A sensor, its preparation method and use

Abstract

The application discloses a sensor and a preparation method and application thereof, and belongs to the technical field of biomedicine. The sensor comprises a working electrode, a reference electrode and an auxiliary electrode; and the working electrode is loaded with an electrocatalyst Cu-Co3O4 with double catalytic active sites on the surface. The sensor can realize real-time feedback of the electrocatalysis process and pathological signals in a bimodal mode while electrocatalysis treatment is carried out, realizes efficient and controllable electrocatalytic decomposition of intracellular hydrogen peroxide, and further promotes the expression of anti-inflammatory factors and effectively inhibits the apoptosis of nerve cells. The application combines electrocatalysis treatment with the feedback of the space-time synchronization treatment process, and provides a controllable, safe and reliable integrated new scheme for the diagnosis and treatment of nervous system diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a sensor, its preparation method, and its application. Background Technology

[0002] Mitochondria produce energy through aerobic glycolysis, which generates the majority of intracellular hydrogen peroxide (H2O2). Physiological levels of H2O2 play a crucial role in maintaining physiological functions such as signal transduction, cell proliferation, differentiation, and migration. However, abnormal accumulation of H2O2 is a core pathological event in neurodegenerative diseases (such as Parkinson's disease and Alzheimer's disease), manifesting as neuronal apoptosis and loss of dopamine release. Correcting this abnormal accumulation by remodeling the oxidative metabolism of H2O2 in nerve cells aligns with theories related to reactive oxygen species (ROS) in oxidative metabolism and demonstrates therapeutic potential.

[0003] In recent years, catalytic metabolism regulating H2O2 levels has become an important strategy for treating a range of diseases. Key advances have been made in treating various neurological disorders induced by high H2O2 levels through the development of highly catalytically active nanozymes, photothermal kinetic therapy, exploration of sonodynamically driven catalytic metabolism, and clinical integration. However, existing methods still have significant limitations: nanozymes have low biocompatibility; the photothermal effect is difficult to control and exhibits photothermal cytotoxicity; and sonosensitive agents have stringent requirements for ultrasound equipment. Therefore, the application of these methods in neuroscience is still in its early stages of research.

[0004] In contrast, direct electrocatalytic H2O2 metabolism, with its rapid decomposition rate and catalytic voltage far below cellular tolerance levels, shows great potential for efficient and rapid regulation of diseases. However, there are currently no reported strategies for using electrocatalytic H2O2 metabolism to regulate the progression of neurological diseases. Although electrocatalytic H2O2 is a promising therapeutic approach, existing electrocatalytic strategies mainly focus on improving the catalytic efficiency of H2O2, neglecting a key issue in practical applications—how to achieve precise and controllable electrocatalytic H2O2 decomposition. Excessive electrocatalysis can significantly disrupt the intracellular redox balance, while a certain level of H2O2 is fundamental to maintaining normal cellular life activities. This contradiction is particularly prominent in sensitive nerve cells (such as PC12).

[0005] Furthermore, advanced neurotherapeutic electronic devices need to be able to continuously track key biomarkers (such as dopamine) in the disease process to aid in treatment decisions. Electrocatalytic sensing devices capable of spatiotemporally synchronously feeding back electrocatalytic processes and pathological signals hold promise for addressing this challenge, but this requires catalysts with high electrocatalytic activity and high electron transfer rates, necessitating the atomic-level arrangement of active sites and catalytic interfaces. Therefore, developing an integrated sensor that can both precisely and controllably electrocatalyze the decomposition of H₂O₂ to treat neurological diseases and synchronously feed back electrocatalytic processes and pathological signals in real time is a pressing technical problem in this field. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a sensor, its preparation method, and its application, thereby resolving the technical problems of existing electrocatalytic therapy strategies, such as difficulty in achieving precise and controllable H2O2 decomposition, lack of real-time feedback capability on electrocatalytic processes and pathological signals, and the tendency of excessive electrocatalysis to disrupt intracellular redox balance.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a sensor for electrocatalytic therapy and spatiotemporal synchronization feedback is provided, including a working electrode, a reference electrode and an auxiliary electrode; the surface of the working electrode is loaded with an electrocatalyst with dual catalytic active sites; the electrocatalyst is Cu-Co3O4.

