersed and mixed evenly with the PCL solution, forming a β-CD / PCL mixed solution.

[0050] Measure an appropriate amount of graphene powder and add it to the β-CD / PCL mixed solution, ensuring that the mass-volume concentration of graphene in the final mixed solution is 0.1% (w / v). Stir the mixture for 1 hour, and then place it in an ultrasonic cleaner for ultrasonic treatment for 30 minutes to further disperse the graphene and reduce its agglomeration, finally obtaining a uniformly dispersed Gr / β-CD / PCL electrospinning precursor solution.

[0051] S2. Electrospinning film formation: Take about 1 mL of the prepared Gr / β-CD / PCL spinning solution and put it into a 5 mL medical syringe equipped with a 20G flat-tipped needle.

[0052] Fix the syringe to the electrospinning machine and adjust the vertical distance between the needle tip and the flat aluminum foil receiver to 15 cm.

[0053] The spinning voltage was set to +17 kV, and the spinning process was propelled by gravity. All spinning processes were conducted at room temperature, with the relative humidity of the environment controlled.

[0054] Spinning continued for approximately 40 minutes to form a uniform conductive nanofiber film on the receiver aluminum foil. After spinning, the aluminum foil with the attached fiber film was placed in a fume hood and allowed to air dry at room temperature for 24 hours to allow residual solvents to completely evaporate. The dried conductive nanofiber film was carefully peeled off from the aluminum foil to obtain a self-supporting Gr / β-CD / PCL composite conductive nanofiber film for later use.

[0055] The second aspect of this invention provides a method for preparing a conductive nanofiber film with a surface modified with gold nanowires, specifically including the following steps: S1. Preparation of gold seed suspension: Chloroauric acid (HAuCl4) was used as the gold source, and sodium citrate was used as a stabilizer. The chloroauric acid was dissolved in a solvent to form a mixed solution. Sodium borohydride (NaBH4) was then added as a reducing agent to the mixed solution, and the reaction was carried out rapidly at low temperature. Stirring was used to promote the complete reaction, resulting in a well-dispersed gold seed suspension. The obtained gold seed suspension was stored at low temperature for later use.

[0056] S2. Preparation of gold nanowire growth solution: Chloroauric acid (HAuCl4) was used as a gold precursor, mercaptobenzoic acid (MBA) was added as a linker / stabilizer, and L-ascorbic acid (L-AA) was introduced as a mild reducing agent. The above components were dissolved in a mixed solvent of water and ethanol to prepare a growth solution for gold nanowire growth.

[0057] S3. Pretreatment and Surface Functionalization of Conductive Nanofiber Membranes: The conductive nanofiber membranes were subjected to oxygen plasma treatment using a plasma treatment device to improve the hydrophilicity and activity of the scaffold surface. Subsequently, the treated fiber membranes were immersed in an organic solution containing 3-aminopropyltriethoxysilane (APTES) to introduce amino functional groups into the scaffold surface, thereby achieving surface functionalization. After treatment, the membrane surface was cleaned and dried.

[0058] S4. Deposition of Gold Seeds: The surface-functionalized conductive nanofiber membrane is immersed in the gold seed suspension, allowing the negatively charged gold seeds to be adsorbed onto the positively charged amino functional groups on the surface through electrostatic attraction, thereby forming a uniformly distributed gold seed layer on the surface of the conductive nanofiber membrane. After deposition, the membrane surface is cleaned and dried.

[0059] S5. Growth of gold nanowires on the surface of a conductive nanofiber membrane: A conductive nanofiber membrane loaded with gold seeds is immersed in the gold nanowire growth solution. Under the action of a reducing agent, the gold precursor in the growth solution is reduced on the surface of the gold seeds and gradually deposited and grown to form a continuous gold nanowire (Au nanowires, AuNWs) structure. Mercaptobenzoic acid participates in the growth and assembly process of the gold nanowires as a linking or stabilizing molecule. After growth is complete, the conductive nanofiber membrane is removed, rinsed with an organic solvent to remove residual reactants, and dried to obtain a conductive nanofiber membrane with a surface modified with gold nanowires.

