Nanowire pH electrode with miniaturized reference and its preparation method and application

Abstract

The application discloses a kind of nanowire pH electrode with micro reference and its preparation method and application, belong to test electrode technical field.The electrode structure includes insulating shell, working part and reference part;Working part, reference part is respectively by with liquid metal connection working electrode material, reference electrode material;Working part and reference part are separated by insulating shell assembly, and do not communicate with each other;Working electrode material and reference electrode material are core-shell structure nanowire with silicon carbide as core and surface cladding shell layer, wherein the shell layer of working electrode material is the metal oxide with pH response performance, and the shell layer of reference electrode material is silver-silver chloride compound.The application realizes the real-time dynamics of monitoring intracellular or micro area pH, and realizes reliable detection of intracellular pH, and provides a simple and easy preparation method, breaks the limitation that metal oxide is difficult to be miniaturized and used for intracellular pH detection in electrode preparation, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of test electrode technology, and in particular to a nanowire pH electrode with a miniaturized reference, its preparation method, and its application. Background Technology

[0002] Intracellular H + Ion levels (pHi) play a crucial role in regulating cellular metabolism and are a key indicator of cellular metabolic behavior. Therefore, in situ monitoring of pH changes within single cells has attracted widespread research interest. Currently, various methods for detecting pHi have been established, including colorimetric methods, fluorescence methods, surface-enhanced Raman spectroscopy (SERS), and electrochemical methods. In particular, methods using H+... + Electrochemical sensors, represented by ion-selective electrodes, have been widely used as the primary tool for pH measurement. However, for intracellular H+... + For ion level measurement, miniaturization of sensors remains necessary, but this still presents challenges.

[0003] In recent years, single nanowire electrodes (NWEs) have been widely used in intracellular amperometric measurements due to their ultra-small size, flexible sensing materials, and advantages such as high spatiotemporal resolution, high sensitivity, and minimal cell damage after insertion. Currently, micro / nanoscale pH sensors mainly utilize H... + Sensitive conductive polymers or ion carrier / polymer matrix blends are used, but poor conductivity may limit their dynamic response performance. Existing technologies, based on a simple and universal "one-pot" reaction, have achieved the mass production of functionalized core-shell nanowires by uniformly coating a polyEDOT (PEDOT)-noble metal nanoparticle composite coating on the surface of non-conductive nanowires through the oxidative polymerization reaction between 3,4-ethylenedioxythiophene (EDOT) and noble metal complexes, by controlling the types of core and shell materials. Using these core-shell nanowires as electrode materials, functionalized nanoelectrodes with excellent electrochemical performance can be assembled, simplifying the nanoelectrode preparation process and overcoming the limitations of existing nanoelectrode preparation materials, enabling real-time quantitative monitoring of biological signal molecules within single living cells (Patent Document 1).

[0004] In comparison, H + Sensitive metal oxides (MO) x ), such as iridium oxide (IrO) x ) and ruthenium oxide (RuO) x This technology has attracted increasing attention in fields such as biochemical science, environmental monitoring, and industrial applications, and shows great potential in intracellular pH sensing. Due to its application in MO... x Reversible H atoms can occur at the oxygen atom sites on the surface. + Coupled electron transfer occurs, resulting in pH-dependent electrode potentials. Simultaneously, these MOs...x The inherently low resistivity (~60 μΩ·cm) prevents H from + The charge retention within the electrode during the binding process enables rapid pH signal transduction. Furthermore, the tunable thickness (nm to μm) allows for sensor miniaturization, but based on MO... x Intracellular pH detection using electrodes has not yet been reported.

[0005] Electrochemical detection methods based on single nanowire electrodes (NWEs) enable real-time monitoring of various intracellular active substances. However, for potential-based measurement methods, such as pH monitoring, cell membrane potential introduces interference. Although this interference can be circumvented by inserting a reference electrode into the cell to form a localized intracellular detection loop, current intracellular references are often micrometer-sized and separate from the working electrode, requiring independent operation to bring the two electrodes close together for localized pH detection. Therefore, there is still a high demand and significant potential benefits in developing a simple method for fabricating nanoscale pH electrodes with miniaturized references, but no such method has been reported to date.

