Electrochemical nanosensor for detecting nitric oxide in macrophages and preparation method and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to an electrochemical nanosensor for detecting NO in macrophages and a preparation method and application thereof. The electrochemical nanosensor comprises a working electrode and gold nanoparticle layers, platinum nanoparticle layers and 5-amino-1-naphthol polymer layers successively modified on the surface of the working electrode; the 5-amino-1-naphthol polymer layers have a porous structure and can selectively permeate NO molecules and block interfering substances. The application constructs an electrochemical nanosensing platform for in-situ detection of NO in single cells, which is used for evaluating the polarization phenotype of macrophages. Meanwhile, the platform is applied to a co-culture system of macrophages and cancer cells under different stress states, and the regulation of cell immunogenic death and ferroptosis on the polarization of macrophages is discussed, thereby providing technical support and experimental basis at the single cell level for the research on the tumor immune microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to an electrochemical nanosensor for detecting NO in macrophages, its preparation method, and its application. Background Technology

[0002] As core immune cells in the tumor microenvironment, macrophages' dynamic polarization of the M1 / M2 phenotype directly determines their immunosuppressive or antitumor effects. Nitric oxide (NO), as a key effector molecule, is an important marker for distinguishing the activation state of macrophages.

[0003] Existing NO detection technologies (such as the Griess method, fluorescent probes, chemiluminescence, and electron paramagnetic resonance) are widely used in bioanalysis, but none can simultaneously achieve directness, high spatiotemporal resolution, and in vivo applicability. The Griess method can only indirectly reflect the overall level of a population of cells; the fluorescence method is limited by photobleaching and inaccurate quantification; and chemiluminescence and paramagnetic resonance technologies cannot meet the real-time monitoring needs at the single-cell level due to expensive instruments, complex pretreatment, and difficulty in miniaturization. Although electrochemical methods have the potential for direct response and miniaturization, they still face the core contradiction of balancing cell damage and detection stability. Microelectrode insertion can easily trigger cellular stress responses, interfering with normal metabolic activities. At the same time, the complex reducing environment inside the cell can easily lead to electrode surface contamination and signal drift. Moreover, existing studies are mostly limited to the extracellular or pericellular regions, and truly deep intracellular direct measurements with low damage, interference resistance, and good reproducibility are still extremely scarce, limiting their application in resolving delicate biological processes such as macrophage polarization heterogeneity. Summary of the Invention

[0004] The purpose of this invention is to provide an electrochemical nanosensor for NO detection in macrophages, its preparation method, and its application, thereby overcoming the shortcomings of existing technologies. This invention constructs an electrochemical nanosensor for in-situ NO detection in single cells, enabling in-situ NO detection and analysis in single cells and providing a novel detection tool for macrophage polarization evaluation. This research combines nanosensing technology with tumor immune microenvironment research, providing a new tool for rapidly assessing macrophage polarization status.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention is an electrochemical nanosensor for detecting NO in macrophages, comprising a working electrode and a gold nanoparticle layer, a platinum nanoparticle layer and a 5-amino-1-naphthol (5A1N) polymer layer sequentially modified on its surface; the 5-amino-1-naphthol polymer layer has a porous structure and can selectively permeate NO molecules while blocking interfering substances.

[0006] The gold-platinum bimetallic layer not only significantly reduces the oxidation overpotential of NO, but also greatly improves the electron transport rate and sensitivity; while the outer 5-amino-1-naphthol polymer (PAN), with its unique porous network structure, effectively repels interfering substances such as ascorbic acid and dopamine, while ensuring the rapid and selective permeation of NO.

[0007] In other embodiments, the working electrode is a carbon fiber nanoelectrode (CFNE); the carbon fiber nanoelectrode has needle-like nanotippies with a diameter of 100-200 nm. By using a carbon fiber substrate with a tip diameter of only 100-200 nm as the working electrode, this sensor can minimize physical damage to cells to maintain their activity and overcome the limitations of existing population detection technologies.

[0008] Secondly, the present invention provides a method for preparing an electrochemical nanosensor for detecting NO in macrophages, comprising the following steps: A gold-platinum bimetallic layer modified electrode was prepared by electrochemically depositing gold nanoparticle layers and platinum nanoparticle layers sequentially on the surface of carbon fiber nanoelectrodes. An electrode modified with a gold-platinum bimetallic layer was immersed in a 5-amino-1-naphthol deposition solution, and a 5-amino-1-naphthol polymer layer was formed on the electrode surface by cyclic voltammetry.

[0009] By utilizing the stepwise deposition of Au / Pt bimetallic layers, a highly conductive network was constructed, which significantly reduced the NO oxidation overpotential and provided abundant catalytic active sites through the alloying effect. The in-situ polymerized 5-amino-1-naphthol (PAN) porous coating acted as a "molecular sieve," which, while physically blocking large molecular interferences and resisting biofouling, ensured rapid mass transfer of NO by utilizing its pore size advantage.

[0010] In some other embodiments, the carbon fiber nanoelectrode is composed of carbon fiber, copper wire and capillary glass tube. The carbon fiber and copper wire are connected by conductive silver paste. One end of the capillary glass tube is drawn into a pointed shape. Two copper wires with carbon fiber attached are inserted into the flat end of the capillary glass tube in sequence, and the carbon fiber passes out of the pointed end of the capillary glass tube. After the electrode position is adjusted, the flat end is first sealed with resin glue, and then the pointed end is melted and solidified with the outer flame of an alcohol lamp.

[0011] In some other embodiments, the preparation steps of the gold nanoparticle layer are as follows: a three-electrode system is constructed using a carbon fiber nanoelectrode as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode; the working electrode is immersed in a chloroauric acid deposition solution with a concentration of 0.1-1.0 mM, and a chronoamperometry method is used to deposit the gold nanoparticle layer at a constant potential for 10-20 s at a deposition voltage of -0.2 to -0.5 V. For example, the concentration of the chloroauric acid deposition solution is 0.5 mM, the deposition voltage is -0.4 V, and the deposition time is 15 s.

[0012] In some other embodiments, the preparation steps of the platinum nanoparticle layer are as follows: An electrode modified with a gold nanoparticle layer was used as the working electrode and immersed in a chloroplatinic acid deposition solution with a concentration of 0.5-1.5 mM. Cyclic voltammetry was used within a potential window of -0.5 V to 0 V at a rate of 80-120 mV·s. - ¹ The scan rate was cyclically scanned 1-2 times. For example, the concentration of the chloroplatinic acid deposition solution was 1 mM, and the scan rate was 100 mV·s. -1 , scan in a loop once.

[0013] In some other embodiments, during the preparation of the 5-amino-1-naphthol polymer layer, the concentration of the 5-amino-1-naphthol deposition solution is 0.1-1.0 mM, the voltage of the cyclic voltammetry is 0 V-1.0 V, and the scan rate is 8-12 mV·s. -1 Cyclic scanning was performed for 3-6 cycles. For example, the concentration of the 5-amino-1-naphthol deposition solution was 0.5 mM, and the scan rate was 10 mV·s. -1 Scan in a loop for 5 cycles.