[0060] Example 2 This embodiment provides a specific method for preparing a conductive nanofiber film with a surface modified with gold nanowires, which specifically includes the following steps: S1. Preparation of gold seed suspension: Prepare 20 mL of an aqueous solution containing 0.25 mM chloroauric acid and 0.5 mM sodium citrate in a 50 mL Erlenmeyer flask.

[0061] Take another 2 mL of deionized water and dissolve an appropriate amount of sodium borohydride in an ice bath to prepare a 6 mM NaBH4 aqueous solution (prepare fresh before use).

[0062] Under vigorous stirring, a pre-cooled NaBH4 solution was rapidly injected into the above chloroauric acid-sodium citrate mixture. The solution immediately turned wine-red, indicating the formation of gold nanoparticles (gold seeds).

[0063] After stirring for another 5 minutes, transfer the resulting gold seed suspension to a sample vial and store it in a 4°C refrigerator for later use (shelf life not exceeding one week).

[0064] S2. Preparation of gold nanowire growth solution: Weigh 6 mM chloroauric acid, 1 mM mercaptobenzoic acid and 30 mM L-ascorbic acid, and dissolve them together in a mixed solvent of water and ethanol. The volume ratio of water to ethanol in the mixed solvent is 1.2:1.

[0065] Stir until all reagents are completely dissolved to obtain a gold nanowire growth solution.

[0066] S3. Surface pretreatment and functionalization of fiber membrane: The conductive nanofiber membrane prepared in Example 1 was cut to the required size and placed in an oxygen plasma cleaner for 40 seconds to improve the hydrophilicity of the membrane surface and introduce active groups.

[0067] The plasma-treated fiber membrane was immersed in a 5 mM APTES ethanol solution for 2 hours at room temperature. This step aims to introduce amino functional groups onto the fiber surface. After immersion, the fiber membrane was gently rinsed three times with anhydrous ethanol to remove physically adsorbed APTES molecules, and then dried with nitrogen gas.

[0068] S4. Gold seed adsorption and in-situ growth of gold nanowires: The surface-aminated fiber membrane was immersed in a gold seed suspension and incubated at 4°C for 2 hours. The negatively charged gold seeds were adsorbed onto the positively charged aminated fiber surface through electrostatic attraction.

[0069] Remove the fiber membrane, gently rinse it three times with deionized water, and dry it with nitrogen to obtain a fiber membrane loaded with gold seeds.

[0070] The gold-seeded fiber membrane was immersed in a prepared growth solution and reacted at room temperature for 5 minutes. During this process, L-ascorbic acid reduced Au in the solution. 3+ They are then directionally deposited and grown on gold seeds to form gold nanowires.

[0071] After the growth reaction was complete, the fiber membrane was removed and thoroughly rinsed with anhydrous ethanol and deionized water in sequence to remove residual reactants and byproducts. Finally, it was dried with nitrogen gas. This yielded an AuNWs nanofiber membrane with uniformly loaded gold nanowires on its surface.

[0072] A third aspect of this invention provides a three-electrode detection chip, comprising an insulating substrate and a working electrode, a reference electrode, and a counter electrode disposed on the insulating substrate. The surface of the working electrode is loaded with a conductive nanofiber film with gold nanowires surface-modified, prepared by the above-described method. Specifically, a miniaturized circular disk array structure of a three-electrode independent detection system, with the working electrode, reference electrode, and counter electrode constructed entirely of homogeneous gold material, achieves stable and repeatable electrochemical detection of nitric oxide.

[0073] The three-electrode system uses borosilicate glass (BF33) as an insulating substrate and integrates a complete three-electrode detection unit on a single chip, including: Working electrode (WE): Located in the central region of the chip, it has a circular disk-like structure and is used to support subsequent biofunctionalization materials and serve as the detection interface for the electrochemical oxidation reaction of nitric oxide. The working electrode is connected to an external pad via an independent metal lead for accessing the working electrode interface of the electrochemical workstation.