[0006] Existing technical documents

[0007] Patent Document 1: CN113152081A Summary of the Invention

[0008] In view of the above-mentioned deficiencies of the prior art, in a first aspect of the present invention, a nanowire pH electrode with a miniaturized reference is provided, capable of monitoring real-time pH kinetics in intracellular or micro-regions and achieving reliable intracellular pH detection. The electrode comprises an insulating shell, a working part, and a reference part; the working part and the reference part are respectively composed of a working electrode material and a reference electrode material connected to a liquid metal; the working part and the reference part are separated by an insulating shell and are not interconnected; the working electrode material and the reference electrode material are core-shell structured nanowires with silicon carbide (SiC) as the core and a surface-coated shell, wherein the shell of the working electrode material is a metal oxide (MO) with pH-responsive properties. x The shell of the reference electrode material is a silver-silver chloride complex (Ag / AgCl). During operation, the working electrode and reference electrode nanowire at one end of the working part and the reference part are placed inside the cell, and the other end is directly connected to the potential detection device through an extended wire to input a potential signal to the device, forming a complete detection circuit.

[0009] The liquid metal is a low-melting-point conductive metallic material. Preferably, the liquid metal includes at least one of gallium, gallium-indium alloy, gallium-tin alloy, and gallium-indium-tin-zinc alloy.

[0010] Preferably, the metal oxide includes iridium-ruthenium bimetallic oxide (IrRuO₂). xIt is one of the following: bismuth oxide, tungsten oxide, titanium oxide, and tin oxide. Compared to other metal oxides, iridium-ruthenium bimetallic oxide (IrRuO) is... x Due to its excellent conductivity, it is more conducive to the rapid transduction of pH signals, making it a very suitable metal oxide for use as a working electrode shell.

[0011] The nanowire pH electrode uses working electrode and reference electrode materials with specific structures. The two electrode materials can be produced in-house, for example, by using hydrolysis and deposition to attach the aforementioned metal oxides to the surface of silicon carbide nanowires to form core-shell structured nanowires; or electrode materials with the aforementioned structure can be prepared by other methods.

[0012] More preferably, the metal oxide is an iridium-ruthenium bimetallic oxide (IrRuO₂). x The working electrode material is prepared by the following method: silicon carbide nanowires are dispersed in an organic solvent; hydrated ruthenium chloride and hydrated iridium chloride powders are added to the resulting dispersion, mixed and reacted to form a coating layer on the surface of silicon carbide; after the reaction is completed, the insoluble matter is collected, purified, and the working electrode material is obtained.

[0013] Furthermore, the addition relationships of each raw material are as follows: the addition amount of silicon carbide nanowires is 1-3 mg, the addition amount of hydrated ruthenium chloride is 6-10 mg, the addition amount of hydrated iridium chloride is 7-11 mg; the organic solvent includes at least one of ethylene glycol, glycerol, and propylene glycol, and its addition amount is 6-9 mL.

[0014] Furthermore, the iridium and ruthenium elements added to the raw materials are in an equimolar ratio. At this ratio, the resulting working electrode material exhibits a uniform and dense nanoscale morphology, providing excellent conductivity and electrochemical performance.

[0015] Furthermore, the reaction temperature is 180–210°C, and the reaction time is 3–6 hours.

[0016] Preferably, the reference electrode material is prepared by the following method: dispersing silicon carbide nanowires in a reducing organic solvent; adding soluble silver salt and polymer surfactant to the resulting dispersion, mixing and carrying out a reduction reaction to form a single silver layer on the silicon carbide surface; after the reduction reaction is completed, collecting the insoluble matter, purifying it to obtain silver-coated silicon carbide nanowires; subjecting the silver-coated silicon carbide nanowires to chlorination treatment, partially chlorinating the silver layer to silver chloride to form a silver-silver chloride complex, and purifying it after treatment to obtain the reference electrode material.

[0017] Further preferred, the addition relationship of each raw material is as follows: the addition amount of silicon carbide nanowires is 1-3 mg, the addition amount of soluble silver salt is 80-120 mg, the addition amount of polymer surfactant is 3-6 mg, and the addition amount of reducing organic solvent is 4-7 mL.

