A sensing electrode, sensor and method for detecting H2O2 secreted by living cells

Abstract

The present application belongs to the technical field of biosensors, and particularly relates to a sensing electrode for detecting H2O2 secreted by living cells, a sensor and a method. The present application uses carbon cloth as a substrate to in-situ grow cobalt-based metal organic framework precursors, utilizes a phosphate solution to in-situ etch the Co MOF precursors to form cobalt phosphate, and then electro-deposits platinum nanoflowers to obtain a PtNFs / CoPi@CC sensing electrode. On this basis, a non-enzyme biosensor based on cobalt phosphate-platinum nanoflowers derived from Co MOF grown on carbon cloth is constructed to realize in-situ real-time detection of H2O2 secreted by living cells. The flexible electrode has excellent sensitivity, selectivity and reproducibility. The non-enzyme sensor for detecting H2O2 established by the present application has good practicability in biomedical detection and analysis, and provides a new idea and direction for the construction of a new type of device for real-time detection of H2O2 in the field of biomedical research.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a sensing electrode, sensor, and method for detecting H2O2 secreted by living cells. Background Technology

[0002] Hydrogen peroxide (H2O2) is an important component of reactive oxygen species (ROS) and plays a crucial role in physiological and pathological processes. In biological systems, adequate H2O2 is essential for intracellular signal transduction, cell proliferation, and protein synthesis. When H2O2 concentration exceeds a certain limit, it is closely related to a range of diseases, including cell damage, oxidative stress, Alzheimer's disease, and cancer. Therefore, reliable measurement of H2O2 concentration or relative levels is essential. Currently, the main methods for measuring H2O2 include electron paramagnetic resonance (EPR), fluorescence staining, chemiluminescence, chromatography, and spectrophotometry. However, most of these methods require expensive equipment, are complex to operate, and are difficult to widely apply. Electrochemical sensors, on the other hand, have attracted much attention due to their advantages such as low cost, speed, convenience, and applicability for both in vitro and in vivo monitoring. However, the intracellular microenvironment is complex, contains many interfering substances, and the endogenous H2O2 content is extremely small with a short half-life, making the detection of endogenous H2O2 within cells a significant challenge.

[0003] Organometallic frameworks (MOFs) offer a large surface area and numerous catalytic sites, making them an effective strategy for developing superior electrocatalytic materials. Among these, Co-MOFs have garnered significant attention due to their simple and energy-efficient preparation process and unique leaf-like structure, which may provide even more active sites for catalysis. However, there are no reports of using Co-MOF materials alone for substance detection, presumably due to the unique properties of the ligand 2-methylimidazole. 2-methylimidazole is protonated under acidic conditions and deprotonated under alkaline conditions, resulting in insufficient stability under varying pH conditions. To improve the stability of Co-MOFs, many efforts have been made by those skilled in the art, such as high-temperature carbonization and ion exchange methods. Among these, ion exchange methods have attracted considerable attention due to their mild reaction conditions and energy efficiency.

[0004] In the prior art, invention patent CN113433188A discloses a method for preparing a nanoenzyme H2O2 sensor using a nanoflower composite membrane and its application. This invention uses bimetallic nanoflowers with a core-shell structure attached to MOF materials to assemble and prepare an ultrasensitive and rapid nanoenzyme sensor for detecting hydrogen peroxide, with a detection limit of 86 nM. This nanoenzyme sensor incorporates exogenous enzymes, which may interfere with biological systems and are susceptible to enzyme inactivation and degradation. Summary of the Invention

[0005] In view of this, this invention proposes a strategy to combine a flexible CC conductive substrate composed of interlaced carbon fibers suitable for cell growth with a micro / nano array (PtNFs / CoPi) to form a flexible electrode. This invention employs in-situ growth, room temperature etching, and electrochemical deposition methods to prepare the PtNFs / CoPi@CC electrode, achieving cell adhesion growth on the sensing electrode, shortening the reaction distance, and reducing H2O2 loss. Based on this, an enzyme-free biosensor based on Co-MOF-derived cobalt phosphate-platinum nanoflowers (PtNFs / CoPi@CC) grown on carbon cloth is constructed, enabling in-situ real-time detection of H2O2 secreted by living cells.

[0006] One of the objectives of this invention is to provide a PtNFs / CoPi@CC sensing electrode. This invention modifies Co-MOF to synthesize a stable MOF derivative, cobalt phosphate. The sensing electrode prepared in this way has excellent sensitivity, selectivity, and reproducibility, providing technical support for in-situ real-time detection of H2O2 secreted by cells.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The PtNFs / CoPi@CC sensing electrode is prepared by: growing a Co-MOF precursor in situ on a carbon cloth substrate, etching the Co-MOF precursor in situ with a phosphate solution to form CoPi, and then modifying the electrode with PtNFs by electrodeposition to obtain the PtNFs / CoPi@CC sensing electrode.