[0014] Thirdly, this invention provides the application of an electrochemical nanosensor for detecting NO in macrophages in the construction of an in vitro macrophage polarization model.

[0015] In some other embodiments, the method for constructing an in vitro macrophage polarization model includes the following steps: inducing macrophage polarization, inserting an electrochemical nanosensor for detecting NO within the macrophage into the macrophage, detecting the electrical signal generated by NO in the cell using amperometry or differential pulse voltammetry, determining the NO content based on the signal intensity, thereby distinguishing the polarization state of M1 pro-inflammatory macrophages and M2 immunosuppressive macrophages.

[0016] Fourthly, this invention provides the application of an electrochemical nanosensor for detecting NO within macrophages in the preparation of diagnostic and targeted cancer therapy products, whereby the products are selected from pharmaceuticals, reagent kits, or biosensor devices. The sensor assesses the immune status of the tumor microenvironment by detecting NO levels within tumor-associated macrophages.

[0017] The beneficial effects of this invention are: (1) The present invention modifies the working electrode surface sequentially with a gold nanoparticle layer, a platinum nanoparticle layer and a 5-amino-1-naphthol polymer layer. The Au / Pt bimetal not only accelerates electron transport and reduces the NO oxidation overpotential due to its excellent conductivity and catalytic activity, but also endows the electrode with excellent chemical stability and anti-aging ability, ensuring that it maintains stable performance in long-term complex intracellular monitoring. The outer 5-amino-1-naphthol polymer coating constructs a highly selective barrier through a triple synergistic mechanism of size screening, charge repulsion and hydrophobic interaction, which not only effectively inhibits the interference of electroactive substances such as nitrite, but also significantly improves the sensor's anti-fouling ability and detection reliability in complex biological matrices.

[0018] (2) The preparation method of this invention is simple, mild, and easy to control. The entire process eliminates the cumbersome chemical synthesis and complex post-processing steps, and relies entirely on electrochemical deposition technology to complete the in-situ construction of AuNPs, Pt NPs, and 5AlN polymer layers in an aqueous system at room temperature and pressure in one step. By precisely controlling the electrochemical reaction parameters such as voltage, time, and solution concentration, the morphology and film thickness of the nanomaterials can be precisely customized at the nanoscale without the need for expensive vacuum equipment or photolithography processes.

[0019] (3) The highly selective nanoelectrode prepared in this invention enables in-situ detection and analysis of NO in single cells, providing a novel detection tool for evaluating macrophage polarization. This study combines nanosensing technology with the study of the tumor immune microenvironment, providing a new tool for rapidly assessing macrophage polarization status. Attached Figure Description

[0020] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 The image shows the morphological characterization of Au / Pt / 5AlN / CFNE, where A represents CFNE, B represents Au / CFNE, C represents Au / Pt / CFNE, and D represents Au / Pt / 5AlN / CFNE. Figure 2 The images show the EDX characterization of elements in Au / Pt / 5AlN / CFNE, including the merged EDX of multiple elements (C, Au, Pt, N, and O) in Au / Pt / 5AlN / CFNE and the EDX of individual elements C (red), Au (yellow), Pt (purple), N (green), and O (blue). Figure 3The diagram shows the construction process of Au / Pt / 5AlN / CFNE, where A represents the CV response of CFNE modified with different materials in K3[Fe(CN)6], B represents the statistical results corresponding to A, n = 3, and n represents the number of electrodes used. Error bars represent the mean ± standard deviation. Figure 4 The diagram shows the optimization of experimental conditions. In this diagram, A represents the CV curves of Au / Pt / 5AlN / CFNE modified with different cycle numbers for detecting 100 μM NO, B represents the anti-interference effect of Au / Pt / 5AlN / CFNE modified with different cycle numbers (n = 3), C represents the it response of Au / Pt / 5AlN / CFNE to 100 μM NO at different potentials, and D represents the statistical results corresponding to C (n = 3). Here, n represents the number of electrodes used, and the error bars represent the mean ± standard deviation. Figure 5 The graph shows the electrochemical response of Au / Pt / 5AlN / CFNE to NO. In this graph, A represents the CV curves of Au / Pt / 5AlN / CFNE for NO and PBS; B represents the it response of Au / Pt / 5AlN / CFNE at +0.85 V to different concentrations of NO (1 ~ 13 represent 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 30 μM, 50 μM, 70 μM, 100 μM, 130 μM, 150 μM, 170 μM, 200 μM); C represents the CV response of Au / Pt / 5AlN / CFNE to low concentrations; and D represents the CV response of Au / Pt / 5AlN / CFNE to high concentrations of NO (unit: μM). Figure 6 This chart shows the stability, reproducibility, and selectivity studies of Au / Pt / 5AlN / CFNE. A represents the stability study of 3 Au / Pt / 5AlN / CFNE electrodes over 7 days; B represents the reproducibility study of 4 electrodes; C represents the reproducibility study of 4 batches of electrodes; and D represents the selectivity study of Au / Pt / 5AlN / CFNE. n = 3, where n represents the number of electrodes used. Error bars represent the mean ± standard deviation, and statistical differences are shown below. **** P ≤ 0.0001; Figure 7The diagram shows the characterization of cell viability after electrode insertion. A represents the CV response of Au / Pt / 5A1N / CFNE to Ru(NH3)6Cl3 solution at different depths of insertion into RAW264.7 cells. B represents cell fluorescence imaging before and after Au / Pt / 5A1N / CFNE insertion, after staining with calcein-AM and PI. C represents the comparison of calcein-AM fluorescence intensity before and after Au / Pt / 5A1N / CFNE insertion. D represents the statistical analysis corresponding to C. The inset is a schematic diagram of fiber optic detection (scale bar: 10 μm). n = 7, where n represents the number of randomly selected cells in the experiment. Error bars represent mean ± standard deviation. Statistical difference: ns represents no significant difference. Figure 8 This is a validation image of macrophage polarization. A shows bright-field images of M0, M1, and M2 states; B shows the immunoblotting results of iNOS in RAW264.7 cells treated with different concentrations of LPS; C shows the immunoblotting results of Arg-1 in RAW264.7 cells treated with IL-4 + IL-13 for different time periods; D shows the statistical results of multiple replicates corresponding to C, n = 3, where n represents the number of replicates of an independent experiment. Error bars represent mean ± standard deviation. Statistical differences: * P ≤ 0.05 and ns, no significant difference, E is the immunofluorescence result of iNOS and Arg-1 staining before and after RAW264.7 cell polarization, F is the flow cytometry result of related marker protein expression before and after RAW264.7 cell polarization; Figure 9 This diagram shows the electrochemical response of NO after Au / Pt / 5AlN / CFNE insertion into cells. A represents the current change at different locations within a single cell after Au / Pt / 5AlN / CFNE insertion; B represents the current response of NO in cells M0, M1, and M2; C represents the current response of NO in 30 macrophages in the M0 polarized state; D represents the current response of NO in 30 macrophages in the M1 polarized state; E represents the current response of NO in 30 macrophages in the M2 polarized state; and F represents the statistical results of the current response of NO in 50 macrophages in different polarization states. Error bars represent the mean ± standard deviation. Statistical differences are: * P ≤ 0.05, **** P ≤ 0.0001; Figure 10This is a graph showing the molar rate of NO detected by Au / Pt / 5A1N / CFNE after insertion into cells. A represents the molar rate of NO detected by Au / Pt / 5A1N / CFNE in 30 macrophages with M0 polarization; B represents the molar rate of NO detected by Au / Pt / 5A1N / CFNE in 30 macrophages with M1 polarization; C represents the molar rate of NO detected by Au / Pt / 5A1N / CFNE in 30 macrophages with M2 polarization; and D represents the statistical results of the molar rate of NO detected by Au / Pt / 5A1N / CFNE in 50 macrophages with different polarization states. Error bars represent mean ± standard deviation. Statistical differences are: * P ≤ 0.05, **** P ≤ 0.0001. Detailed Implementation