[0018] More preferably, the soluble silver salt includes at least one of silver nitrate and silver fluoride.

[0019] More preferably, the polymer surfactant includes at least one of polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO).

[0020] More preferably, the reducing organic solvent includes at least one of ethylene glycol, glycerol, and propylene glycol.

[0021] More preferably, the chlorinating agent used in the chlorination includes one of sodium hypochlorite, ferric chloride, and copper chloride.

[0022] More preferably, the temperature of the reduction reaction is 180–210°C, and the reaction time is 6–12 h.

[0023] Chlorination treatment can be performed using commercially available chlorinating agent solutions of the types described above. These solutions should be diluted with pure water before use. For example, a commercially available sodium hypochlorite solution with a chlorine content greater than 5% should be used, diluted 100 times with pure water to a concentration of 0.05%. The volume of the diluent is not limited, but generally 5-8 mL is used. The silver-coated silicon carbide nanowires are added to the sodium hypochlorite solution, and the mixture is shaken and reacted at room temperature for 3 minutes. The chlorination step is a standard procedure in the art. Given the specific types of chlorinating agents available, those skilled in the art can select appropriate parameters to complete this operation without special limitations.

[0024] In this invention, the insulating shell serves to separate the working part and the reference part, ensuring a complete detection circuit during testing and preventing short circuits. Therefore, while meeting this design objective, the shape of the insulating shell and the selection of insulating materials are diverse and can be chosen based on the actual processing conditions and requirements in the field. Considering the convenience, cost, and stability of raw material sources, glass materials (such as borate glass, quartz glass, etc.) are highly suitable for preparing the insulating shell of this invention. Furthermore, under industrial precision machining conditions, insulation between the working part and the reference part can be directly achieved through glass; while under laboratory conditions, a θ-type double-channel glass tube can be used to house the working part and the reference part, with an insulating material that is solid at room temperature and has a relatively low melting point (such as sealing wax) used for auxiliary sealing and fixation.

[0025] In a second aspect of the present invention, a simple method for preparing a nanowire pH electrode with a miniaturized reference, as described in the first aspect of the present invention, is provided, comprising the following steps:

[0026] (1) Disperse the working electrode material and the reference electrode material in a solvent to form corresponding dispersions;

[0027] (2) The obtained dispersions are dropped onto the surface of the substrate. After the solvent evaporates, the substrate is cut to expose the nanowires of the working electrode material or the reference electrode material at the edge of the substrate.

[0028] (3) Pull the θ-shaped double-channel glass tube into a tapered tube with a tip diameter of 3-5 μm;

[0029] (4) Inject liquid metal into the two channels of the obtained conical tube, push the liquid metal to a distance of 5-10 μm from the tip of the conical tube, and seal the tip with sealing wax;

[0030] (5) Heat the sealing wax to melt it. Insert the nanowires of the working electrode material and the reference electrode material into the liquid metal of the conical tube and then stop heating. The sealing wax solidifies to complete the sealing and fixation, and a nanowire pH electrode with a miniaturized reference is obtained.

[0031] To facilitate rapid solvent evaporation, common low-boiling-point organic solvents in the field can be used, such as methanol, ethanol, isopropanol, acetone, and tetrahydrofuran, all of which are suitable for this process step.

[0032] This method presents a laboratory-scale fabrication process for nanowire pH electrodes with miniaturized references. The θ-shaped dual-channel glass tube has a cylindrical glass capillary shell with a diameter of approximately 2 mm. An internal glass septum of the same material divides the cylindrical cavity into two non-conductive parts, with a θ-shaped cross-section. After heating and drawing using a laser drawing apparatus, a tapered capillary tip is obtained, maintaining the θ-shaped cross-section. Liquid metal is then poured in and sealed with insulating wax. Two different functional conductive nanowires can be assembled into different channels, independently performing their respective functions to form a complete detection circuit. The unsealed end of the electrode is directly connected to a potential detection device via an extended wire, inputting a potential signal to the device.

[0033] In a third aspect of the invention, an application is provided for a nanowire pH electrode with a miniaturized reference prepared by the method of the first aspect of the invention or by the method of the second aspect of the invention, specifically as a test electrode for monitoring intracellular or micro-region pH levels and dynamic changes.