[0074] Reference electrode (RE): This electrode employs an arc-shaped strip structure, arranged around the working electrode and maintaining an insulating distance from it. It is used to form a stable reference electrode layer in subsequent processing. The reference electrode is also connected to the corresponding pad via independent leads.

[0075] Counter electrode (CE): A ring structure is positioned outside the working electrode, forming a symmetrical electric field system with the working and reference electrodes to provide a stable current loop. The counter electrode is connected to the corresponding pad via an independent lead.

[0076] The lead paths of the three electrodes are isolated from each other to avoid crossing and short circuits; all pads are arranged linearly along the edge of the substrate to facilitate subsequent probe contact or wire bonding.

[0077] More specifically, this embodiment also provides a method for fabricating the above-mentioned three-electrode detection chip: S1. Substrate Cleaning and Pretreatment: A borosilicate glass substrate was selected as the substrate and chemically cleaned using a strong oxidizing cleaning solution to remove surface organic contaminants and activate the substrate surface. After cleaning, the substrate was rinsed with deionized water, dried with nitrogen, and then heat-treated to remove residual moisture.

[0078] S2. Photoresist spin coating and pre-baking: The treated glass substrate is placed on a spin coater and positive photoresist is spin-coated onto its surface to form a uniform photoresist layer; then the substrate is placed on a heated plate for pre-baking to remove solvents from the photoresist and enhance adhesion.

[0079] S3. One-step photolithography of three-electrode patterns: A photomask containing a complete working electrode, reference electrode, counter electrode, and alignment marks is used to expose the photoresist-coated substrate in an ultraviolet lithography machine, allowing the desired three-electrode areas to be patterned. After exposure, the substrate is placed in a developing solution to remove the photoresist in the exposed areas, forming the patterned windows corresponding to the three electrodes.

[0080] S4. Magnetron sputtering deposition of metal thin films: The developed substrate is placed in a magnetron sputtering deposition system. The substrate surface is first cleaned, followed by the sequential deposition of a metal adhesion layer and a gold functional layer to form a continuous, dense metal thin film structure. The substrate is rotated during deposition to ensure uniform metal layer thickness.

[0081] S5. Stripping Process and Electrode Forming: The deposited substrate is placed in an organic solvent, and the metal layer in the non-electrode areas is removed by a stripping process, retaining only the three-electrode structure located within the pattern window. After stripping, the substrate is cleaned and dried sequentially to obtain the complete three-electrode pattern.

[0082] S6. Post-processing and chip separation: The prepared triple-metal electrode substrate is heat-treated to release the internal stress of the thin film and enhance the adhesion between the metal layer and the glass substrate. Subsequently, a precision dicing machine is used to separate the entire substrate along a preset cutting path to obtain a single independent triple-metal electrode detection chip.

[0083] S7. Surface treatment is performed on the reference electrode region to form a stable reference electrode layer; at the same time, AuNWs modified nanofiber membrane is introduced into the working electrode region to construct a nitric oxide electrochemical sensing interface.

[0084] Example 3 This embodiment provides a specific method for constructing a three-electrode system, which includes at least a working electrode, a reference electrode, and a counter electrode. The working electrode is an electrode structure loaded with a conductive nanofiber membrane with gold nanowires modified on its surface, obtained in Example 2. The reference electrode and the counter electrode are disposed in an electrolyte solution and together with the working electrode, form a complete electrochemical detection circuit.

[0085] S1, Three-electrode circular disc structure design This embodiment employs a "three-electrode independent detection system + miniaturized circular disk array" structure. Using 4-inch borosilicate glass (BF33) as the substrate, a single chip integrates a complete three-electrode detection channel, including a working electrode (WE), a reference electrode (RE), and a counter electrode (CE), all of which are made of gold (Au) material, forming a homogeneous integrated electrochemical sensing interface.

[0086] Working electrode (WE): Located in the central region of the chip, it has a circular disk structure with a diameter of 10.00 mm; it extends to the edge of the substrate through a 2.5 mm wide metal lead and connects to an independent 3 mm × 3 mm Au pad for accessing the WE interface of the electrochemical workstation.