[0009] This invention selects carbon cloth (CC) as the substrate for the working electrode. A major challenge in detecting endogenous H2O2 in this field lies in its low concentration and short half-life. Therefore, the sensor needs to possess sufficiently rapid and sensitive detection capabilities for H2O2. Carbon cloth, with its large specific surface area, can enhance detection sensitivity, and its fibrous structure can provide more sites for modification of other catalytic materials. Therefore, carbon cloth can significantly improve detection sensitivity and achieve a lower detection limit, providing the possibility for in-situ real-time detection of H2O2 in living cells.

[0010] Furthermore, the CoPi has a coral-like microarray structure.

[0011] Furthermore, the PtNFs are uniformly distributed on the surface of the PtNFs / CoPi@CC sensing electrode.

[0012] This invention proposes a novel Co-MOF modification and stabilization scheme, employing a one-step etching method to synthesize a stable MOF derivative, cobalt phosphate (CoPi), which is then used in subsequent research. It is well known that some noble metals exhibit excellent electrocatalytic activity towards hydrogen peroxide. Therefore, to further improve the sensor's sensitivity and other detection performance, we used electrodeposition to modify the electrode with platinum nanoflowers (Pt nanoflowers, PtNFs). The unique coral-like microarray structure of CoPi not only provides more growth sites for PtNFs, promoting the rapid diffusion of H2O2 on the sensing electrode and thus better leveraging the electrocatalytic activity of PtNFs / CoPi@CC; but also, the unique coral-like structure of CoPi facilitates the adhesion of living cells, shortening the distance between the catalytic material and the cell releasing H2O2, reducing hydrogen peroxide loss, and providing an important guarantee for in-situ real-time detection of cell-secreted H2O2.

[0013] Furthermore, the X-ray diffraction pattern of the PtNFs / CoPi@CC sensing electrode shows two diffraction peaks at 25.6° and 43.4°, corresponding to the 002 and 100 crystal planes of carbon in the carbon cloth; the XRD pattern of the PtNFs / CoPi@CC sensing electrode shows nine diffraction peaks with 2θ values ​​of 39.3°, 45.7°, 66.6°, 80.1°, 84.5°, 20.4°, 25.9°, 36.8°, and 54.4°, corresponding to the 111, 200, 220, 311, and 222 crystal planes of platinum and the -101, 210, 031, and 312 crystal planes of cobalt phosphate, respectively.

[0014] Furthermore, the growth method of the Co-MOF precursor is as follows: a pretreated hammer-shaped carbon cloth electrode with an effective area of ​​1cm×1cm is immersed in a mixture of 2-methylimidazole (2-MIM) aqueous solution and cobalt nitrate hexahydrate (Co(NO3)2·6H2O) aqueous solution and left to stand for 4h, and Co-MOF is grown on the carbon cloth by room temperature crystallization.

[0015] Furthermore, in the mixture, the volume ratio of the 2-methylimidazole aqueous solution and the cobalt nitrate hexahydrate aqueous solution is 1:1, the concentration of the 2-methylimidazole aqueous solution is 0.4M, and the concentration of the cobalt nitrate hexahydrate aqueous solution is 0.05M.

[0016] Furthermore, the carbon cloth pretreatment method is as follows: commercial flexible carbon cloth (CC, 1×1cm) 2To remove impurities, the carbon cloth was ultrasonically treated for 2 minutes each in acetone, ethanol, and ultrapure water. After cleaning, the carbon cloth was immersed in a 1:1 mixture of concentrated nitric acid and concentrated sulfuric acid by volume and boiled for 1 hour to improve its hydrophilicity. After cooling, it was rinsed with a large amount of ultrapure water until deacidified, and the pH of the eluent was measured to be 7.00. The cleaned carbon cloth was dried in a vacuum drying oven at 60°C for later use. The commercially available carbon cloth was cut into hammer-shaped carbon cloth electrodes with an effective area of ​​1 cm × 1 cm using scissors.

[0017] Furthermore, the etching time is 24 hours.

[0018] Furthermore, in the electrodeposition method, the electrodeposition solution is a mixed solution of Na2SO4 and H2PtCl6 with a pH of 7; the concentration of Na2SO4 is 0.01M and the concentration of H2PtCl6 is 2mM.

[0019] Furthermore, the pH value of the mixed solution is adjusted using NaOH.