[0008] Furthermore, the reference electrode is an Ag / AgCl electrode; the auxiliary electrode is a platinum electrode.

[0009] This invention provides a method for manufacturing the above-mentioned sensor, comprising the following steps: (1) Preparation of Cu-Co3O4: S1. Dissolve 2-methylimidazole and a surfactant in water to prepare an imidazole solution; then add a cobalt salt solution to the imidazole solution, let it stand to react, then centrifuge to collect the precipitate, wash and dry the precipitate to obtain the organometallic precursor. S2. Add the organometallic precursor from step (1) to the sulfuric acid solution, stir the reaction, centrifuge to collect the precipitate, wash and dry the precipitate to obtain TA-Co NPs; S3. Add the TA-Co NPs from step (2) to the copper salt solution, stir the reaction and centrifuge to collect the precipitate, wash and dry the precipitate and then perform stepwise pyrolysis to obtain Cu-Co3O4. (2) Sensor integration: The working electrode, reference electrode and auxiliary electrode are integrated into the microneedle, and the electrodes are connected to the screen-printed electrode leads using silver paste to obtain the sensor.

[0010] Further, in step (1), the cobalt salt is at least one of Co(NO3)3·6H2O, CoCl2·6H2O, CoSO4·7H2O and Co(CH3COO)2·4H2O; the surfactant is hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide or octadecyltrimethylammonium bromide; the copper salt is at least one of CuCl2, Cu(NO3)2 and CuSO4; and the stepwise pyrolysis temperatures are 200℃ and 300℃ respectively.

[0011] Furthermore, in step (2), the working electrode is a microneedle electrode; the microneedle electrode includes a microneedle substrate, an insulating layer located on the surface of the microneedle substrate, a conductive carbon / silver paste layer filled inside the microneedle, and an electrocatalyst supported on the surface of the conductive carbon / silver paste layer.

[0012] Furthermore, the method for preparing the insulating layer is as follows: microneedles are immersed in a polyurethane solution, purged with N2, and then cured to obtain the insulating layer.

[0013] Furthermore, the conductive carbon / silver paste layer is plasma-treated and then loaded with an electrocatalyst; the electrocatalyst is loaded by ultrasonically treating microneedles in a chitosan suspension containing Cu-Co3O4.

[0014] This invention provides an application of the above-mentioned sensor in the preparation of a device or instrument for electrocatalytic treatment of nervous system diseases and / or real-time monitoring of electrocatalytic processes or pathological signals.

[0015] Furthermore, electrocatalytic therapy includes the catalytic decomposition of hydrogen peroxide; real-time monitoring includes the detection of dopamine.

[0016] The present invention also provides a device or instrument for electrocatalytic therapy of nervous system diseases and / or real-time monitoring of electrocatalytic processes or pathological signals, including the aforementioned sensor.

[0017] The present invention has the following beneficial effects: (1) This invention provides a sensor (CCHMS) that integrates electrocatalytic therapy and spatiotemporal synchronous feedback. A nanocomposite electrocatalyst (Cu-Co3O4) with high dual catalytic sites of Cu-Co was designed through interface engineering. The low electronegativity of Cu and O forms a stronger orbital hybridization, which allows Cu to accept electrons from Co through bridging oxygen and form an electron-rich low-valence oxidation state. This spontaneous charge redistribution enhances the adsorption of oxygen-containing intermediates by Co sites and promotes the transfer of electrons to the highly catalytic H2O2 decomposition reaction, thereby realizing a wide linear range and the most sensitive dopamine detection probe known to date.

[0018] (2) The sensor of this invention can efficiently catalyze the decomposition and metabolism of H2O2 in nerve cells (such as PC12) containing high levels of H2O2, remodel the intracellular environment, restore normal cellular physiological functions, and simultaneously achieve spatiotemporal synchronous dual-modal feedback of the H2O2 catalytic process and the concentration of dopamine, a key pathological marker in neurodegenerative diseases. This innovation enables precise and controllable cell electrocatalytic therapy, making up for the shortcomings of existing electrocatalytic schemes in real-time feedback of the electrocatalytic process and tracking changes in in vivo biological signals, and effectively avoiding adverse effects such as redox imbalance caused by excessive electrocatalysis. Therefore, this invention provides a new technical solution for the development of next-generation electrocatalytic diagnostic and therapeutic tools and neuroscience research platforms. Attached Figure Description