[0087] Reference electrode (RE): It has an arc-shaped strip structure, which is arranged around WE and maintains an insulating gap of 2 mm with the edge of WE; the arc length is 2.47 mm and the width is 3 mm; it is connected to a 3 mm × 3 mm independent Au pad through a 2.5 mm wide metal lead for subsequent construction of Ag / AgCl reference layer.

[0088] Counter electrode (CE): It has an outer ring structure, which is arranged around the outside of WE and maintains an insulation gap of 2 mm with the edge of WE; the inner diameter of the ring is 14.00 mm and the outer diameter is 20.00 mm; it is connected to a 3 mm × 3 mm independent Au pad through a 2.5 mm wide metal lead.

[0089] The lead paths of the three electrodes are isolated from each other, the pads are arranged linearly along the edge of the substrate, and the center-to-center spacing between adjacent pads is 4.5 mm, which avoids short circuits and facilitates subsequent bonding or probe station testing.

[0090] S2, Substrate Cleaning and Pretreatment A 4-inch BF33 glass substrate was immersed in freshly prepared Piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3:1, volume ratio) at 120°C for 15 min to remove organic contaminants and activate the surface. After treatment, it was rinsed thoroughly with deionized water until neutral, dried with nitrogen, and then dried in a 120°C oven for 30 min before use.

[0091] S3, Photoresist Spin Coating and Pre-Baking The pretreated substrate was placed on a spin coater and a positive photoresist AZ5214 was spin-coated at 3000 rpm for 30 s to form a uniform photoresist layer with a thickness of about 1.5 μm. Then, it was pre-baked on a hot plate at 95°C for 60 s to remove residual solvent.

[0092] S4, Exposure and Development (Three-electrode simultaneous patterning) A photomask containing the complete structure of WE, RE, CE and alignment marks is used for hard contact alignment with the resist-coated substrate. Exposure is performed using 365 nm ultraviolet light in a lithography machine with an exposure energy of 100 mJ / cm². 2 The exposure time is approximately 8 seconds.

[0093] After exposure, the substrate was immersed in AZ 400K developer (diluted with deionized water at a ratio of 1:4) for 40 seconds. After development, it was immediately rinsed with deionized water and dried with nitrogen to obtain the patterned window of the three electrodes.

[0094] S5, Magnetron Sputtering Deposition of Metal Layer The developed substrate was placed into a magnetron sputtering system and backwashed for 5 min under an argon atmosphere. The following metal layers were then deposited sequentially: Cr adhesive layer: 50 nm thick; Au functional layer: 200 nm thick.

[0095] The substrate is kept rotating during sputtering to ensure the uniformity and consistency of the metal film.

[0096] S6, Lift-off and Tri-electrode Forming The sputtered substrate was immersed in acetone solution and sonicated for 20–30 min to remove the photoresist and the overlying metal layer in the non-patterned areas, leaving only the three-electrode structure. It was then rinsed sequentially with fresh acetone, ethanol, and deionized water, and dried with nitrogen gas.

[0097] S7, Post-processing and Chip Separation The prepared electrode array substrate was placed in a rapid annealing furnace and annealed at 300°C for 30 min under a nitrogen protective atmosphere to eliminate sputtering stress and enhance the adhesion between the gold layer and the glass substrate. Subsequently, a dicing machine was used with a resin grinding wheel blade to dice along a preset cutting path to separate individual independent three-gold disk-type three-electrode sensor chips.

[0098] This invention also provides an electrochemical detection method for nitric oxide, which specifically includes the following steps: Step S1: Prepare a saturated nitric oxide solution; Step S2: Use phosphate buffer solution with a pH of 7.4 to perform gradient dilution of the saturated solution prepared in S1 to obtain working solutions of nitric oxide with different concentrations. Step S3: Introduce the nitric oxide working solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal to obtain a standard curve between the oxidation current response and the nitric oxide concentration. Step S4: Introduce the nitric oxide test solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal. Obtain the concentration of the nitric oxide test solution through the standard curve obtained in step S3. In steps S3 and S4, the working electrode surface is loaded with a conductive nanofiber film with gold nanowires modified on the surface, as prepared in Example 2.