[0020] Furthermore, the PtNFs electrodeposition time is 4800s and the operating voltage is -0.5V.

[0021] The second objective of this invention is to provide an enzyme-free electrochemical biosensor for in-situ real-time detection of H2O2 secreted by living cells. This living cell sensor has a wide linear detection range for H2O2, with a detection limit as low as 0.222 μM.

[0022] To achieve the above objectives, the present invention adopts the following technical solution:

[0023] An enzyme-free electrochemical biosensor for in-situ real-time detection of H2O2 secreted by living cells, wherein the working electrode of the sensor is the PtNFs / CoPi@CC sensing electrode.

[0024] The third objective of this invention is to provide a method for in situ real-time detection of H2O2 secreted by living cells.

[0025] To achieve the above objectives, the present invention adopts the following technical solution:

[0026] A method for in situ real-time detection of H2O2 secreted by live cells involves attaching the cells to be tested to the PtNFs / CoPi@CC sensing electrode as described in any one of claims 1-7, placing the electrode in an electrochemical detection system, performing electrochemical detection using a chronoamperometry method, and performing qualitative and / or quantitative analysis of the H2O2 secreted by the live cells based on the measured current response.

[0027] Furthermore, the electrochemical detection system is a three-electrode system, with a Pt wire as the counter electrode, an Ag / AgCl electrode as the reference electrode, and a PtNFs / CoPi@CC sensing electrode as the working electrode.

[0028] Furthermore, when the concentration of H2O2 is in the range of 10 μM-26.64 mM, Y = 0.2744X + 0.0636, R 2 =0.9968, where Y is the Y-axis, representing the current signal; X is the X-axis, representing the H2O2 concentration.

[0029] The beneficial effects of this invention are as follows:

[0030] 1. In-situ real-time detection of cellularly secreted hydrogen peroxide (H2O2) is of great significance for exploring the metabolic state of cancer cells and even for biomedical research. This invention utilizes a simple room-temperature crystallization method to in-situ grow Co-MOF on a carbon cloth with a three-dimensional network structure, followed by etching to form a vertically grown cobalt phosphate coral-like array. Then, platinum nanoflowers are uniformly electrodeposited on the surface using a potentiostatic method to fabricate a PtNFs / CoPi@CC flexible electrode, thus establishing a non-enzymatic sensor for H2O2 detection. The unique structure formed by the three-dimensional network of the CC substrate and the cobalt phosphate coral-like array grown on the carbon cloth surface exhibits a large specific surface area and more active sites. This unique structure not only provides excellent catalytic effects but also allows cells to attach to the material, reducing the loss of hydrogen peroxide released by cells and enabling in-situ real-time detection. Furthermore, the addition of Pt nanoflowers enhances the catalytic performance of this composite material for H2O2 detection.

[0031] 2. The PtNFs / CoPi@CC sensor designed in this invention has a superior material structure, exhibiting a low detection limit, good selectivity, and stability. Under optimal conditions, due to the good catalytic efficiency of CoPi and Pt nanoflowers, this sensor demonstrates a wide linear detection range for H2O2 in the concentration range of 10 μM–26.64 mM, with a detection limit as low as 0.222 μM.

[0032] 3. Fluorescence staining and in situ cell growth experiments showed that the PtNFs / CoPi@CC sensing electrode constructed in this invention has good biocompatibility and cell adhesion, and can promptly capture H2O2 released by cells stimulated by drugs. This sensor has good practicality in biomedical detection and analysis, providing a reliable method for the application of bioelectrochemical sensors in biomedical research, and may be a novel device for real-time H2O2 detection in the biomedical research field in the future.

[0033] 4. The PtNFs / CoPi@CC sensing electrode of the present invention does not show a significant current response to other interfering substances such as dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), uric acid (UA), adrenaline (E), norepinephrine (NE), and serotonin (5-HT), and can effectively detect intracellular secreted H2O2 without being affected by other substances. Attached Figure Description

[0034] Figure 1 A is a scanning electron microscope (SEM) image of bare carbon cloth (magnified 4000 times); Figure 1 B is a scanning electron microscope (SEM) image of PtNFs / CoPi@CC (magnified 2500 times); Figure 1 C is a scanning electron microscope (SEM) image of PtNFs / CoPi@CC (6000x magnification); Figure 1 D is the energy dispersive spectroscopy (EDS) image of PtNFs / CoPi@CC; Figure 1 E represents the elemental distribution images (overlay) of the PtNFs / CoPi@CC electrode; Figure 1 F is the distribution image of various elements (including Pt, Co, P, O, C) in PtNFs / CoPi@CC;

[0035] Figure 2 X-ray diffraction pattern of PtNFs / CoPi@CC electrode;

[0036] Figure 3 XPS spectra of PtNFs / CoPi@CC electrodes;

[0037] Figure 4 XPS spectrum of Pt 4f;

[0038] Figure 5 XPS spectrum of Co 2p;

[0039] Figure 6 XPS spectrum of P 2p;

[0040] Figure 7 The XPS spectrum of O1s;

[0041] Figure 8 The cyclic voltammetry curves of bare CC, CoPi@CC, and PtNFs / CoPi@CC electrodes are shown in a 5mM potassium ferricyanide solution containing 0.1M KCl at a scan rate of 50mV / s.