[0019] Figure 1 A diagram illustrating the working mechanism of CCHMS in regulating neurons through electrocatalysis; Figure 2 The flowchart shows the preparation process of Cu-Co3O4 nano-electrocatalytic composite material. Figure 3 Images are SEM images; image A is an SEM image of the metal-organic framework; image B is an SEM image of the metal-organic framework after sulfuric acid etching; image C is an SEM image of the metal-organic framework after etching with Cu adsorbed on it. 2+ SEM image; Figure 4 XRD image of Cu-Co3O4 nano-electrocatalytic composite material; Figure 5 The images show TEM and EDS images of the Cu-Co3O4 nano-electrocatalytic composite material; where image A is the TEM image and image B is the EDS image. Figure 6 HR-TEM images of Co3O4 and Cu-Co3O4; Figure 7 XPS full spectrum scans of ZIF-67, T-Co, Co3O4 and Cu-Co3O4 composite materials; Figure 8 These are XPS scan images; where image A is the XPS scan image of Co 2p in the Co3O4 and Cu-Co3O4 composite materials; image B is the XPS scan image of Cu 2p in the Cu-Co3O4 composite material. Figure 9 Figure A shows the CV and EIS plots of CCHMS; where Figure A is the CV plot and Figure B is the EIS plot. Figure 10 The specific surface area diagram of CCHMS electrocatalytic activity; Figure 11 DPV diagram of CCHMS electrocatalytic dopamine oxidation; Figure 12The graphs show the it response curve and linear calibration curve of the CCHMS sensor in 100 pM-1 mM dopamine solution; where Figure A is the it response curve and Figure B is the linear calibration curve. Figure 13 Cycle curves of 1, 100, and 500 it scans were obtained for the CCHMS dopamine sensor in PBS, serum, and DMEM medium, respectively. Figure 14 The graph shows the it response of CCHMS after the addition of all interfering substances and then 100 μM dopamine. Figure 15 Figure A shows the reproducibility and repeatability of the CCHMS sensor; Figure B shows the repeatability. Figure 16 The figures show the Ampere current response curve and the linear fitting curve for the electrocatalytic decomposition of H2O2 by CCHMS; Figure A shows the Ampere current response curve, and Figure B shows the linear fitting curve. Figure 17 This is a concentration distribution diagram of H2O2 after low-voltage periodic electrocatalytic decomposition by CCHMS. Figure 18 The graph shows the H2O2 removal efficiency after different electrocatalytic cycles of CCHMS. Figure 19 The correlation between CCHMS and UV-Vis spectroscopy in determining H2O2 concentration in PC12 cells is shown in the figure. Figure 20 A statistical analysis of the mean fluorescence intensity of DCFH-DA after treating PC12 cells with different sensors; Figure 21 This is a comparison chart of the response current increments of three different batches of Co3O4 sensors or CCHMS during the spatiotemporal synchronous feedback process of H2O2 concentration. Figure 22 The correlation between CCHMS and commercial kits in determining H2O2 concentration in PC12 cells is shown in the graph. Figure 23 The diagrams show the DPV and it graphs for KCl-induced dopamine release from PC12 cells; Figure A is the DPV diagram, and Figure B is the it graph. Figure 24 The graph shows the linear fitting curves of the response current of dopamine released by different numbers of PC12 cells under different concentrations of KCl stimulation and the KCl concentration. Figure 25 The correlation between CCHMS and HPLC in determining dopamine release levels in PC12 cells is shown in the figure. Detailed Implementation

[0020] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0021] Example 1: Preparation of Cu-Co3O4 electrocatalyst This embodiment provides a method for preparing a Cu-Co3O4 electrocatalyst with dual catalytic active sites (the preparation process is as follows). Figure 2 (As shown), including the following steps: (1) Dissolve 0.73g Co(NO3)3·6H2O in 25mL of water to prepare a cobalt salt solution; dissolve 11.35g 2-methylimidazole and 0.02g hexadecyltrimethylammonium bromide in 175mL of water to prepare an imidazole solution; add the cobalt salt solution to the imidazole solution, stir at room temperature for 5min, then let stand at room temperature for 24h, then centrifuge the obtained product at 8000rpm for 5min, collect the precipitate, wash it 5 times with ethanol, and dry it in a vacuum oven at 60℃ for 12h to obtain the organometallic precursor.