[0034] This invention primarily aims to provide an electrochemical detection tool for monitoring the rapid kinetics of intracellular pH. Because methods such as fluorescent labeling and surface-enhanced Raman spectroscopy (SERS) cannot provide rapid and accurate quantitative pH responses, electrochemical methods are the most widely used pH detection techniques. However, currently reported intracellular pH detection electrodes are based on hydrogen ion-sensitive polymer membranes, which have extremely poor conductivity, making it difficult to achieve rapid kinetic monitoring of intracellular pH. Meanwhile, metal oxides (MO) such as iridium oxide and ruthenium oxide... x It possesses excellent conductivity, which is beneficial for real-time monitoring of rapid pH changes and provides kinetic information that cannot be measured by other methods, but intracellular pH detection electrodes based on iridium oxide and ruthenium oxide have not yet been realized.

[0035] Secondly, existing micro- and nano-electrodes are based on current-based detection of intracellular substances. However, since pH detection requires potentiometric monitoring, if the monitoring process is carried out as before (with the reference electrode placed outside the cell and the working electrode fully inserted inside the cell), the detection circuit will be connected in series with the cell membrane. Potentiometric detection will be interfered with by the unstable potential difference across the cell membrane, resulting in large fluctuations in the obtained potential detection signal, making accurate and reliable quantification impossible.

[0036] Based on the above-described technical solutions, the inventive concept and principle of this invention lie in using an intracellular miniaturized reference electrode, allowing both the working electrode and the reference electrode to be located within the cell simultaneously, forming a complete detection circuit within the same cell, thus greatly reducing membrane potential interference. This is achieved using iridium-ruthenium bimetallic oxide (IrRuO₂). x Taking silicon carbide-coated nanowires as an example, the structures of the working electrode and reference electrode have significant advantages. The working electrode surface IrRuO... x As a highly conductive hydrogen ion-sensitive layer, it has a uniform and dense morphology and excellent electrochemical performance, which is conducive to the rapid transduction and output of pH signals. This electrode breaks through the limitations of metal oxides (MO). x The limitations of miniaturization and application for intracellular pH detection are significant. Silver-silver chloride complexes, however, are commonly used reference electrodes in electrochemistry. Their electrode potential in solution is not easily altered by other substances, contributing to the accuracy and stability of micro-area monitoring. The advantage of intracellular references lies in eliminating interference from cell membrane potential changes during detection, enabling the measurement of pH levels and their dynamic changes under physiological and pathological conditions within a single living cell.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] This invention provides a nanowire pH electrode with a miniaturized reference. Based on the rapid pH transduction properties of metal oxides, it provides a tool for monitoring the real-time kinetics of intracellular or micro-region pH, solving the problem that intracellular pH detection by potentiometric methods is easily interfered with by membrane potential changes, and realizing reliable detection of intracellular pH.

[0039] This invention provides a method for preparing a nanowire pH electrode with a miniaturized reference. This method is simple and easy to implement, overcoming the limitations of metal oxide (MO) methods in electrode preparation. x The limitation is that it is difficult to miniaturize and use it for intracellular pH detection.

[0040] The present invention also provides an application of a nanowire pH electrode with a miniaturized reference, which has broad prospects as a detection tool for intracellular or micro-region pH levels and dynamic changes. Attached Figure Description

[0041] Figure 1 This is a schematic diagram and elemental mapping diagram of the preparation method of the working electrode material used in the example;

[0042] Figure 2 This is a schematic diagram and elemental mapping diagram of the preparation method of the reference electrode material used in the example;

[0043] Figure 3 A schematic diagram of the fabrication of a nanowire pH electrode with an attached miniaturized reference and its electron microscopy characterization.

[0044] Figure 4 A schematic diagram illustrating the signal and response principle of a nanowire pH electrode with a miniaturized reference for monitoring intracellular pH changes in human breast cancer cells (MCF-7) under different conditions.