[0042] Figure 9 Electrochemical impedance spectroscopy (EIS) spectra of bare CC, CoPi@CC, and PtNFs / CoPi@CC electrodes in a 5 mM potassium ferricyanide solution containing 0.1 M KCl.

[0043] Figure 10 The graph shows the results of comparing the differences in current-ampere response after continuous addition of 0.5 mM H2O2 to different electrodes, namely bare CC, CoPi@CC and PtNFs / CoPi@CC;

[0044] Figure 11 Cyclic voltammetry curves of the PtNFs / CoPi@CC electrode in the absence of H2O2 (0 mM H2O2) and in the presence of different concentrations of H2O2 (2, 4, 6, 8 mM);

[0045] Figure 12 The it current response curves are shown for the continuous addition of 500 μM H2O2 (4 times) under different PtNFs electrodeposition times.

[0046] Figure 13 A bar chart showing the it current response when 500 μM H2O2 is added continuously (4 times) under different PtNFs electrodeposition times;

[0047] Figure 14 The it current response curves when 500 μM H2O2 is added continuously (4 times) at different operating potentials;

[0048] Figure 15 A bar chart showing the it current response when 500 μM H2O2 is added continuously (4 times) at different operating potentials;

[0049] Figure 16 The figure shows the amperometric response of PtNFs / CoPi@CC when H2O2 is continuously added at -0.5V in 0.1M PBS, with the inset showing the current response of the PtNFs / CoPi@CC electrode at low H2O2 concentration.

[0050] Figure 17 The graph shows the linear relationship between the ampere response and H2O2 concentration (from 10 μM to 26.64 mM).

[0051] Figure 18 The amperometric response curves of the PtNFs / CoPi@CC electrode to the selective addition of 0.5 mM H2O2 and 0.1 mM interfering substance are shown.

[0052] Figure 19 A bar chart showing the selectivity of PtNFs / CoPi@CC electrode to the successive addition of 0.5 mM H2O2 and 0.1 mM interfering agent;

[0053] Figure 20The amperometric response results for five PtNFs / CoPi@CC electrode pairs when 0.5 mM H2O2 was added are shown in the figure. Each electrode was tested four times.

[0054] Figure 21 The bar chart shows the amperometric response of the PtNFs / CoPi@CC electrode to H2O2 on days 3, 6, 9, and 12.

[0055] Figure 22 A microscope image showing PC12 cells growing in a culture dish;

[0056] Figure 23 For the CCK8 experiment: a statistical graph of cell viability obtained by incubating PC-12 cells with naked CC and PtNFs / CoPi@CC electrodes for 24 hours;

[0057] Figure 24 Bright-field and fluorescence imaging and overlay images of PC12 cells stained with Calcein-AM (green) and PI (red);

[0058] Figure 25 A microscope image showing PC12 cells grown on the surface of a PtNFs / CoPi@CC electrode;

[0059] Figure 26 The current response of the PtNFs / CoPi@CC electrode to H2O2 released by PC12 cells under drug stimulation. Detailed Implementation

[0060] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0061] Example 1. Fabrication of PtNFs / CoPi@CC sensing electrode

[0062] Commercial flexible carbon fiber cloth (1×1cm) 2 To remove impurities, the carbon cloth was ultrasonically treated for 2 minutes each in acetone, ethanol, and ultrapure water. After cleaning, the carbon cloth was immersed in a 1:1 mixture of concentrated nitric acid and concentrated sulfuric acid by volume and boiled for 1 hour to improve its hydrophilicity. After cooling, it was rinsed with plenty of ultrapure water until deacidified, and the pH of the eluent was measured to be 7.00. The cleaned carbon cloth was then dried in a vacuum drying oven at 60°C for later use.