[0022] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0023] To address the limitations of existing methods in simultaneously achieving directness, selectivity, spatiotemporal resolution, and in vivo applicability, this invention constructs a nanoelectrode for in-situ detection of NO in single cells. Its electrocatalytic activity towards NO is enhanced through material optimization and surface functionalization. The electrode's performance is characterized using NO standard solutions, demonstrating excellent detection performance, along with good anti-interference ability, reproducibility, stability, and cell compatibility. It can specifically recognize NO signals within single cells in complex cellular environments. The successful construction of this electrode provides a reliable technical platform for subsequent dynamic analysis of NO in macrophages.

[0024] Example 1 I. Experimental Materials 1. The experimental reagents and chemicals are shown in Table 1.

[0025] Table 1 Reagents and Medicines

[0026] 2. The experimental instruments and equipment are shown in Table 2.

[0027] Table 2 Experimental Equipment

[0028] 3. Preparation of main experimental reagents / solutions (1) Preparation of 10 mM pH = 7.4 phosphate buffer solution (1×PBS): Weigh 1.45 g of Na2HPO4, 0.27 g of KH2PO4, 8 g of NaCl and 0.2 g of KCl and dissolve them in 800 mL of ultrapure water. Then adjust the pH of the solution with sodium hydroxide solution. Note that the adjustment should be done in small amounts and multiple times. Finally, adjust the pH of the solution to 7.4 and then add ultrapure water to make up to 1000 mL.

[0029] (2) 10 mM potassium ferricyanide (K3[Fe(CN)6]) solution: Weigh 0.1317 g potassium ferricyanide and 0.2982 g potassium chloride into a beaker, then dilute to 40 mL with ultrapure water and stir with a glass rod until completely dissolved. This solution should be prepared fresh for use.

[0030] (3) 5% skim milk powder blocking solution: Weigh 0.5 g of skim milk powder, dissolve it fully with 1×TBST, and finally adjust the volume to 10 mL with 1×TBST. Stir until completely dissolved.

[0031] (4) 5×Running Buffer: Weigh 15.1 g Tris, 94 g glycine, and 5 g SDS, add a small amount of deionized water and stir until the solids are completely dissolved, then bring the volume to 1 L and mix well. It needs to be diluted to 1× for use in experiments and can be stored at room temperature.

[0032] (5) 5× Transfer Buffer: Weigh 15.1 g Tris and 75.1 g glycine, add a small amount of deionized water and stir until the solid is completely dissolved, then bring the volume to 1 L and stir well. It needs to be diluted to 1× before use and stored at 4℃.

[0033] (6) 4% BSA solution: Weigh 0.4 g of BSA powder, dissolve it completely in 1×PBS solution, make up to 10 mL, stir and mix well, and filter it through a 0.45 mm filter membrane filter. Prepare and use immediately or store at -20℃.

[0034] (7) Saturated KCl solution: Weigh a certain amount of KCl and dissolve it in deionized water. Then add KCl while stirring until no more KCl is added, thus obtaining a saturated KCl solution.

[0035] (8) 1 mM Ru(NH3)6Cl3 solution: Weigh 3 mg Ru(NH3)6Cl3 powder and dissolve it in 10 mL KCl solution to obtain 1 mM Ru(NH3)6Cl3 solution.

[0036] II. Experimental Methods 1. Preparation of Au / Pt / 5AlN / CFNE (1) Place the borate glass capillary on a microelectrode drawing instrument and draw it. Adjust the parameters of the drawing instrument to control the tip diameter at 10 ~ 20 μm.

[0037] (2) Cut the copper wire to about 15 cm, then connect the copper wire and carbon fiber with conductive gel, let it dry overnight until completely dry, and then carefully insert it into the borosilicate capillary.

[0038] (3) To etch carbon fibers using flame etching, first seal the glass tube, and then use a microscope to repeatedly observe in order to control the time and position of flame etching, so as to prepare CFNE with a suitable carbon fiber tip size.

[0039] 2. Modification of nanoelectrodes (1) The calcined CFNE was used as the working electrode. A three-electrode system was used. The working electrode, counter electrode and reference electrode were connected to the electrochemical workstation. Au NPs were deposited using the it method. At a deposition voltage of -0.4 V, the CFNE was immersed in 0.5 mM HAuCl4 deposition solution for 15 s to obtain Au NPs modified CFNE (Au / CFNE).

[0040] (2) Platinum nanoparticles (Pt NPs) were then deposited using the CV method. A three-electrode system was used, in which Au / CFNE was immersed in a 1 mM H2PtCl6 deposition solution and one cycle was performed in the range of -0.5 ~ 0 V with a scan rate of 100 mV. s -1 , thus obtaining Au / Pt / CFNE.

[0041] (3) Au / Pt / CFNE was immersed in 0.5 mM 5AlN (5-amino-1-naphthol) deposition solution. A three-electrode system was used, and the CV method was employed to perform 5 cycles in the range of 0 ~ +1 V, with a scan rate of 10 mV·s. 1 Au / Pt / 5A1N / CFNE was obtained.

[0042] 3. Preparation of NO solution The basic principle of NO preparation is the disproportionation reaction of NaNO2 in a strongly acidic solution.

[0043] (1) Before preparing NO, the entire reaction device needs to be purged with N2 under good airtight conditions for at least 20 minutes to remove O2 from the entire device. The entire preparation process needs to be carried out in a fume hood.

[0044] (2) First, assemble the entire apparatus, connect the round-bottom flask to the separatory funnel, add saturated NaNO2 solution to the round-bottom flask, and add 25 mL of 2 M H2SO4 solution to the separatory funnel. During the reaction, the H2SO4 dripping rate is uniform and slow, about 5 to 6 drops per minute, to carry out the disproportionation reaction and ensure the stability and continuous generation of NO gas flow.

[0045] (3) Next, after connecting the gas washing bottle to the round-bottom flask, add 4 M KOH solution to remove NO2 gas. Finally, pass the purified NO gas into a gas collecting bottle containing 10 mL PBS solution to collect and prepare a saturated NO solution. In order to prevent excessive NO gas from diffusing into the environment and causing pollution, a tail gas collecting device (0.4 M KMnO4 + 1.2 M KOH solution) is connected at the end of the entire reaction system.