[0099] Example 4 This embodiment provides a specific electrochemical detection method for nitric oxide. A nitric oxide solution is prepared using phosphate-buffered saline (PBS, pH 7.4) simulating physiological conditions. By applying a predetermined potential to the working electrode, nitric oxide undergoes an electrochemical oxidation reaction on the surface of a conductive nanofiber membrane. The corresponding current signal is collected, and a quantitative relationship is established between the current signal and the nitric oxide concentration. The specific steps include: S1. System deoxygenation pretreatment (key step): Before introducing NO gas, use high-purity nitrogen (N2) to continuously purge the PBS solution, purification and collection pipeline system for 30 min to thoroughly remove dissolved oxygen and residual oxygen in the system and prevent NO from being oxidized to NO2 during the preparation process as much as possible.

[0100] Preparation of S2 and NO saturated solutions: 99.9% pure NO gas at a pressure of 3 MPa was passed into PBS (pH 7.4) and the aeration was continued for 30 min at room temperature to obtain a NO saturated PBS solution. After the aeration was completed, the solution was kept under a NO atmosphere for future use as a nitric oxide stock solution.

[0101] S3. Storage of NO saturated solution: Transfer the NO saturated stock solution to a sealed glass bottle with a rubber septum and store it under light-protected conditions to reduce the rate of spontaneous oxidation and decomposition of NO.

[0102] S4. Preparation of NO working solutions of different concentrations: The NO saturated stock solution obtained in step S3 was serially diluted with PBS (pH 7.4) that had been pre-treated with nitrogen deoxygenation to prepare nitric oxide working solutions of different concentrations for subsequent electrochemical detection experiments.

[0103] S5. Electrochemical detection and quantitative analysis of nitric oxide: The NO working solution is introduced into a three-electrode electrochemical detection system. The working electrode modified with a conductive nanofiber membrane is used as the sensing interface. Under PBS simulated physiological conditions, a predetermined potential is applied to the working electrode to cause an electrochemical oxidation reaction of nitric oxide on the electrode surface. The corresponding oxidation current signal is collected, and a quantitative relationship between the oxidation current response and the nitric oxide concentration is established.

[0104] The present invention also provides an application of a conductive nanofiber membrane with gold nanowires on its surface in evaluating the function of vascular endothelial cells. The working electrode loaded with the conductive nanofiber membrane is integrated with a microfluidic structure to construct a biomimetic vascular chamber. During the endothelial cell culture process, the functional status or response to stimuli is evaluated by detecting the nitric oxide signal released by the cells.

[0105] Example 5 like Figures 1-6 As shown in the figure, this embodiment specifically illustrates the application of a conductive nanofiber membrane with a surface modified with gold nanowires in evaluating vascular endothelial cell function, including the following: Cell culture and detection are performed simultaneously. The core technology involves directly culturing endothelial cells on a working electrode made of gold nanowires (AuNWs) functionalized nanofiber membranes. This electrode is bonded to PDMS microfluidic channels to form a closed, biomimetic vascular chamber. Microfluidic technology allows for precise control of the fluid environment, dynamically introducing pathological stimuli such as Ox-LDL or drugs to simulate the atherosclerotic process. NO released by stimulated cells is instantly captured by the sensing interface beneath the cells and converted into an electrical signal. The entire process requires no cell destruction or sample transfer, enabling stable, continuous, and in-situ real-time monitoring of endothelial cell NO release dynamics under pathophysiological conditions.

[0106] Preparation of the patterned male mold for metal mold S1. Selection and pretreatment of mold material High-strength stainless steel or aluminum alloy is selected as the mold substrate. First, the substrate is precision ground and polished to make its surface flatness (Ra) better than 0.1 µm. Then, it is ultrasonically cleaned with acetone, ethanol and deionized water in sequence to remove oil and particulate matter, and then dried with nitrogen gas for later use.