[0063] Commercial carbon cloth was cut into hammer-shaped carbon cloth electrodes with an effective area of ​​1cm × 1cm using scissors. Then, 10 mL of 2-methylimidazole aqueous solution (0.4 M) was quickly poured into 10 mL of cobalt nitrate hexahydrate aqueous solution (0.05 M) while stirring. After stirring thoroughly for 5 minutes, the pretreated and trimmed carbon cloth was immersed in the mixture and allowed to stand for 4 hours. Co-MOF was grown on the carbon cloth using a room temperature crystallization method. After removing the carbon cloth, it was washed with ultrapure water and then dried in a vacuum drying oven at 60°C for 1 hour to obtain Co-MOF@CC. The prepared Co-MOF@CC was then etched in phosphate buffered saline (PBS) at room temperature for 24 hours to achieve the conversion of Co-MOF to CoPi on the conductive carbon cloth substrate. The prepared electrode was then washed multiple times with ultrapure water and dried in a 60°C oven to obtain the electrode CoPi@CC. The prepared CoPi@CC electrode was fixed with a platinum sheet clamp and placed in an electrodeposition solution containing 0.01 M Na₂SO₄ and 2 mM H₂PtCl₆, with the pH adjusted to neutral (pH = 7.00) using NaOH. Electrodeposition was performed for 4800 s using chronoamperometry (it) at a working voltage of -0.2 V. After rinsing with ultrapure water, the PtNFs / CoPi@CC sensing electrode was successfully prepared.

[0064] Example 2. Characterization and electrochemical testing of the PtNFs / CoPi@CC sensing electrode

[0065] Morphological observation of PtNFs / CoPi / @CC materials was performed using a Hitachi S-8010 field emission scanning electron microscope (Hitachi, Tokyo, Japan). Energy-dispersive X-ray spectroscopy (EDX) images of PtNFs / CoPi@CC were obtained using an X-MaxN (Oxford Instruments, UK). X-ray photoelectron spectroscopy (XPS) measurements were performed using a K-Alpha (Thermo Fisher Science) spectrometer. X-ray diffraction patterns of PtNFs / CoPi / @CC were obtained using an X-ray diffractometer (XRD-6100, SHIMADZU). Fluorescence imaging was performed using an inverted fluorescence microscope (ECLIPSE Ti2, Nikon).

[0066] A classic three-electrode system (CHI-660E, Shanghai, China) was constructed using Pt wire as the counter electrode, an Ag / AgCl electrode as the reference electrode, and PtNFs / CoPi@CC prepared in Example 1 as the working electrode. The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in a 5 mM potassium ferricyanide solution containing 0.1 M KCl. The CV characterization conditions were: potential range -0.1 V to 0.6 V, scan rate 100 mV / s, and EIS frequency range 100 kHz to 0.1 Hz. Chromatographic and CV methods were performed in 0.1 M PBS (pH = 7.4) solution, with an optimal operating voltage of -0.5 V. The CV operating voltage range was -1.0 V to 1.0 V, and the scan rate was 100 mV / s.

[0067] result:

[0068] (1) Physical characterization of PtNFs / CoPi@CC: such as Figure 1 As shown in Figure A, the carbon cloth is composed of a large number of smooth carbon fibers interwoven together. Scanning electron microscopy (SEM) images show that the smooth surface of the carbon fibers is ideal for modifying other catalytic materials. This 3D network structure of the carbon cloth provides a larger surface area for catalytic material modification and cell growth. After uniform in-situ growth of Co-MOF on the carbon fibers, an in-situ etching process was used to form a coral-like structure. This unique coral-like structure provides a huge specific surface area, which is conducive to the deposition of a large number of Pt nanoflowers and also provides a favorable environment for cell adhesion and growth. Simultaneously, the special coral-like structure allows metabolic products such as H2O2 produced by cells to be closer to the electrode material, shortening the distance from H2O2 to the electrode and reducing concentration loss. Electrodeposition results show that a large number of Pt nanoflowers are uniformly distributed on the surface of PtNFs / CoPi@CC. (See Figure A for details.) Figure 1 B and Figure 1 C. Energy dispersive spectroscopy (EDS) images of the composite material surface show that Co, O, P, C, and Pt elements are uniformly distributed on the electrode surface, such as... Figure 1 D- Figure 1 As shown in Figure F, these data strongly validate the successful preparation of PtNFs / CoPi composites on CC surfaces.