[0046] (4) The final saturated NO concentration is approximately 1.8 mM. NO standard solutions of different concentrations can be obtained by continuously diluting the saturated NO solution with PBS. To ensure the NO solution is as accurate as possible, the NO solution should be prepared fresh for each use.

[0047] 4. Electrochemical Measurement (1) CFNE, Au / CFNE, Au / Pt / CFNE and Au / Pt / 5AlN / CFNE were prepared according to the above modification method. Then, using a three-electrode system, the four electrodes were immersed in different electrolyte solutions for detection, such as PBS, 10 mM K3 [Fe(CN)6] solution and 100 μM NO solution, to explore the electrochemical response and performance characteristics of the layer-by-layer modified electrodes.

[0048] (2) To further investigate the catalytic performance of the constructed nanoelectrode for NO, the CV method and the it method were used to verify the electrochemical response of Au / Pt / 5AlN / CFNE in NO solutions of different concentrations. When using the it method to investigate the catalytic performance of the electrochemical sensor for NO, a certain amount of NO solution needs to be added to the reaction system every 10 s, so that the final concentration of each addition forms a NO concentration gradient, and the final NO concentration of the entire system reaches the target concentration. At the same time, in order to ensure the uniformity of the addition to the entire reaction system, the experiment was carried out under stirring conditions.

[0049] 5. Cell Culture (1) Cell resuscitation: Before the experiment, the water bath was heated to 37°C. First, the frozen RAW264.7 cells were thawed by heating in the water bath. The cell suspension was transferred to a 15 mL centrifuge tube. 1 mL of 10% complete culture medium (5 mL FBS + 45 mL DMEM) was added to the centrifuge tube and gently pipetted to mix. Then, the cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cell pellet was retained. 1 mL of culture medium was added to resuspend the cells. Finally, the cells were transferred to a culture dish and cultured in a CO2 constant temperature incubator (5% CO2, 37°C).

[0050] (2) Cell passage: For RAW264.7 cells, after the cell confluence rate reaches 80-90%, the culture medium is aspirated, fresh DMEM culture medium is added, the cells are gently blown off the culture dish and collected into a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min, discard the supernatant, add fresh culture medium and gently mix. Add the cells to several culture dishes according to the cell concentration and continue to culture in a CO2 constant temperature incubator.

[0051] (3) Cell cryopreservation: The initial process is the same as cell passage. After centrifugation, discard the supernatant, add 1 mL of cryopreservation solution, mix well by pipetting, transfer to cryopreservation tubes, seal, write the corresponding label, transfer the cryopreservation tubes to cryopreservation boxes, and store at -80℃ or in liquid nitrogen for a long time after 24 hours.

[0052] 6. Immunoblot analysis (1) Preparation of protein samples: After cells were passaged and adhered to the 6-well plate overnight, and the cells were observed to be growing well, 1 μg·mL⁻¹ of protein samples were added to each well. -1 LPS or 20 ng / mL -1 Incubate IL-4 and IL-13 in a CO2 incubator for 24 h. Remove the cell plate, discard the original culture medium, and gently wash the surface with cooled PBS to remove drug residues and cell debris. Add an appropriate amount of pre-prepared cell lysis buffer (lysis buffer: protease inhibitor: phosphatase inhibitor = 100:2:1), and repeatedly pipette on ice for 15 min until complete lysis. Collect the lysate in 1.5 mL Eppendorf tubes, centrifuge at 4°C, 15000 rpm for 15 min, collect the supernatant, discard the precipitate, add SDS buffer, label and store for later use.

[0053] (2) Gel preparation: iNOS has a molecular weight of 130 kDa, and Arg-1 has a molecular weight of 37 kDa, so a 10% stacking gel was prepared. After cleaning the gel plate, the prepared stacking gel was added, and then a small amount of isopropanol was added to flatten the gel surface to prevent unevenness of the stacking gel surface from affecting the bonding with the separating gel. After the stacking gel solidified, an appropriate amount of separating gel was added, and the comb was gently inserted to wait for all the gel to solidify.

[0054] (3) Electrophoresis: Remove the solidified stacking gel and separating gel, place them in the electrophoresis tank and add 1×Running Buffer. Slowly pull out the comb in the buffer to prevent the gel wells from breaking. Add an appropriate amount of pre-prepared protein sample and marker protein. Adjust the voltage and run at 80 V for 30-60 min. When the protein sample reaches the separating gel and the marker bands are clear, adjust the voltage to 120 V to separate all the proteins and make them reach the bottom of the glass plate. The electrophoresis can then be stopped.

[0055] (4) Transfer: Pour the pre-cooled 1×Transfer Buffer into the tray, remove the gel and cut off the excess part, assemble according to the sandwich method, place the gel in the position of "black gel and white membrane", insert it into the transfer tank and add enough 1×Transfer Buffer. Place a lot of ice next to it to prevent the large amount of heat generated during the transfer from damaging the protein structure. Adjust the current to a constant current of 260 mA for the transfer, and set an appropriate time according to the size of the protein.

[0056] (5) Blocking: After transfer, remove the PVDF membrane, cut off the excess, retain the required protein, and wash with 1×TBST for 10 min. Add 5% skim milk powder that has been dissolved in advance to the blocking container to block the non-specific sites on the membrane. Place the membrane on a decolorizing shaker at room temperature for 80 min. After the block is completed, wash the PVDF membrane with 1×TBST multiple times for 10 min each time.

[0057] (6) Incubation with primary antibody: Cut the PVDF membrane according to the protein molecular weight and place them in boxes containing the corresponding diluted antibody. Incubate overnight (about 12 h) at 4°C. After incubation on the second day, wash the membrane multiple times with 1×TBST for 10 min each time.

[0058] (7) Incubation of secondary antibody: Place the PVDF membrane in the corresponding diluted secondary antibody and incubate it at room temperature for 60-90 min. After the incubation, wash it with 1×TBST multiple times for 10 min each time.

[0059] (8) Exposure: Prepare the color development solution at a ratio of 1:1, take 100 μL of the color development solution and drop it evenly onto the PVDF membrane, set an appropriate time, and expose it on a chemiluminescence analyzer.

[0060] 7. Immunofluorescence staining experiment (1) Add the required number of sterile coverslips to a 24-well plate, remove the cells from the incubator, add an appropriate amount of PBS to the laminar flow hood to wash away dead cells and cell debris, centrifuge in the same cell passage steps, collect the precipitate and discard the supernatant, add 1 mL of culture medium to mix the cells and count the cells, take the required cell concentration, add 300 μL of complete culture medium and an appropriate amount of mixed cell suspension to each well of the 24-well plate, and culture overnight.

[0061] (2) After the cells adhere to the plate and are evenly distributed on the coverslip, add the appropriate concentration of drug for treatment. After the appropriate treatment time, remove the 24-well plate, discard the original culture medium, wash three times with pre-cooled PBS, add 300-500 μL of pre-cooled 4% PFA, and fix the cells at room temperature for 20 min. After removing paraformaldehyde, wash three times with PBS for 5 min each time. Prepare 4% BSA with sterile water as blocking solution, add 300-500 μL of blocking solution to each well, and block at room temperature for 60 min.