[0022] (2) Take 15 mg of the organometallic precursor from step (1) and disperse it in 10 mL of ethanol. Then inject it into 1 M 80 mL of sulfuric acid solution. Stir at room temperature for 10 min and centrifuge at 8000 rpm for 5 min. Collect the precipitate, wash it 5 times with ethanol, and then dry it in a vacuum oven at 60 °C for 12 h to obtain TA-Co NPs.

[0023] (3) Disperse the TA-Co NPs from step (2) in 20 mL of ethanol by ultrasonication, then add 10 mL of CuSO4 aqueous solution (2 mg CuSO4 dissolved in 10 mL of water), and stir at room temperature for 1 h. Centrifuge at 8000 rpm for 5 min, collect the precipitate, wash it 5 times with ethanol, and then dry it in a vacuum oven at 60 °C for 12 h.

[0024] (4) The product obtained in step (3) is pyrolyzed in muffle furnaces at 200℃ and 300℃ (heating rate of 1℃ / min) for 30 min respectively. After natural cooling, an electrocatalyst with high dual catalytic activity sites is obtained, denoted as Cu-Co3O4.

[0025] Example 2: Fabrication of CCHMS Sensor This embodiment provides a method for preparing a sensor (CCHMS) for electrocatalytic therapy and spatiotemporal synchronization feedback, including the following steps: (1) Preparation of microneedle working electrode: ① Quickly immerse the acetone-cleaned microneedles in a 5wt% polyurethane (PU) solution for 5 seconds, then immediately remove them and purge with N2 to prevent the PU from clogging the microneedles during the curing process. Then cure at 80℃ and 60% relative humidity for 4 hours to form an insulating layer.

[0026] ② Fill the microneedles with conductive carbon / silver paste and cure it to obtain a microneedle working electrode substrate filled with conductive carbon / silver paste. Perform plasma treatment on the cured conductive paste to introduce hydrophilic and highly reactive functional groups on the substrate surface.

[0027] ③ The plasma-treated microneedle tip was placed in a 0.1wt% chitosan suspension in which Cu-Co3O4 (5mg / mL) was uniformly dispersed, and ultrasonically treated at a frequency of 40kHz for 30min to ensure that the Cu-Co3O4 electrocatalyst was uniformly dispersed on the filling material, thus obtaining a working electrode loaded with Cu-Co3O4.

[0028] (2) Preparation of reference electrode and auxiliary electrode: The silver wire was soaked in 0.1M FeCl3 solution for 6 hours to convert it into Ag / AgCl reference electrode, and a platinum wire with a diameter of 50µm was selected as the auxiliary electrode.

[0029] (3) Sensor integration: The above working electrode, Ag / AgCl reference electrode and platinum wire auxiliary electrode are integrated into the microneedle, and silver paste is used to connect each electrode to the screen-printed electrode leads to obtain a complete CCHMS sensor.

[0030] Comparative example: Co3O4 was used instead of Cu-Co3O4 in Example 2 to prepare a Co3O4 electrode, and then a sensor was prepared according to the preparation method of Example 2 as a control for subsequent experiments.

[0031] Experimental Example 1: Morphology and Property Characterization of Cu-Co3O4 Electrocatalyst The morphology and properties of the Cu-Co3O4 electrocatalyst prepared in Example 1 were characterized. The crystal structure of the catalyst was characterized using X-ray diffraction (XRD, Rigaku Ultima IV, Japan). The morphology of the samples was observed using scanning electron microscopy (SEM, ZEISS GeminiSEM 360, Germany) and transmission electron microscopy (TEM, Electron F200, Japan). Elemental analysis of the nanomaterials was performed using energy-dispersive X-ray spectroscopy (EDS, Super-X G2, Thermo Fisher Scientific, USA). Surface composition and chemical state were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo Fisher ESCALAB Xi+, USA).