[0069] This embodiment uses X-ray photoelectron spectroscopy (XPS) to study the elemental composition of PtNFs / CoPi@CC. See [link to relevant documentation]. Figure 3 The signals for P, Co, O, C, and Pt were consistent with expectations, such as... Figures 4-7 As shown. Furthermore, the X-ray diffraction pattern of the PtNFs / CoPi@CC electrode is as follows: Figure 2As shown, two distinct diffraction peaks are observed at 25.6° and 43.4°, which correspond precisely to the (002) and (100) crystal planes of carbon contained in the carbon cloth serving as the conductive substrate. Five very weak diffraction peaks in the XRD image, with 2θ values ​​of 39.3°, 45.7°, 66.6°, 80.1°, and 84.5°, correspond to the (111), (200), (220), (311), and (222) crystal planes of platinum, consistent with previous reports. Additionally, four very weak diffraction peaks with 2θ values ​​of 20.4°, 25.9°, 36.8°, and 54.4° match the (-101), (210), (031), and (312) crystal planes of cobalt phosphate, consistent with previous reports. Characterization results from SEM, EDS, XPS, and XRD all indicate the successful construction of the PtNFs / CoPi@CC electrode.

[0070] (2) Electrochemical characterization of PtNFs / CoPi@CC: In this embodiment, the electrochemical properties of bare CC, CoPi@CC, and PtNFs / CoPi@CC electrodes were studied using cyclic voltammetry and AC impedance spectroscopy. Figure 8 As shown, in potassium ferricyanide solution, the bare CC electrode exhibits a small redox peak. After modification with CoPi and PtNFs, the redox peak current of the electrode increases significantly. This is mainly because the coral-like structure of CoPi, derived from the unique metal-organic framework, provides a large number of loading sites for PtNFs, accelerating electron transport and thus improving conductivity. AC impedance spectroscopy is an effective tool for studying the interfacial properties of modified electrodes. Figure 9 As shown, the bare CC impedance is 5.802Ω, the CoPi@CC impedance is 2.966Ω, and the PtNFs / CoPi@CC impedance is 0.647Ω. This demonstrates the varying impedance values ​​of the electrodes (R...). CT The gradual decrease in impedance indicates that PtNFs / CoPi@CC possesses good conductivity, a result of both the unique structure of CoPi and the modification with PtNFs. Furthermore, the trend of impedance change is consistent with the cyclic voltammetry curve, indicating the successful fabrication of the PtNFs / CoPi@CC electrode. Figure 10As shown, the electrocatalytic activity of bare CC, CoPi@CC, and PtNFs / CoPi@CC sensing electrodes for H2O2 was investigated using chronoamperometry. It can be clearly seen that with the continuous addition of H2O2, the current signals of the bare CC, CoPi@CC, and PtNFs / CoPi@CC electrodes all exhibit a stepwise response. The reduction current signal of the PtNFs / CoPi@CC electrode is significantly greater than that of the CoPi@CC electrode, indicating that the catalytic performance of the electrodes for H2O2 is significantly improved with the layer-by-layer modification of the catalytic material. Furthermore, after adding different concentrations of H2O2 (0, 2, 4, 5, 6, and 8 mM) to the detection system (0.1 M PBS, pH = 7.40), the cyclic voltammetry curves showed significant changes around -0.2 V, and the reduction peak potential shifted slightly negatively. Figure 11 As shown.

[0071] (3) Optimization of conditions for H2O2 detection using PtNFs / CoPi@CC: To obtain better catalytic and detection performance, the effects of voltage and PtNFs electrodeposition time on catalytic performance were investigated. Figure 12 The it current response curves of the sensor are shown when 500 μM H₂O₂ is added continuously (4 times) at different PtNF electrodeposition times. Upon addition of H₂O₂, the sensing electrode rapidly generates a current response. Figure 13 As shown in the bar chart, as the electrodeposition time increases from 1200s to 6000s, the current gradually increases accordingly, reaching its peak at 4800s. Figure 14 The it current response curves of the sensor when 500 μM H₂O₂ was continuously added (4 times) at different operating potentials are shown. Figure 15 As shown in the bar chart, the current response of the PtNFs / CoPi@CC electrode to H2O2 gradually increases with the operating potential from -0.1V to -0.6V, reaching a maximum at -0.5V. When the operating potential is further increased to -0.6V, the catalytic current signal weakens. Clearly, the flexible PtNFs / CoPi@CC electrode exhibits the best catalytic performance for H2O2 at -0.5V. These results indicate that when the PtNFs electrodeposition time is 4800 s, the optimal reduction current signal can be obtained when tested in a PBS solution with 0.1 MPa = 7.40 and a voltage of -0.5V.