[0062] (3) Incubation with primary antibody: After blocking, do not wash. Dilute the corresponding antibody with 4% BSA, add 200-300 μL of antibody to each well, and incubate at room temperature for more than 2 hours. After incubation, wash several times with PBS.

[0063] (4) Incubation with fluorescent secondary antibody: Dilute the secondary antibody with 4% BSA, add 200-300 μL of diluted antibody to each well, and incubate at room temperature in the dark for at least 2 hours. All subsequent steps are performed in the dark. After incubation, wash several times with PBS.

[0064] (5) Incubation of DAPI dye: Dilute with PBS at a certain ratio, add 200 ~ 300 μL of diluted DAPI into a 24-well plate, incubate for 3 min, and wash with PBS multiple times.

[0065] 8. Flow cytometry Surface protein staining: (1) Collect cells in advance and seed them into 12-well plates overnight. After the cells adhere and grow well, add the corresponding drug at the preset concentration to each well. After culturing for the required time, digest and collect the cells and wash them twice with PBS.

[0066] (2) Staining: Resuspend the cells in FACS buffer, take 10 µL for cell counting, dilute PEAnti-Mouse CD80 and FITC Plus Anti-Mouse CD86 antibodies according to cell concentration, mix them with the cells and incubate at 4°C in the dark for 30 to 60 min.

[0067] (3) After incubation, wash twice with FACS buffer, discard the supernatant, resuspend the cells with an appropriate amount of FACS buffer, and then perform detection.

[0068] Intracellular protein staining: (1) Collect cells in advance and seed them into 12-well plates overnight. After the cells adhere and grow well, add the corresponding drug at the preset concentration to each well. After culturing for the required time, digest and collect the cells and wash them twice with PBS.

[0069] (2) Fixation: Resuspend the cells in 200-300 µL of fixative and fix them at room temperature in the dark for 10-20 min. After fixation, wash once with FACS buffer and collect the pellet by centrifugation.

[0070] (3) Permeabilization: Add 300 ~ 500 µL of permeabilization solution to each cell sample to resuspend the cells, centrifuge for 5 min, discard the supernatant and collect the precipitate, centrifuge twice, collect the precipitate and add 100 µL of permeabilization solution to resuspend the cells.

[0071] (4) Intracellular protein staining: Cell counts were performed separately. PE Anti-Mouse Arg-1 and APC Anti-Mouse CD206 antibodies were diluted according to cell concentration, mixed with cells, and incubated at 4°C in the dark for 30-50 min.

[0072] (5) After incubation, wash twice with FACS buffer, discard the supernatant, resuspend the cells with an appropriate amount of FACS buffer, and then perform detection.

[0073] 9. Electrochemical detection within single cells Au / Pt / 5AlN / CFNE was used as the working electrode, platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. All electrochemical detections were performed on a vibration-damped table and in a Faraday cage, and all apparatus was grounded. The Au / Pt / 5AlN / CFNE was connected to a micromanipulator, and the cell was placed under an inverted microscope. The Au / Pt / 5AlN / CFNE was moved near the cell using the micromanipulator, its position adjusted to contact the cell membrane, and further advanced until the tip was fully inserted into the cell. Electrochemical detection was performed in the cytoplasm of RAW264.7 cells. Using the iterative method, a working potential of +0.85 V was applied, and all amperometric responses were transmitted to the workstation and recorded by a CHI 630E electrochemical analyzer, obtaining the current I measured at a specific time. According to Faraday's law, the time-dependent formation rate of NO (denoted as f) can be calculated from the time change of a single current, called the molar rate, expressed as ΔI = Fnf. Where F represents the Faraday constant (9,6500 C / mol), and n represents the electron stoichiometry related to electrochemical oxidation on Au / Pt / 5AlN / CFNE (n NO = 1).

[0074] III. Results and Discussion 1. Characterization of Au / Pt / 5AlN / CFNE To improve the electrochemical performance of the nanoelectrode, Au NPs and Pt NPs were modified onto the bare CFNE surface. These noble metal nanoparticles exhibit excellent conductivity, high catalytic activity, and strong stability, resisting oxidation and deactivation, and maintaining stable performance during long-term use. Furthermore, they provide abundant surface active sites and enhance electron transport capabilities, further optimizing the electrode's interfacial properties and ensuring good performance for electrochemical detection. This facilitates rapid and reliable detection applications in complex real-world samples.

[0075] The electrode tip was sealed using flame etching, and its morphology was observed and manipulated under an optical microscope to form a fine conical structure. To gain a deeper understanding of the morphological characteristics of the nanoelectrode tip, scanning electron microscopy (SEM) was used to characterize four types of electrode tips: CFNE, Au / CFNE, Au / Pt / CFNE, and Au / Pt / 5AlN / CFNE. Figure 1 ).like Figure 1 As shown in Figure A, the exposed CFNE tip surface is smooth. The electrode surface modified with Au NPs exhibits dense and uniformly distributed particles. Figure 1 (B in the text); further modification of Pt NPs, the tip surface is completely covered ( Figure 1(C in the text). After modification with 5A1N, a uniform and dense thin film can be observed covering the electrode surface (C in the text). Figure 1 The results (D) confirm that a porous polymer coating was successfully formed on the electrode surface.

[0076] To further investigate the chemical state and microscopic distribution characteristics of each element in Au / Pt / 5AlN / CFNE, SEM-EDX was used to characterize the presence and distribution of various elements in Au / Pt / 5AlN / CFNE. Figure 2 In this composition, carbon (C) is derived from carbon fibers, Au belongs to Au NPs, Pt belongs to Pt NPs, and N and O mainly originate from the 5AlN polymer porous coating. Analysis further confirmed the elemental distribution of Au / Pt / 5AlN / CFNE, showing that C, Au, Pt, N, and O are uniformly distributed across the entire electrode tip. These results demonstrate the successful construction of Au / Pt / 5AlN / CFNE, indicating a significant increase in the electrochemical active sites and catalytic area of ​​the modified electrode, effectively improving the detection sensitivity and catalytic efficiency of the nanoelectrode, and providing strong support for subsequent analysis of NO within single cells.

[0077] 2. Construction and performance optimization of Au / Pt / 5AlN / CFNE Based on morphological characterization, this study constructed an Au / Pt / 5AlN / CFNE that can specifically detect NO. Figure 3 The fabrication process of the nanoelectrode was demonstrated and validated using CV technology. Figure 3 Figure A shows the CV curves in 10 mM K3[Fe(CN)6] during the layer-by-layer modification of the electrode. After modification with Au NPs and Pt NPs, the current signal generated by Au / Pt / CFNE is significantly higher than that of Au / CFNE, and the redox current of K3[Fe(CN)6] is significantly increased, reflecting the synergistic effect of the bimetallic layer, indicating that the conductivity of the electrode is significantly improved after modification with Au and Pt. After modification with 5AlN polymer, the electrochemical response decreases due to the conductivity of 5AlN and charge repulsion, but overall it presents a classic "S"-shaped curve, and the statistical results of multiple electrodes also conform to this trend. Figure 3 (B in the text). This also confirms the successful preparation of Au / Pt / 5AlN / CFNE.