[0045] Figure 5 Signals and working images of a nanowire pH electrode with a miniaturized reference for monitoring intracellular pH changes in human cervical cancer cells (HeLa) in different states;

[0046] Figure 6 Signals and working images of a nanowire pH electrode with a miniaturized reference for monitoring pH changes at the tips of cotton ovule fiber cells in different states;

[0047] Figure 7 This diagram illustrates the difference in intracellular pH detection results and the underlying principle when using a potentiometric method to compare results without an intracellular reference with those using an intracellular miniaturized reference. Detailed Implementation

[0048] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0049] In the following embodiments:

[0050] The working electrode material was prepared in the laboratory, using iridium-ruthenium bimetallic oxide (IrRuO₂). xNanowires with a shell encapsulating a silicon carbide (SiC) core are prepared using the following method:

[0051] (1) Place 1 mg of silicon carbide nanowires in a 25 mL sample bottle, add 8 mL of ethylene glycol as a solvent, and use ultrasound to disperse the silicon carbide nanowires evenly.

[0052] (2) Under stirring, 8 mg of ruthenium chloride trihydrate and 9 mg of iridium chloride hydrate were added to the obtained silicon carbide nanowire dispersion to make the two aqueous metal chlorides uniformly dispersed and dissolved.

[0053] (3) Transfer the obtained mixture into a 10 mL polytetrafluoroethylene liner and seal it inside a stainless steel hydrothermal reactor.

[0054] (4) Heat the reactor to 198°C, keep the reaction system at the temperature for 4 hours and then stop heating to hydrolyze the water-containing metal chloride in the mixture into metal oxides, which are deposited and coated on the surface of silicon carbide nanowires to form a metal oxide conductive layer, thus completing the reaction.

[0055] (5) After the hydrothermal reactor has cooled to room temperature, the reaction solution is centrifuged and washed three times with water and ethanol respectively to obtain the working electrode material.

[0056] Figure 1 A schematic diagram of the fabrication method and elemental mapping of the working electrode material are shown. The image indicates that the working electrode surface is completely coated with a metal oxide layer, and the characteristic elements Ru and Ir are uniformly distributed throughout the core-shell nanowire; while the characteristic elements Si and C are uniformly distributed in the central region of the core-shell nanowire, indicating that IrRuO x Completely and uniformly coated on the surface of silicon carbide nanowires.

[0057] The reference electrode material was prepared in-house using the following method:

[0058] (1) Place 1 mg of silicon carbide nanowires in a 25 mL sample bottle, add 6 mL of ethylene glycol as a solvent, and use ultrasound to disperse the silicon carbide nanowires evenly.

[0059] (2) Under stirring, add 5 mg of polyvinylpyrrolidone (PVP) and 101 mg of silver nitrate to the obtained silicon carbide nanowire dispersion to make the two drugs evenly dispersed and dissolved.

[0060] (3) Transfer the obtained mixture into a 10 mL polytetrafluoroethylene liner and seal it inside a stainless steel hydrothermal reactor.

[0061] (4) Heat the reactor to 200°C, keep the reaction system at the temperature for 10 hours and then stop heating. Under the control of polyvinylpyrrolidone, the silver ions in the mixture are reduced from ethylene glycol to elemental silver and coated on the surface of silicon carbide nanowires to form a silver conductive layer. After the hydrothermal reactor cools down to room temperature, the reaction solution is centrifuged and washed three times with water and ethanol respectively to obtain silver core-shell nanowires.

[0062] (5) Add sodium hypochlorite solution to the silver core-shell nanowires and shake at room temperature for 3 min to complete chlorination, so that the silver layer is partially chlorinated to silver chloride, forming a silver-silver chloride complex. Wash with water and ethanol three times respectively to obtain the reference electrode material.

[0063] Figure 2 A schematic diagram of the preparation method and elemental mapping of the reference electrode material are shown. The image shows that the surface of the reference electrode is completely coated with a silver-silver chloride composite layer, and the characteristic elements Ag and Cl are uniformly distributed throughout the core-shell nanowire, indicating that the silver-silver chloride composite is completely and uniformly wrapped on the surface of the silicon carbide nanowire.