[0072] Example 3. In situ detection of hydrogen peroxide secreted by cells

[0073] The PtNFs / CoPi@CC prepared in Example 1 was irradiated with ultraviolet light to obtain sterilized PtNFs / CoPi@CC. PC12 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (5% CO2, 37°C). Cells in the exponential growth phase were digested with trypsin, and the digestion was terminated by adding culture medium. After thorough mixing, a cell suspension was obtained. 1 ml of the cell suspension was transferred to a 24-well plate containing sterilized PtNFs / CoPi@CC material. After culturing in an incubator for 24 h, the electrodes attached to the PC12 cells were placed in an electrochemical detection system to measure the H2O2 secreted by the cells in situ. Then, 500 μM ascorbic acid was added to the detection system as a stimulant to stimulate the release of H2O2 from the PC12 cells, and electrochemical detection was performed using chronoamperometry at the optimal potential (-0.5 V). Under the same experimental conditions, 500 U / ml of peroxidase (CAT) was added to the detection system to remove hydrogen peroxide released by cells as a control group.

[0074] Results: Under optimal conditions (PtNFs electrodeposition time 4800 s, operating voltage -0.5 V), a typical current-time curve is shown below. Figure 16 As shown in the figure. The results indicate that the rapid, stepwise current response was obtained by continuously adding H₂O₂ of progressively increasing concentration to 10 ml of 0.1 M PBS solution. Furthermore, Figure 16 The inset shows the it curves for the low concentration region (10-90 μM). This indicates that the electrode can detect response currents down to 10 μM H₂O₂ concentrations. Figure 17 This represents the linear relationship between the current signal and the H₂O₂ concentration. The linear range is from 10 μM to 26.64 mM, and the corresponding linear equation is: I = 0.2744 C + 0.0636 (R 2 =0.9968), where C: H2O2 concentration, in mM; I: current signal, in mA, and the lowest detection limit is calculated to be 0.222 μM (S / N = 3). The detection limit of the PtNFs / CoPi@CC sensor proposed in this invention is comparable to the performance of previously reported sensors for determining H2O2, while its linear range exceeds that of all previously reported sensors of the same type for determining H2O2.

[0075] Example 4. Selectivity, repeatability, and stability study of PtNFs / CoPi@CC electrode

[0076] To evaluate the performance of the constructed electrode, this embodiment demonstrated its selectivity, repeatability, and stability using it curves at an operating voltage of -0.5V. Figure 18The addition of 0.5 mM H₂O₂ showed a significant increase in reduction current, exhibiting a stepwise response. However, the electrode showed no significant current response to other interfering substances (0.1 mM), including dopamine, dihydroxyphenylacetic acid, ascorbic acid, uric acid, adrenaline, noradrenaline, and serotonin. After adding multiple interfering substances, the addition of 0.5 mM H₂O₂ again resulted in almost no change in the current response compared to before the addition of the interfering substances. Meanwhile, Figure 19 The histogram of the current response also demonstrates the good selectivity of this electrode. Furthermore, repeatability and stability are also important indicators for evaluating sensor performance. Five PtNFs / CoPi@CC electrodes were measured with 0.5 mM H₂O₂ added, with each electrode tested four times. Results Figure 20 and Figure 21 As shown, the relative standard deviation (RSD) between electrodes is 2.28%; the electrode constructed in this invention can maintain 93.3% of the initial current response within 12 days, exhibiting good reproducibility and acceptable stability.

[0077] Example 5. Practical application of in situ detection of hydrogen peroxide secreted by cells

[0078] To explore further applications of the sensor of this invention and address the challenge of real-time in-situ detection of H2O2 released from living cells, this invention uses an it curve to monitor and record in real-time the reduction current response generated after H2O2 is released from living cells directly grown and attached to the electrode surface. This strategy can promptly capture and detect hydrogen peroxide released by cancer cells, avoiding the diffusion of H2O2 from the solution to the PtNFs / CoPi@CC electrode, thus ensuring rapid and sensitive detection of the H2O2 signal by the electrode.

[0079] Figure 22 This is a microscope image of PC12 cells growing in a culture dish. You can see the spindle-shaped PC12 cells adhering to the wall of the dish. Figure 23 The image shows a statistical graph of cell viability obtained after incubating PC-12 cells with naked CC and PtNFs / CoPi@CC electrodes for 24 hours. Figure 23 It can be seen that the viability of PC12 cells remained basically unchanged before and after the material modification (cell viability was 86% and 84.2%, respectively), indicating that the electrode material has low toxicity to cells.