[0078] After constructing Au / Pt / 5AlN / CFNE, the experimental conditions were optimized. Figure 4The optimization mainly includes the selective modification of the electrode and the detection voltage. 5AlN is widely used due to its efficient suppression of nitrite interference. It effectively eliminates interfering substances and improves sensor selectivity through a triple selection mechanism of size screening, charge repulsion, and hydrophobic synergy. Since the polymer film itself has poor conductivity, it affects the electrochemical response of the electrode and increases detection error. Therefore, the number of deposition cycles for the 5AlN polymer film was optimized. Electrodes were modified with 3, 5, and 10 deposition cycles respectively. The electrochemical response of different modified Au / Pt / 5AlN / CFNE in 100 μM NO was verified using CV technology. Figure 4 (A) Next, the anti-interference effects of the modified electrodes with good response for 3 and 5 cycles were compared ( Figure 4 (B) Even though the electrode modified in 5 cycles showed some loss in response current for NO detection, it also showed resistance to interfering substances H2O2 and NO2. - The response of AA decreased significantly, the shielding effect was better, and the anti-interference effect was superior to that of electrodes modified for three cycles. For subsequent testing, Au / Pt / 5AlN / CFNE modified with five cycles of 5AlN polymer was selected.

[0079] Furthermore, the choice of detection voltage is also a crucial factor affecting electrode detection performance. Voltage optimization is performed based on the NO response on the it curve. Figure 4 As can be seen from C, in the range of +0.7 ~ +0.95 V, the current response of the electrode to NO increases with increasing voltage. After statistical analysis of the corresponding currents within this range (…),… Figure 4 In the D section, it was found that the electrode response amplitude changed more rapidly before +0.85 V; after this voltage, the response amplitude changed more slowly. Considering that excessive voltage can easily damage cells, +0.85 V was chosen as the detection voltage for the it method in subsequent experiments.

[0080] 3. Electrochemical response of Au / Pt / 5AlN / CFNE to NO Next, to verify the sensitivity of the constructed Au / Pt / 5AlN / CFNE to NO, freshly prepared saturated NO solution was diluted to create NO standard solutions of different concentrations for subsequent electrochemical detection of NO. First, it was verified that Au / Pt / 5AlN / CFNE exhibited a significant current response to NO, while no similar peak change was observed in PBS solution. Figure 5 (A in the original text). Next, at a potential of +0.85 V, the electrochemical response to changes in NO concentration was investigated using IT techniques. Figure 5(B in the text). As the NO concentration increases, the response signal of Au / Pt / 5AlN / CFNE to NO also gradually increases. Meanwhile, the CV method was also used at low concentrations (…). Figure 5 C in the middle) and high concentration ( Figure 5 The electrochemical peak response of Au / Pt / 5AlN / CFNE was detected in a NO standard solution (D) of the assay. As the NO concentration increased, the peak response of Au / Pt / 5AlN / CFNE also increased. These results indicate that the constructed Au / Pt / 5AlN / CFNE exhibits excellent catalytic activity for NO, demonstrating a clear concentration dependence. This provides a reliable tool for subsequent analysis of NO in single cells.

[0081] 4. Performance verification of Au / Pt / 5AlN / CFNE Next, the stability, reproducibility, and selectivity of Au / Pt / 5AlN / CFNE were verified. To verify the stability of the electrode, the modified electrode was placed for 1–7 days and detected in 100 μM NO. After 5 days, its electrochemical activity still retained more than 90% of the original signal. Figure 6 (A in the original text) On day 7, the electrochemical signal remained at 85%, demonstrating the good stability of the constructed Au / Pt / 5AlN / CFNE. Four other electrodes were used for CV detection of NO, and the resulting electrochemical signals were highly consistent. Figure 6 The relative standard deviation (RSD) was 4.6%, indicating good reproducibility of the electrode. Four batches of electrodes were prepared, and the current values ​​were analyzed and statistically processed at a NO concentration of 100 μM. Figure 6 The results of the tests were basically consistent with those of the C in the test, which again shows that the constructed electrode has good reproducibility.

[0082] The intracellular environment of a single cell is complex, and many substances within it interfere with the detection of NO. Therefore, the selection of electrodes was explored. Cysteine ​​(Cys), L-Arg, glucose (Glc), histidine (His), hydrogen peroxide (H2O2), and nitrite (NO2) were selected respectively. - Common biomolecules such as ascorbic acid (AA) act as interfering agents, and the results are as follows: Figure 6 As shown in Figure D, the electrochemical response of Au / Pt / 5AlN / CFNE to NO is much higher than that of other interfering substances, proving that the constructed Au / Pt / 5AlN / CFNE can be applied to the selective detection of NO.

[0083] 5. Feasibility assessment of single-cell analysis Given the excellent detection performance of Au / Pt / 5AlN / CFNE in NO standard solutions, this paper further applies it to intracellular electrochemical monitoring. Before detecting NO in single cells, the feasibility of using Au / Pt / 5AlN / CFNE for single-cell analysis was first evaluated to verify whether electrode insertion into cells would cause significant cell damage. Since Ru... 3+ Unable to penetrate the intact cell membrane, Ru(NH3)6Cl3 was used as an electrochemical molecular probe. A PBS solution containing 1 mM Ru(NH3)6Cl3 was added to the culture medium to further verify the cell membrane encapsulation effect after electrode insertion. Figure 7 As shown in Figure A, Ru(NH3)6Cl3 exhibited the largest redox current when the electrode was not inserted into the cell (insertion depth of 0%). As the electrode was gradually inserted into the cell, the current response continuously decreased; when the electrode was fully inserted, the current response reached its lowest point. This result indicates that during electrode insertion, the cell membrane effectively coats the electrode surface, maintaining the overall integrity of the cell. This demonstrates that Au / Pt / 5AlN / CFNE possesses good mechanical properties and biocompatibility, meeting the application requirements for electrochemical detection within single cells.

[0084] Cell viability was characterized using commercially available fluorescent probes Calcein-AM and Propidium iodide (PI). Calcein-AM, with its lipophilic methyl acetate group, can cross intact cell membranes to enter living cells and emits green fluorescence after hydrolysis by intracellular esterases. PI, on the other hand, is a nuclear dye that binds to nuclear DNA to produce red fluorescence, but it can only cross damaged cell membranes and cannot enter living cells. Therefore, it is often used in combination with Calcein-AM for cell viability assessment. Figure 7 As shown in Figure B, after staining cells with Calcein-AM and PI, Au / Pt / 5A1N / CFNE was inserted into the cells under the assistance of a micromanipulator and then gently removed. The cells were then observed under a fluorescence microscope. The results showed that all cells exhibited green fluorescence, with no obvious red fluorescence, indicating that the cells were not significantly damaged. These results demonstrate that the insertion of Au / Pt / 5A1N / CFNE into cells does not significantly affect cell viability and exhibits good cell compatibility.