[0032] Uniform (approximately 200 nm) and well-defined cubic metal-organic framework (MOF) nanoparticles were prepared via a simple room-temperature coordination reaction (see [link to original text]). Figure 3 (Figure A). Subsequently, etching was performed using monophosphonic acid (T-Co) and Cu. 2+ An adsorption-exchange process yields a pyrolytic bimetallic nanobox precursor (T-CoCu NPs) (see...). Figure 3 (See Figure BC). Next, the particles were pyrolyzed at 300℃ for 30 min in air atmosphere. Through a carbothermic reduction-metal transfer mechanism, a large number of hollow nanoparticles (Cu-Co3O4) with a complete rough surface structure were generated. These nanoparticles are characterized by Cu-Co sites with high dual catalytic activity on their framework.

[0033] Depend on Figure 4 It can be seen that MOF nanosubstrates were successfully assembled into structures with sodalite topology and A cubic crystal with space group 43m. After etching and pyrolysis, the electrocatalyst formed a typical spinel oxide structure. Figure 5 It is evident that Cu-Co3O4 inherits the physical morphology of the MOF framework, but its surface becomes rough due to gas release, metal migration, and aggregation during pyrolysis. TEM visually demonstrates that Cu-Co3O4 exhibits good dispersibility in aqueous media, and its hollow structure remains intact, which is highly advantageous for preparing uniform, high-quality electrocatalyst coatings. EDS elemental analysis results indicate that Cu atoms are uniformly dispersed on the hollow Co3O4 framework.

[0034] High-resolution TEM (HRTEM) images of both Co3O4 and Cu-Co3O4 show highly oriented and clear lattice fringes. Significantly different from Co3O4, Cu-Co3O4 exhibits both (111) and (002) crystal planes belonging to CuO (lattice spacings of 0.23 nm and 0.25 nm, respectively), and (002) and (100) crystal planes belonging to Co3O4 (lattice spacings of 0.26 nm and 0.27 nm, respectively). These crystal planes together form a tight bimetallic site interface (see...). Figure 6 This is the direct reason for enhancing the catalytic activity and charge transfer of electrocatalysts.

[0035] This Cu-Co dual-site enhanced charge transfer was confirmed by XPS energy dispersive spectroscopy fitting analysis. Figure 7 It can be seen that with the introduction of Cu atoms, vibrational signals of Cu, Co, and O appeared in both the MOF substrate and the XPS full spectrum of Cu-Co3O4. After peak fitting, the Co2p high-resolution spectrum showed three peaks at ~779.8 eV, 781.6 eV, and 788.6 eV, which were attributed to Co, respectively. 3+ Co 2+And its satellite peaks. Compared with Co3O4, the binding energy of Co2p in Cu-Co3O4 is negatively shifted by about 1.6 eV (see... Figure 8 Figure A shows that Cu enhances electron transfer from Co atoms to Cu atoms. The high-resolution Cu2p peak spectrum identifies Cu... + and Cu 2+ The presence of this mixed valence state facilitates the formation of electron-rich centers and electron-deficient states, thereby enhancing the electrocatalytic activity towards the substrate (see [link to relevant documentation]). Figure 8 (Figure B in the middle)

[0036] The above results indicate that the Cu-Co3O4 electrocatalyst prepared in this invention possesses a tightly coupled Cu-Co interface with highly bimetallic sites and a highly active catalytic redox pair (Co... 2+ / Co 3+ and Cu + / Cu 2+ These are precisely the characteristic structures of Cu-Co3O4.

[0037] Experimental Example 2: Electrochemical Characterization of CCHMS Sensor (1) Characterization of electron transfer capability: In a solution containing 5 mM [Fe(CN)6] 3- / 4- In 0.1 M KCl solution, the electron transfer capabilities of different modified electrodes were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CV was performed at a potential range of -0.2–0.6 V with a scan rate of 100 mV / s. EIS was conducted at open-circuit potentials with frequencies ranging from 0.1 to 1 × 10⁻⁶ mV / s. 5 Hz, amplitude 5mV. From Figure 9 It can be seen that Cu 2+ The high number of dual catalytic sites formed by doping in Cu-Co significantly improves the electron transfer capability of Cu-Co3O4NPs, manifested as an increase in the redox peak current and a significantly lower EIS resistance in the CV spectrum. The CCHMS electron transfer coefficient (calculated based on the linear relationship between Ep and lnν) is... k s (1.2×10) -1 s -1 Compared to Co3O4 (1.4×10⁻⁶), -3 s -1 The significant 60-fold increase indicates that the Cu-Co high dual catalytic sites achieved a faster electron conversion rate and increased the electrocatalytic active surface area at the CCHMS electrode interface (see...). Figure 10 The control group consisted of unmodified blank microneedle electrodes.