[0080] This invention further investigated the biocompatibility of the electrode through live / dead cell staining experiments. PC12 cells grown on PtNFs / CoPi@CC electrodes were fluorescently stained using a Calcein-AM / PI dual staining kit to observe their growth and adhesion. Specifically, 1.5 μL of Calcein-AM and 1.5 μL of PI were added to 1.5 mL of analytical buffer, cells were washed with PBS, and then 250 μL of the Calcein-AM / PI staining solution was added to a 24-well plate and incubated at 37°C in the dark for 30 min. Finally, the growth of PC12 cells on PtNFs / CoPi@CC was recorded under a fluorescence microscope. Calcein-AM is a fluorescent labeling agent for live cells. Once inside the cell, Calcein-AM (which is not luminescent itself) is cleaved by intracellular esterases to form the membrane-impermeable polar molecule Calcein, which remains inside the cell and emits strong green fluorescence. Propidium iodide (PI) stains only dead cells, reaching the nucleus only by penetrating the disordered region of the dead cell membrane. It then embeds itself into the cell's DNA double helix, producing red fluorescence. The results are as follows... Figure 24 As shown, PC12 cells grow stably on the PtNFs / CoPi@CC electrode, and the stained PC12 cells grow well. Figure 24 The red fluorescence representing dead cells was less observed, while a large amount of green fluorescence representing living cells was observed, indicating that the constructed electrode has good biocompatibility and cell adhesion.

[0081] In the following experiments, PtNFs / CoPi@CC electrodes and PC12 cells were cultured in 24-well plates for 24 hours to allow the cells to attach to the carbon fibers. Figure 25 As shown. Figure 26 The current response of the PtNFs / CoPi@CC electrode to H2O2 released from PC12 cells under drug stimulation is shown in the figure. Figure 26 As can be seen, at an operating voltage of -0.5V, the PtNFs / CoPi@CC electrode recorded the real-time current response of PC12 cells to 500μM ascorbic acid (AA). However, when no cells grew on the electrode surface, the same 500μM ascorbic acid (AA) was added as a control, and no current response was observed. Therefore, the novel sensing platform constructed in this invention possesses excellent characteristics for in-situ real-time detection of H2O2 secreted by cells, demonstrating great application potential in future biomedical research.

Claims

1. A PtNFs / CoPi@CC sensing electrode for in-situ real-time detection of H2O2 secreted by living cells, characterized in that, The preparation method of the PtNFs / CoPi@CC sensing electrode is as follows: 1) In-situ growth of Co-MOF precursor on carbon cloth substrate: Specifically, a hammer-shaped carbon cloth electrode with an effective area of ​​1cm×1cm after pretreatment was immersed in a mixture of 2-methylimidazole aqueous solution and cobalt nitrate hexahydrate aqueous solution and left to stand for 4h. Co-MOF was grown on carbon cloth by room temperature crystallization method. 2) CoPi was formed by in-situ etching of the Co-MOF precursor using phosphate solution for 24 hours; the CoPi had a coral-like microarray structure. 3) PtNFs were modified on the electrode by electrodeposition to obtain the PtNFs / CoPi@CC sensing electrode; the electrodeposition solution was a mixed solution of Na2SO4 and H2PtCl6 with a pH of 7; the concentration of Na2SO4 was 0.01M and the concentration of H2PtCl6 was 2mM; the PtNFs electrodeposition time was 4800s and the operating voltage was -0.5V.

2. The sensing electrode according to claim 1, characterized in that, The PtNFs are uniformly distributed on the surface of the PtNFs / CoPi@CC sensing electrode.

3. The sensing electrode according to claim 1, characterized in that, The X-ray diffraction pattern of the PtNFs / CoPi@CC sensing electrode shows two diffraction peaks at 25.6° and 43.4°, corresponding to the 002 and 100 crystal planes of carbon in the carbon cloth. The XRD pattern of the PtNFs / CoPi@CC sensing electrode shows nine diffraction peaks with 2θ values ​​of 39.3°, 45.7°, 66.6°, 80.1°, 84.5°, 20.4°, 25.9°, 36.8°, and 54.4°, corresponding to the 111, 200, 220, 311, and 222 crystal planes of platinum and the -101, 210, 031, and 312 crystal planes of cobalt phosphate, respectively.

4. An enzyme-free electrochemical biosensor for in-situ real-time detection of H2O2 secreted by living cells, characterized in that, The working electrode of the sensor is the PtNFs / CoPi@CC sensing electrode as described in any one of claims 1-3.

5. A method for in situ real-time detection of H2O2 secreted by living cells, characterized in that, The cells to be tested are attached to the PtNFs / CoPi@CC sensing electrode according to any one of claims 1-3, the electrode is placed in an electrochemical detection system, electrochemical detection is performed using chronoamperometry, and qualitative and / or quantitative analysis of H2O2 secreted by live cells is performed based on the measured current response.

6. The method according to claim 5, characterized in that, When the concentration of H2O2 is in the range of 10 μM-26.64 mM, Y = 0.2744X + 0.0636, R 2 =0.9968, where Y is the Y-axis, representing the current signal; X is the X-axis, representing the H2O2 concentration.

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