[0085] After observation under an inverted fluorescence microscope, the Au / Pt / 5A1N / CFNE electrode was connected to a real-time in situ single-cell analyzer. The electrode was gently inserted into the cells and then removed. A fiber optic probe was used to quantitatively detect changes in fluorescence intensity and photon count of calcein-AM staining in single cells before and after insertion, to assess the effect of electrode insertion on cell viability. Figure 7As shown in C, the number of fluorescence photons was monitored within 10 seconds before and after electrode insertion, and there was almost no difference between the two; statistical analysis results of multiple cells ( Figure 7 Similarly, D) indicates that there was no significant change in calcein-AM fluorescence intensity before and after Au / Pt / 5A1N / CFNE insertion. These results demonstrate that Au / Pt / 5A1N / CFNE has minimal impact on cell viability when used for single-cell analysis, allowing for analysis while maintaining normal cell physiological conditions.

[0086] 6. Construction of an in vitro macrophage polarization model Macrophages, as core effector cells of the innate immune system, play a crucial role in the body's inflammatory response, tissue repair, and disease progression. Different microenvironmental signals can induce macrophages to differentiate into opposite polarization phenotypes, with the anti-tumor M1 type and the pro-tumor M2 type being the most typical. In vitro induction of directed macrophage polarization is a common method for elucidating activation mechanisms and screening regulatory molecules at the cellular level. M1 macrophages highly express iNOS, thereby catalyzing the production of large amounts of NO from substrates, while M2 macrophages typically express low levels of iNOS and tend to express Arg-1, producing almost no NO. Therefore, changes in NO levels serve as a key biomarker for assessing macrophage polarization status, providing important evidence for determining macrophage polarization and studying subsequent immune regulatory mechanisms.

[0087] First, RAW264.7 cells were induced to form different macrophage polarization states in vitro, and preliminary judgment was made based on changes in cell morphology. For example... Figure 8 As shown in Figure A, M0 cells in the resting state are mostly round, small in size, and have a low degree of spreading. After LPS stimulation, the cell volume increases significantly, and pseudopodia-like extension occurs, resulting in an overall spreading state, which is significantly different from M0 cells. After co-stimulation with IL-4 and IL-13, the cell boundaries are smoother, the cell body widens, and the cells become spindle-shaped or fusiform. Subsequently, the expression of iNOS and Arg-1, which are related to NO synthesis, was detected by Western blotting to determine the optimal induction conditions. According to literature reports, M1 macrophages are often induced by LPS stimulation for 24 h, therefore, 0.1, 0.5, 1, 5, and 10 μg / mL were used. -1 Macrophages were treated with LPS for 24 h. Immunoblotting results showed ( Figure 8 In the control group (B), iNOS expression was almost nonexistent, while it gradually increased with increasing LPS concentration; when the LPS concentration was higher than 1 μg / mL... -1 At that time, the increase in iNOS expression was not significant, therefore 1 μg / mL was selected. -1 LPS was used as the induction conditioned condition for M1 macrophages. For M2 polarization, referring to relevant reports, 20 ng / mL was selected. -1Induction was achieved using IL-4 + IL-13. Immunoblotting results showed that ( Figure 8 In the control group (C), Arg-1 expression was low, while after 20 ng / mL... - 1 After 24 h of treatment with IL-4 and IL-13, Arg-1 expression significantly increased, but the change was not significant after 48 h; the statistical results of subsequent repeated experiments ( Figure 8 D) in the data also confirms this trend. Therefore, 20 ng / mL was ultimately determined. -1 Treatment with IL-4 and IL-13 for 24 h was the induction condition for M2 macrophages.

[0088] Next, immunofluorescence was used to further verify the expression levels of iNOS and Arg-1 in RAW264.7 cells induced by LPS or IL-4+IL-13 for 24 h. Green 488 dye was used to label iNOS, and red 647 dye was used to label Arg-1. Figure 8 As shown in Figure E, LPS-induced macrophages tended towards M1 polarization, characterized by upregulated iNOS expression and downregulated Arg-1 expression; while after induction with IL-4+IL-13, macrophages tended towards M2 polarization, characterized by upregulated Arg-1 expression and downregulated iNOS expression. Furthermore, in addition to iNOS and Arg-1, M1 macrophages also exhibited high expression of CD80 and CD86, while CD206 expression was significantly increased in M2 macrophages. Therefore, flow cytometry was further used to validate the relevant biomarkers. The results showed ( Figure 8 In the control group (F), after LPS induction, the positive rates of M1 markers CD80 and CD86 in macrophages were significantly higher than those in the control group; after induction with IL-4 and IL-13, cells specifically expressed high levels of M2 markers Arg-1 and CD206. These results were consistent with the results of immunofluorescence and Western blotting analysis, further demonstrating the successful induction of macrophage polarization.

[0089] 7. Nanoelectrodes for in-situ detection of NO in single macrophages Building upon the successful construction of Au / Pt / 5A1N / CFNE and the induction of macrophage polarization, we further performed electrochemical in situ detection of intracellular NO in macrophages under different polarization states. First, at +0.85 V, electrodes were inserted at random locations within the same cell of M0, M1, and M2 macrophages using IT technology. Figure 9 (A in the original text). The results showed that the detection signal difference at different locations within the same cell was small, indicating that Au / Pt / 5A1N / CFNE has good stability in single-cell detection, and also indicating that the NO distribution within a single cell is relatively uniform. Subsequently, Au / Pt / 5A1N / CFNE was used for in situ detection of intracellular NO levels (…). Figure 9 In the study of macrophages (B cells), compared to M0 cells, M1 macrophages showed a significantly increased electrochemical response, while M2 macrophages exhibited only a weaker current response. These results indicate that there are significant differences in NO levels within macrophages under different polarization states.

[0090] Further electrochemical signal statistical analysis was performed on macrophages in 30 different polarization states to compare the differences in NO levels among the groups. Figure 9 As shown in Figure C, the current response of M0 macrophages is low and relatively stable, with an average current value of 8.41 pA corresponding to NO, indicating that unstimulated macrophages only maintain basal levels of NO production. In contrast, after LPS induction, macrophages undergo M1 polarization, with a significantly enhanced electrochemical signal and an average current value increasing to 41.07 pA. Figure 9 (D in the text). This result indicates that under inflammatory stimulation, M1 macrophages can produce large amounts of NO, consistent with their pro-inflammatory phenotype. Conversely, M2 macrophages induced by IL-4+IL-13 exhibited a weaker electrochemical response, with an average current of only 1.13 pA (D in the text). Figure 9 The NO levels in M2 macrophages were significantly lower than those in the M0 and M1 groups, indicating a lower NO level in M2 macrophages. This result is consistent with the biological functions of M2 macrophages, which are mainly involved in anti-inflammatory responses, tissue repair, and immune regulation. Further statistical analysis of the cell detection results in the three groups was performed (…). Figure 9 The NO current response of macrophages in different polarization states (F) showed significant differences, exhibiting clear grouping characteristics. These results indicate that Au / Pt / 5A1N / CFNE can sensitively and accurately distinguish the differences in intracellular NO levels in macrophages with different polarization states.