[0038] (2) Electrocatalytic detection of dopamine: The electrocatalytic activity of different electrodes for dopamine (DA) was analyzed using differential pulse voltammetry (DPV) in 0.01M PBS (pH 7.2-7.4), serum, or DMEM medium. The potential range was -0.1-0.6 V, the pulse width was 0.06 s, the pulse period was 0.5 s, and the amplitude was 50 mV. Figure 11 It can be seen that (the control group is a blank microneedle electrode without any modification, and the blank group is without the addition of dopamine), the DPV oxidation current of Cu-Co3O4 is significantly enhanced compared with Co3O4, indicating that its electrocatalytic product generation ability is more outstanding.

[0039] At 0V, the sensor's response to dopamine was characterized using time-current curves (it) in DMEM solutions containing varying concentrations of dopamine. The sampling interval was 0.1 s, and the settling time was 0 s. Linear calibration curves were constructed. CCHMS can linearly detect DA over a wide concentration range of 100 pM–1 mM, with a detection limit of 0.01 nM (see [link to relevant documentation]). Figure 12 ).

[0040] (3) Anti-interference performance, repeatability and reproducibility test: The anti-interference performance of the CCHMS sensor was evaluated under the same IT conditions by adding 0.1 M of interfering substances (MgSO4, NaCl, L-tryptophan, L-valine, CaCl2, glucose, L-ascorbic acid, uric acid, L-cysteine, ibuprofen, L-glutathione, cocaine, KCl, and piperacillin-tazobactam) to a 0.01 M DMEM solution containing 100 μM dopamine. Figure 13 The CCHMS sensor exhibited a stable DA amperometric response in biological sample matrices (PBS, serum, and culture medium), demonstrating its excellent anti-biofouling properties. Furthermore, even with the same combination of interfering agents, the current response consistency to the same DA concentration remained high at 92.6%, confirming that the CCHMS sensor can adapt to the PC12 cell environment and output accurate DA values ​​(see [link to relevant documentation]). Figure 14 ).

[0041] The repeatability and reproducibility of CCHMS sensors prepared using seven different batches were evaluated under the same test conditions. Figure 15 As shown in Figure A, the CCHMS sensors prepared from seven different batches exhibited highly consistent electrode characteristics and robust redox signal changes when assessing cellular DA levels. The amperometric signal remained stable above 98.3% after seven consecutive DA measurements using the same CCHMS sensor, indicating that the CCHMS sensor can stably track cellular DA changes in real time (see Figure A). Figure 15 (Figure B in the middle)

[0042] Experimental Example 3: Electrocatalytic and Scavenging Efficiency Test of H2O2 by CCHMS Sensor (1) Electrocatalytic ability test of H2O2: The electrocatalytic activity of different electrodes for H2O2 was analyzed using DPV in 0.01M PBS (pH 7.2–7.4), with a potential range of -0.6 to -0.1 V, pulse width of 0.06 s, pulse period of 0.5 s, and amplitude of 50 mV. At 0.05 V, the sensor's response to H2O2 was characterized using it in PBS (pH 7.2–7.4) solutions containing a series of H2O2 concentrations, with a sampling interval of 0.1 s and a settling time of 0 s. Linear calibration curves were constructed. Figure 16 It is known that CCHMS can linearly electrocatalyze the oxidative decomposition of H2O2 at levels of 1 nM to 500 μM, completely covering the concentration range of nerve cells from normal to highly pathological conditions.

[0043] (2) H2O2 removal efficiency test: The CCHMS sensor was placed in PBS (pH 7.2-7.4) containing 500 μM H2O2, and its H2O2 removal efficiency was evaluated with an electrocatalytic cycle of 1 min. The H2O2 concentration was determined using the Ti(SO4)2 colorimetric method: for each electrocatalytic cycle, 667 μL of electrolyte was added to 1333 μL of Ti(SO4)2 solution, incubated at room temperature for 10 min, and the absorbance of the solution at 405 nm was measured using a UV-Vis spectrophotometer (Evolution 220, Thermo Scientific). The removal rate was calculated using the following formula: Removal rate (%) = (A1–A2) / A1 × 100%, where A1 is the absorbance of the sample before electrocatalysis, and A2 is the absorbance of the sample after electrocatalysis.