[0107] S2. Precision machining of the mold cavity: Based on the graphic design of the target microchannel, detection chamber, and inlet / outlet, precision CNC milling technology is used to directly machine a recessed cavity with complementary geometry onto the pre-treated metal substrate. During the machining process, a micro-diameter milling cutter (up to 0.1 mm in diameter) and optimized cutting parameters are used to ensure that the perpendicularity of the cavity sidewalls and the surface roughness of the bottom surface meet the requirements for microchannel replication.

[0108] S3. Surface Treatment of the Mold: The machined mold cavity is mirror-polished to further reduce its surface roughness (Ra < 0.025 µm). Subsequently, a low surface energy anti-stick coating is applied to the cavity surface using physical vapor deposition technology to significantly improve the subsequent demolding performance of PDMS and extend the mold's service life.

[0109] (II) Fabrication of PDMS microfluidic layer S4. PDMS Preparation and Degassing: Mix PDMS prepolymer and curing agent at a ratio of 10:1 (by mass), stir thoroughly, and then place in a vacuum dryer for degassing for 40 minutes until no obvious bubbles are visible.

[0110] S5. Pouring and Curing: Slowly pour the degassed PDMS mixture onto the surface of the male mold of the metal mold to completely cover the graphic structure; then place it in an 80°C oven for 1 hour to complete curing.

[0111] S6. Peeling and Drilling: After the PDMS cools, peel it off from the metal mold male to obtain a PDMS layer with a recessed microchannel structure; use a biopsy drill with a diameter of 2 mm to drill holes at the inlet and outlet to form liquid channel interfaces.

[0112] (III) Integration and bonding of PDMS microchannels and three-electrode chips S7. Surface Plasma Activation: The PDMS microfluidic layer (channel surface) and the three-metal electrode chip (electrode surface) are simultaneously placed in an oxygen plasma treatment machine and treated under the same conditions for 45 s to generate hydroxyl groups on the surface and enhance hydrophilicity.

[0113] S8. Precise alignment and permanent bonding: After plasma treatment, the PDMS microchannel and the three-electrode chip are precisely aligned under a microscope with the help of alignment marks, so that the detection chamber completely covers the working electrode area modified with AuNWs-nanofiber membrane; after bonding is completed, irreversible covalent bonding is achieved to form a closed biomimetic blood vessel chamber.

[0114] (iv) Realization of biomimetic blood vessel function S9. Endothelial Cell Culture and Real-time NO Detection: The prepared endothelial cell suspension is injected into the chip's culture chamber at the desired density and directly cultured on the surface of the AuNWs functionalized nanofiber membrane working electrode, forming a stable endothelial layer under microfluidic conditions. Ox-LDL or drug stimulants are dynamically introduced through the microfluidic system to simulate the pathological process of arteriosclerosis. Nitric oxide (NO) released by the stimulated cells is instantly captured by the electrochemical sensing interface below at its production location and converted into an electrical signal, enabling real-time monitoring of the vascular endothelial functional status.

[0115] S10. Three-Electrode Chip and Overall Microfluidic Structure Design: The microfluidic chip is made of PDMS (polydimethylsiloxane) material and includes, in sequence, an inlet, a microchannel, a detection chamber, and an outlet. The microchannel and the three-electrode chip are precisely aligned using alignment marks and then bonded together as a single device after plasma treatment, constructing a closed, blood vessel-like cavity structure. The working electrode, modified with AuNWs functionalized nanofiber membrane, is located at the bottom of the detection chamber, serving as a biomimetic culture interface for endothelial cells and a NO sensing interface.

[0116] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for preparing a conductive nanofiber membrane, characterized in that, The preparation method specifically includes the following steps: Step S1: Preparation of composite spinning solution: A composite spinning solution is prepared using polycaprolactone, β-cyclodextrin and graphene as raw materials; Step S2: Preparation of conductive nanofiber membrane: Conductive nanofiber membrane is prepared by electrospinning using the composite spinning solution obtained in step S1 as raw material.