[0064] Example 1

[0065] The fabrication method of a nanowire pH electrode with a miniaturized reference is as follows: Figure 3 As shown, the steps are as follows:

[0066] (1) Take an appropriate amount of working electrode material and reference electrode material and add them to 10 mL of ethanol and disperse them by ultrasonication to obtain a uniform dispersion of working electrode material and reference electrode material;

[0067] (2) The working electrode material dispersion and the reference electrode material dispersion were dropped onto a glass slide and dried at 90°C. Then, the back of the glass slide was cut with a glass cutter and the glass slide was pried open from the front to expose the nanowires of the two electrode materials at its edges.

[0068] (3) The θ-shaped double-channel borate glass tube is drawn into a tapered tube with a tip diameter of about 3 to 5 μm using a laser drawing instrument;

[0069] (4) Inject gallium indium tin zinc alloy into the borate conical tube, centrifuge to push the liquid metal to a distance of 5μm from the tip of the conical tube, and then seal and insulate it with paraffin.

[0070] (5) Under a 40x objective lens, the sealed borate conical tube was placed into a quartz tube with a heating wire wrapped around it. The heating wire was connected to a DC power supply and heated under a DC power of 0.25A to melt the paraffin at the tip of the conical tube. Then, the working electrode material and the reference electrode material exposed at the edge of the glass slide were inserted into the liquid metal in different channels of the borate conical tube in sequence. Then, the heating was stopped and the paraffin solidified to complete the sealing and fixation, thus obtaining a nanowire pH electrode with a miniaturized reference.

[0071] Depend on Figure 3 Measurements of the magnified partial image show that the exposed working electrode and reference electrode have a length of approximately 4 μm and diameters of approximately 400 nm and 1 μm, respectively.

[0072] Example 2

[0073] The nanowire pH electrode with miniaturized reference prepared in Example 1 was applied to the intracellular pH detection of human breast cancer cells (MCF-7) to evaluate its performance.

[0074] The specific steps and procedures for this test are as follows:

[0075] (1) MCF-7 cells were seeded into small dishes containing small round glass slides (7 mm in diameter), and DMEM medium was added and cultured for 12 h for cell electrochemical detection experiments. All cell experiments were performed on an inverted microscope equipped with an electrochemical workstation.

[0076] (2) Connect the nanowire pH electrode with miniaturized reference to the patch clamp amplifier probe. Under a 40x objective lens, use a micromanipulator (TransferMan NK2, Eppendorf) to move the nanowire pH electrode to the vicinity of the cell membrane. Then, slowly move the nanowire pH electrode forward and insert it into the cell. When the open circuit potential signal measured after insertion is stable, drug treatment is performed.

[0077] (3) The nanowire pH electrode outputs different open-circuit potentials based on the hydrogen ion concentration of the surrounding environment; a lower open-circuit potential is output when the hydrogen ion concentration is low, and vice versa. Figure 4 As shown, when the nanowire pH electrode was inserted into the untreated control group MCF-7 cells from the extracellular pH 7.0 PBS buffer, the measured decrease in open circuit potential corresponded to an intracellular pH of 7.2, which is consistent with the weakly alkaline intracellular pH of cancer cells. The measured intracellular pH of the drug-treated experimental group was 6.7, indicating that the MCF-7 cells treated with the glutaminase inhibitor (BPTES) were acidified. The test results were as expected, indicating that the nanowire pH electrode with miniaturized reference achieved the technical objective of this invention.

[0078] Example 3

[0079] The nanowire pH electrode with miniaturized reference prepared in Example 1 was applied to the intracellular pH detection of human cervical cancer cells (HeLa) to evaluate its performance.

[0080] The specific steps and procedures for this test are as follows:

[0081] (1) HeLa cells were seeded into small dishes containing small round glass slides (7 mm in diameter), and DMEM medium was added and cultured for 12 h for cell electrochemical detection experiments. All cell experiments were performed on an inverted microscope coupled to an electrochemical workstation.

[0082] (2) Connect the nanowire pH electrode with miniaturized reference to the patch clamp amplifier probe. Under a 40x objective lens, use a micromanipulator (TransferMan NK2, Eppendorf) to move the nanowire pH electrode to the vicinity of the cell membrane. Then, slowly move the nanowire pH electrode forward and insert it into the cell. When the open circuit potential signal measured after insertion is stable, drug stimulation is performed.