[0091] Based on this, and further combining standard curves and diffusion-reaction kinetic models, the intracellular NO in macrophages under different polarization states was quantitatively analyzed according to Faraday's law. The molar rate of NO (denoted as f) was converted from the current signal and expressed as ΔI = Fn f, where F represents the Faraday constant (9,6500 C / mol), and n represents the electrochemical stoichiometry related to the electrochemical oxidation of the substance (n... NO = 1), plot the molar rate statistics ( Figure 10 The results showed that there were significant differences in the NO molar rate among macrophages in different polarization states. In M0 type cells, the NO molar rate was mainly concentrated around 80 amol / s. -1 This reflects the characteristics of cells maintaining basal levels of NO production under resting conditions. Figure 10 In contrast, M1 macrophages exhibited a higher molar rate, which is highly consistent with their biological function of synthesizing large amounts of NO in a pro-inflammatory activated state. Figure 10(B in the text); when f reaches approximately 200 amol s -1 At this time, the cells exhibited typical M1-type polarization characteristics. On the other hand, the NO molar rate of M2-type macrophages remained at a low level, generally less than 30 amol / s. -1 ( Figure 10 The presence of C in the figure is consistent with its functional phenotype related to anti-inflammatory, tissue repair, and immune regulation. These results further demonstrate that the constructed Au / Pt / 5A1N / CFNE can achieve highly sensitive, in-situ detection of NO in macrophages under different polarization states. Figure 10 The NO level was characterized by D in the D-score, thus providing a reliable quantitative basis for evaluating the polarization and functional status of macrophages based on electrochemical methods.

[0092] In summary, this invention addresses the core issue of evaluating macrophage polarization status through single-cell electrochemical sensing of NO detection. It successfully constructed a highly sensitive nanoelectrode sensing platform, established an in vitro macrophage polarization model, and applied it to a co-culture system of macrophages with normal CT26 cells, CT26 cells undergoing ICD, and ferroptotic CT26 cells. From both phenotypic identification and real-time single-cell NO detection perspectives, it systematically revealed the regulatory mechanisms of different tumor cell death pathways on macrophage polarization. The main conclusions are as follows: 1. A highly sensitive and selective nanoelectrode suitable for single-cell NO detection was successfully prepared, which can realize in situ, real-time and accurate detection of single-cell NO in complex cellular environments, providing a reliable technical platform for analyzing macrophage functional heterogeneity at the single-cell level.

[0093] 2. In M1 and M2 macrophage models, single-cell electrochemical detection results showed that the NO level in M1 macrophages was significantly higher than that in M0 and M2 macrophages, and significant heterogeneity existed among different cell types. This demonstrates that single-cell NO level can serve as a direct and sensitive indicator for assessing M1 / M2 macrophage polarization, providing a novel detection method for rapid macrophage phenotype evaluation that is independent of cell lysis and allows for real-time dynamic monitoring.

[0094] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An electrochemical nanosensor for detecting NO in macrophages, characterized in that, It includes a working electrode and a gold nanoparticle layer, a platinum nanoparticle layer and a 5-amino-1-naphthol polymer layer sequentially modified on its surface; the 5-amino-1-naphthol polymer layer has a porous structure and can selectively allow NO molecules to pass through while blocking interfering substances. The working electrode is a carbon fiber nanoelectrode; the carbon fiber nanoelectrode has needle-shaped nano-tip, the diameter of which is 100-200 nm. The gold nanolayer was formed using chronoamperometry; the platinum nanoparticle layer was formed using cyclic voltammetry; the 5-amino-1-naphthol polymer layer was formed by electrodeposition on the electrode surface using cyclic voltammetry; during the preparation of the 5-amino-1-naphthol polymer layer, the concentration of the 5-amino-1-naphthol deposition solution was 0.1-1.0 mM, the voltage of the cyclic voltammetry was 0 V-1.0 V, and the scan rate was 8-12 mV·s. -1 Perform 3-6 cyclic scans. The electrochemical nanosensor is used for in-situ detection of NO in single cells.

2. A method for preparing an electrochemical nanosensor for detecting NO in macrophages as described in claim 1, characterized in that, Includes the following steps: A gold-platinum bimetallic layer modified electrode was prepared by electrochemically depositing gold nanoparticle layers and platinum nanoparticle layers sequentially on the surface of carbon fiber nanoelectrodes. An electrode modified with a gold-platinum bimetallic layer was immersed in a 5-amino-1-naphthol deposition solution, and a 5-amino-1-naphthol polymer layer was formed on the electrode surface by cyclic voltammetry.

3. The method for preparing the electrochemical nanosensor for detecting NO in macrophages as described in claim 2, characterized in that, The carbon fiber nanoelectrode is composed of carbon fiber, copper wire and capillary glass tube. The carbon fiber and copper wire are connected by conductive silver paste. One end of the capillary glass tube is drawn into a pointed shape. Two copper wires with carbon fiber attached are inserted into the flat end of the capillary glass tube in sequence, and the carbon fiber passes out of the pointed end of the capillary glass tube. The flat end is first sealed with resin glue, and then the pointed end is melted and solidified with the outer flame of an alcohol lamp.

4. The method for preparing the electrochemical nanosensor for detecting NO in macrophages as described in claim 2, characterized in that, The preparation steps of the gold nanoparticle layer are as follows: a three-electrode system is formed by using a carbon fiber nanoelectrode as the working electrode, a platinum wire as the counter electrode, and an Ag / AgCl electrode as the reference electrode; the working electrode is immersed in a chloroauric acid deposition solution with a concentration of 0.1-1.0 mM, and constant potential deposition is performed for 10-20 s at a deposition voltage of -0.2 to -0.5 V using the chronoamperometry method.

5. The method for preparing the electrochemical nanosensor for detecting NO in macrophages as described in claim 2, characterized in that, The preparation steps of the platinum nanoparticle layer are as follows: An electrode modified with a gold nanoparticle layer was used as the working electrode and immersed in a chloroplatinic acid deposition solution with a concentration of 0.5-1.5 mM. Cyclic voltammetry was used within a potential window of -0.5 V to 0 V at a rate of 80-120 mV·s. -1 The scanning rate is used to perform 1-2 cyclic scans.

6. The application of the electrochemical nanosensor for detecting NO in macrophages as described in claim 1 in constructing an in vitro macrophage polarization model.

7. The application as described in claim 6, characterized in that, The method for constructing an in vitro macrophage polarization model includes the following steps: inducing macrophage polarization, inserting an electrochemical nanosensor for NO detection into the macrophage, determining the NO content by detecting the response electrical signal, and determining the macrophage polarization state.

8. The application of the electrochemical nanosensor for detecting NO in macrophages as described in claim 1 in the preparation of diagnostic and targeted tumor therapy products, characterized in that, The product is selected from pharmaceuticals, reagent kits, or biosensor devices.

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