2. The preparation method according to claim 1, characterized in that, The specific operation of step S1 is as follows: Step S11: Dissolve polycaprolactone in an organic solvent to obtain a polymer solution; Step S12: Add β-cyclodextrin to the polymer solution and stir until homogeneous to obtain a composite polymer solution; Step S13: Graphene is uniformly dispersed in the composite polymer solution by mechanical stirring and ultrasonic dispersion to obtain a composite spinning solution.

3. The preparation method according to claim 1, characterized in that, The specific operation of step S2 is as follows: inject the composite spinning solution into the syringe, connect the metal spinning needle, and install it on the electrospinning device; use a conductive plate as a receiving device, and perform electrospinning operation under the action of an external high voltage electric field, so that the composite spinning solution forms continuous nanofibers under the electric field traction and is deposited on the surface of the receiving device. After spinning is completed, remove it and dry it to obtain a conductive nanofiber membrane.

4. A method for preparing a conductive nanofiber film with a surface modified with gold nanowires, characterized in that, Prepare conductive nanofiber membranes according to any one of claims 1-3; Surface-modified nanowires: First, the pretreated conductive nanofiber membrane is immersed in a gold seed suspension, and a gold seed layer is formed on the surface after a single immersion treatment. The nanofiber membrane with gold nanowires on its surface is then immersed in a gold nanowire growth solution for a second impregnation treatment to obtain a conductive nanofiber membrane with gold nanowires on its surface.

5. The preparation method according to claim 4, characterized in that, The gold seed suspension was prepared by the following method: chloroauric acid as the gold source and sodium citrate as the stabilizer were dissolved together in a solvent to form a mixed solution; then sodium borohydride was added to the mixed solution as a reducing agent, and the mixture was subjected to rapid reaction at low temperature while being stirred to obtain the gold seed suspension.

6. The preparation method according to claim 4, characterized in that, The gold nanowire growth solution was prepared by the following method: chloroauric acid as a gold precursor, mercaptobenzoic acid as a linking / stabilizing molecule, and L-ascorbic acid as a reducing agent were dissolved together in a solvent and stirred evenly to obtain the gold nanowire growth solution.

7. The preparation method according to claim 4, characterized in that, The pretreatment steps are as follows: after oxygen plasma treatment, the conductive nanofiber membrane is immersed in an organic solution containing 3-aminopropyltriethoxysilane, amino functional groups are introduced on the surface, and then the membrane surface is cleaned and dried.

8. A three-electrode detection chip, comprising an insulating substrate and a working electrode, a reference electrode, and a counter electrode disposed on the insulating substrate, characterized in that, The surface of the working electrode is loaded with a conductive nanofiber membrane prepared by any of the preparation methods described in claims 4-7.

9. An electrochemical detection method for nitric oxide, characterized in that, The electrochemical detection method specifically includes the following steps: Step S1: Prepare a saturated nitric oxide solution; Step S2: Use phosphate buffer solution with a pH of 7.4 to perform gradient dilution of the saturated solution prepared in S1 to obtain working solutions of nitric oxide with different concentrations. Step S3: Introduce the nitric oxide working solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal to obtain a standard curve between the oxidation current response and the nitric oxide concentration. Step S4: Introduce the nitric oxide test solution into the three-electrode electrochemical detection system. Using the working electrode as the sensing interface, apply a predetermined potential to the working electrode and collect the corresponding oxidation current signal. Obtain the concentration of the nitric oxide test solution through the standard curve obtained in step S3. In steps S3 and S4, the working electrode surface is loaded with a conductive nanofiber membrane prepared by any of the preparation methods described in claims 4-7.

10. The application of a conductive nanofiber membrane prepared by any one of claims 4-7 in evaluating vascular endothelial cell function, characterized in that, By integrating the working electrode loaded with the conductive nanofiber membrane with the microfluidic structure, a biomimetic blood vessel chamber is constructed. During endothelial cell culture, the functional status or response to stimuli is evaluated by detecting the nitric oxide signal released by the cells.

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