[0044] Depend on Figure 17-18 It was found that at a H2O2 concentration of 500 μM, three cycles with a time interval of 1 min were sufficient to restore H2O2 to a near-normal physiological concentration, achieving an H2O2 clearance rate of 80.7%. Furthermore, quantitative results indicated that subsequent three electrocatalytic cycles could further reduce the H2O2 concentration, ultimately achieving a high clearance rate of 91.4%. Figure 19 It can be seen that the residual H2O2 concentration calculated by the current after each catalytic cycle is completed shows a high degree of consistency with the UV-Vis quantitative results based on titanium sulfate (Ti(SO4)2) (ρ=0.998), providing a basis for real-time determination and feedback of cellular H2O2 levels.

[0045] Experimental Example 4: Verification of Dual-Mode Feedback Performance of CCHMS Sensor The mechanism by which the CCHMS sensor regulates neuronal function through electrocatalysis is as follows (see schematic diagram). Figure 1 (i) H2O2 treatment induced the formation of inflammasomes, triggered the production of pro-inflammatory factors, and ultimately led to PC12 cell apoptosis. (ii) CCHMS reduced the concentration of H2O2 in PC12 cells by electrocatalyzing the decomposition of H2O2 into H2O and O2, thereby reshaping the intracellular environment of PC12 cells. Furthermore, based on the current response generated during the electrocatalytic H2O2 process, CCHMS can spatiotemporally and synchronously provide feedback on the remaining H2O2 level in PC12 cells.

[0046] To verify the ability of the CCHMS sensor to simultaneously monitor the H2O2 catalytic process and dopamine concentration during electrocatalytic therapy, the CCHMS sensor was placed in a system containing PC12 cells. Real-time monitoring was performed using the following two modes while an electrocatalytic voltage was applied: Modal 1 (H2O2 feedback): At a potential of 0.05V, the it curve was continuously recorded, and the H2O2 concentration change was calculated in real time based on the current response, reflecting the electrocatalytic process. After regulating intracellular reactive oxygen species (ROS) levels through electrocatalytic H2O2 scavenging using CCHMS, staining with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was performed. Figure 20 It was found that (the control group was a positive control treated with H2O2), the ROS in PC 12 cells treated with the CCHMS sensor was significantly reduced to a level comparable to that of the blank group (no treatment), confirming that the Cu-Co high dual catalytic site can successfully rescue nerve cells by accelerating the electrocatalytic regulation of cellular oxidative stress by H2O2. Furthermore, the CCHMS sensor, characterized by the Cu-Co high dual catalytic site, obtained a more significant and stable steady-state electrocatalytic response current than the Co3O4 sensing probe (see...). Figure 21 More importantly, the H2O2 content in PC12 cells calculated based on these catalytic currents showed results highly consistent with those of commercial cell H2O2 quantification kits (ρ=0.9954) (see...). Figure 22 The consistent baseline current and H2O2 response current increments of the three CCHMS sensors from different batches validated the stability and repeatability of the sensors (see [link to relevant documentation]). Figure 21 This study confirmed the spatiotemporal synchronicity and robustness of the CCHMS sensor in regulating cellular H2O2 levels and providing feedback on H2O2 concentration through electrocatalytic decomposition.