[0083] (3) The nanowire pH electrode outputs different open-circuit potentials based on the hydrogen ion concentration of the surrounding environment; a lower open-circuit potential is output when the hydrogen ion concentration is low, and vice versa. Figure 5 As shown, when the pH electrode measured the intracellular pH of HeLa cells to be 7.1, cells stimulated with the sodium / hydrogen ion exchanger inhibitor calipamide (CAR) exhibited a rapid increase in open circuit potential, which remained for a relatively long time. In contrast, the intracellular pH signal of HeLa cells stimulated only with PBS buffer showed only a brief disturbance before returning to near the initial state, indicating that rapid and sustained acidification occurred in the intracellular cells of CAR-treated HeLa cells. The test results were in line with expectations, demonstrating that the nanowire pH electrode with miniaturized reference achieved the technical objective of this invention.

[0084] Example 4

[0085] The nanowire pH electrode with miniaturized reference prepared in Example 1 was applied to pH detection in the extracellular microregion at the tip of cotton ovule fiber cells to evaluate its performance.

[0086] The specific steps and procedures for this test are as follows:

[0087] (1) The cotton ovules were attached to a small dish containing a small round glass slide (7 mm in diameter), and PBS buffer was added for cell electrochemical detection experiments; all cell experiments were performed on an inverted microscope coupled to an electrochemical workstation.

[0088] (2) Connect the nanowire pH electrode with miniaturized reference to the patch clamp amplifier probe. Under a 40x objective lens, use a micromanipulator (TransferMan NK2, Eppendorf) to move the nanowire pH electrode to the vicinity of the cell wall. Then, slowly move the nanowire pH electrode forward and attach it to the tip of the fiber cell. When the open circuit potential signal measured after insertion is stable, drug stimulation is performed.

[0089] (3) The pH electrode outputs different open-circuit potentials based on the hydrogen ion concentration of the surrounding environment; a lower open-circuit potential is output when the hydrogen ion concentration is low, and vice versa. Figure 6 As shown, when cotton fiber cells were stimulated with PBS buffer containing rapid alkalizing factor peptide (RALF), the pH electrode measured an increase in extracellular pH-related potential, which returned to normal after the stimulation was removed. The corresponding pH change was from the initial 7.0 to 6.7 after stimulation, indicating that the pH of the extracellular microregion at the tip of cotton ovule fiber cells treated with RALF was acidified. This shows that the nanowire pH electrode with miniaturized reference is not only suitable for monitoring intracellular pH, but also for monitoring extracellular microregions.

[0090] The above embodiments demonstrate that the nanowire pH electrode with miniaturized reference constructed by the method of the present invention can detect minute changes in intracellular and micro-region pH. This provides a powerful tool for in-depth research into the fundamental mechanisms of cellular metabolism in tumor killing, tissue repair, and anti-aging.

[0091] Comparative Example 1

[0092] This comparative test compared the results of potentiometric detection of intracellular pH under the same conditions, without using an intracellular reference versus with using an intracellular miniaturized reference. The specific steps of this test are as follows:

[0093] (1) MCF-7 cells were seeded into small dishes containing small round glass slides (7 mm in diameter), and DMEM medium was added and cultured for 12 h for cell electrochemical detection experiments. All cell experiments were performed on an inverted microscope equipped with an electrochemical workstation.

[0094] (2) Nanowire pH electrodes without and with miniaturized references were connected to patch-clamp amplifier probes. Under a 40x objective lens, the nanowire pH electrodes were moved near the cell membrane using a micromanipulator (TransferMan NK2, Eppendorf). The nanowire pH electrodes were then slowly moved forward and inserted into the cell. The results are as follows: Figure 7 As shown. By Figure 7It can be seen that the open-circuit potential without using an intracellular reference is >11mV, with greater fluctuations; while with the use of an intracellular miniaturized reference, the open-circuit potential fluctuation is ≤3mV, showing better stability; indicating that potentiometric detection of intracellular pH is easily affected by membrane potential changes, while the nanowire pH electrode with miniaturized reference of the present invention overcomes the above technical problems, and its detection is more accurate and stable.

[0095] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A nanowire pH electrode with a miniaturized reference, characterized in that: The structure includes an insulating shell, a working part, and a reference part; the working part and the reference part are respectively composed of a working electrode material and a reference electrode material connected to liquid metal; the working part and the reference part are assembled and separated by the insulating shell and are not connected to each other; the working electrode material and the reference electrode material are core-shell structured nanowires with silicon carbide as the core and a shell covering the surface, wherein the shell of the working electrode material is a metal oxide with pH response performance, and the shell of the reference electrode material is a silver-silver chloride composite. The metal oxide is an iridium-ruthenium bimetallic oxide. The working electrode material is prepared by the following method: silicon carbide nanowires are dispersed in an organic solvent; hydrated ruthenium chloride and hydrated iridium chloride powder are added to the resulting dispersion, mixed, and reacted to form a coating layer on the silicon carbide surface; after the reaction is completed, the insoluble matter is collected, purified, and the working electrode material is obtained.

2. The nanowire pH electrode with miniaturized reference according to claim 1, characterized in that: The liquid metal includes at least one of gallium, gallium-indium alloy, gallium-tin alloy, and gallium-indium-tin-zinc alloy.

3. The nanowire pH electrode with miniaturized reference according to claim 1, characterized in that, The addition relationships of each raw material are as follows: the addition amount of silicon carbide nanowires is 1~3mg, the addition amount of hydrated ruthenium chloride is 6~10mg, the addition amount of hydrated iridium chloride is 7~11mg, and the organic solvent includes at least one of ethylene glycol, glycerol, and propylene glycol, with an addition amount of 6~9mL; the reaction temperature is 180~210℃, and the reaction time is 3~6h.

4. The nanowire pH electrode with miniaturized reference according to claim 1, characterized in that, The reference electrode material was prepared by the following method: silicon carbide nanowires were dispersed in a reducing organic solvent; soluble silver salt and polymer surfactant were added to the resulting dispersion, and a reduction reaction was carried out after mixing to form a single silver layer on the surface of silicon carbide; after the reduction reaction was completed, the insoluble matter was collected, purified, and silver-coated silicon carbide nanowires were obtained. Silver-coated silicon carbide nanowires are chlorinated, and the silver layer is partially chlorinated to silver chloride, forming a silver-silver chloride complex. After treatment and purification, a reference electrode material is obtained.

5. The nanowire pH electrode with miniaturized reference according to claim 4, characterized in that, The addition ratios of the raw materials are as follows: silicon carbide nanowires are added at 1-3 mg, soluble silver salts at 80-120 mg, polymer surfactants at 3-6 mg, and reducing organic solvents at 4-7 mL; the reduction reaction temperature is 180-210℃, and the reaction time is 6-12 h.

6. The nanowire pH electrode with miniaturized reference according to claim 5, characterized in that: The soluble silver salt includes at least one of silver nitrate and silver fluoride; the polymer surfactant includes at least one of polyvinylpyrrolidone and polyethylene oxide; the reducing organic solvent includes at least one of ethylene glycol, glycerol, and propylene glycol; and the chlorinating agent used in the chlorination includes one of sodium hypochlorite, ferric chloride, and copper chloride.

7. A method for preparing a nanowire pH electrode with a miniaturized reference as described in any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Disperse the working electrode material and the reference electrode material in a solvent to form the corresponding dispersions; (2) The obtained dispersions are dropped onto the surface of the substrate. After the solvent evaporates, the substrate is cut so that the edges of the substrate expose the nanowires of the working electrode material or the reference electrode material. (3) Pull the θ-shaped double-channel glass tube into a tapered tube with a tip diameter of 3~5μm; (4) Inject liquid metal into the two channels of the obtained conical tube respectively, push the liquid metal to a distance of 5~10μm from the tip of the conical tube, and seal the tip with sealing wax; (5) Heat the sealing wax to melt it. Insert the nanowires of the working electrode material and the reference electrode material into the liquid metal of the conical tube and then stop heating. The sealing wax solidifies to complete the sealing and fixation, and a nanowire pH electrode with a miniaturized reference is obtained.

8. The application of a nanowire pH electrode with a miniaturized reference as described in any one of claims 1 to 6, or a nanowire pH electrode with a miniaturized reference prepared using the method described in claim 7, characterized in that: It is used as a test electrode to monitor intracellular or micro-region pH levels and dynamic changes.

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