[0047] Modal 2 (dopamine feedback): At 0V, the it curve of another channel was recorded simultaneously, and the dopamine concentration was calculated in real time based on the current response, reflecting changes in pathological signals. CCHMS DPV scans verified that KCl could stimulate PC12 cells to release DA. After the addition of KCl, PC12 cells showed a significant current response compared to L929 cells, which gradually increased with increasing KCl concentration (see...). Figure 23 (Figure A). The rapid and stable oxidative amperometric response of the chronoamperometry spectrum verifies that the CCHMS sensor can provide real-time and rapid feedback on the release of DA from PC12 cells under KCl stimulation (see Figure A). Figure 23 (Figure B). By comparing the chronoamperometry spectra of cell-free and L929 cells, the robust oxidative amperometric response of PC12 cells demonstrated the specificity of CCHMS. The addition of high concentrations of KCl and L929 cells did not observe a response amperometric current or cause baseline current drift. The same number of PC12 cells (1×10⁻⁶) 6 The response current changes obtained from the concentrations of DA released under different KCl stimuli (units / mL) showed a linear fit with the KCl concentration (R0). 2 =0.9973) (see Figure 24 Furthermore, the CCHMS sensor can linearly reflect the correlation between PC12 cell number and DA concentration (R0). 2 =0.9978). There was no significant difference between the DA release results from PC12 cells reported by the CCHMS sensor and the HPLC results (ρ=0.997), demonstrating the high accuracy and reliability of CCHMS in real-time monitoring and acquisition of DA release from PC12 cells (see [link to CCHMS]). Figure 25 ).

[0048] In summary, by comparing the changes in H2O2 scavenging rate and dopamine secretion before and after electrocatalysis, the ability of the CCHMS sensor to achieve spatiotemporal synchronous dual-modal feedback was demonstrated.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sensor for electrocatalytic therapy and spatiotemporal synchronization feedback, characterized in that, It includes a working electrode, a reference electrode, and an auxiliary electrode; the working electrode surface is loaded with an electrocatalyst having dual catalytic active sites; the electrocatalyst is Cu-Co3O4; the electrocatalytic treatment includes the catalytic decomposition of hydrogen peroxide.

2. The sensor according to claim 1, characterized in that, The reference electrode is an Ag / AgCl electrode; the auxiliary electrode is a platinum electrode.

3. A method for preparing a sensor as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Preparation of Cu-Co3O4: S1. Dissolve 2-methylimidazole and a surfactant in water to prepare an imidazole solution; then add a cobalt salt solution to the imidazole solution, let it stand to react, then centrifuge to collect the precipitate, wash and dry the precipitate to obtain the organometallic precursor. S2. Add the organometallic precursor from step S1 to the sulfuric acid solution, stir the reaction, centrifuge to collect the precipitate, wash and dry the precipitate to obtain TA-Co NPs. S3. Add the TA-Co NPs from step S2 to the copper salt solution, stir the reaction, centrifuge to collect the precipitate, wash and dry the precipitate, and then perform stepwise pyrolysis to obtain Cu-Co3O4. (2) Sensor integration: The working electrode, reference electrode and auxiliary electrode are integrated into the microneedle, and the electrodes are connected to the screen-printed electrode leads using silver paste to obtain the sensor.

4. The preparation method according to claim 3, characterized in that, The cobalt salt mentioned in step (1) is at least one of Co(NO3)3·6H2O, CoCl2·6H2O, CoSO4·7H2O, and Co(CH3COO)2·4H2O; the surfactant is hexadecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, or octadecyltrimethylammonium bromide; the copper salt is at least one of CuCl2, Cu(NO3)2, and CuSO4; and the stepwise pyrolysis temperatures are 200℃ and 300℃ respectively.

5. The preparation method according to claim 3, characterized in that, The working electrode in step (2) is a microneedle electrode; the microneedle electrode includes a microneedle substrate, an insulating layer on the surface of the microneedle substrate, a conductive carbon / silver paste layer filled inside the microneedle, and an electrocatalyst supported on the surface of the conductive carbon / silver paste layer.

6. The preparation method according to claim 5, characterized in that, The insulating layer is prepared as follows: microneedles are immersed in a polyurethane solution, purged with N2, and then cured to obtain the insulating layer.

7. The preparation method according to claim 5, characterized in that, The conductive carbon / silver paste layer is plasma-treated and then loaded with an electrocatalyst; the electrocatalyst is loaded by ultrasonic treatment of microneedles placed in a chitosan suspension containing Cu-Co3O4.

8. A device or instrument for electrocatalytic therapy and / or real-time monitoring of electrocatalytic processes or pathological signals in neurological diseases, characterized in that, The device or instrument includes the sensor described in claim 1 or 2.

9. The apparatus or instrument according to claim 8, characterized in that, The real-time monitoring includes the detection of dopamine.

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