Microelectrode based on PtN1C3 and preparation method and application thereof

Abstract

The invention belongs to the technical field of electroanalytical chemistry, and particularly relates to a PtN1C3-based microelectrode as well as a preparation method and application thereof. The microelectrode comprises an electrode, a compound layer and a polymer layer, wherein the composite layer is modified on the surface of the electrode, and the polymer layer is modified on the surface of the electrode with the modified composite layer; the composite layer includes a PtN1C3 monatomic catalyst. The preparation method comprises the following steps: modifying a PtN1C3 monatomic catalyst on the surface of bare CFE by utilizing electrochemical deposition to obtain PtN1C3 / CFE; the PtN1C3 / CFE is deposited through PEDOT: TFB, and the surface of the PtN1C3 / CFE is modified with PEDOT, so that the PEDOT / PtN1C3 / CFE is obtained. The microelectrode adopts asymmetric coordination PtN1C3 as a catalyst, successfully realizes in-situ dynamic monitoring of hydrogen peroxide in cells and living brain, and has the characteristics of high sensitivity and high selectivity.

Description

Technical Field

[0001] This invention belongs to the field of electroanalytical chemistry technology, specifically relating to a PtN1C3-based microelectrode, its preparation method, and its application. Background Technology

[0002] Hydrogen peroxide (H2O2), as a key reactive oxygen species, plays a dual role in the brain, acting as both a physiological signal transduction agent and a pathological damage agent. Under physiological conditions, H2O2 participates in regulating synaptic plasticity, neurotransmitter release, and glial cell-neuron communication, which is crucial for higher functions such as learning and memory. Pathologically, abnormal accumulation of H2O2 triggers oxidative stress, directly leading to neuronal death, blood-brain barrier disruption, and a neuroinflammatory cascade, serving as a common pathological hub in diseases such as Alzheimer's disease, Parkinson's disease, and stroke. Precise and specific in-situ dynamic monitoring of neurochemicals such as H2O2 in the brain is helpful in revealing physiological and pathological changes in the brain.

[0003] Electrochemical sensing platforms based on implantable microelectrodes offer an effective means of tracking neurochemical substances in vivo due to their excellent spatiotemporal resolution and mechanical adaptability. However, due to the complexity of the brain environment, microelectrodes inevitably undergo cross-reactions with other active substances, leading to changes in current intensity. Unlike background currents that can be removed by filtering algorithms or machine learning, unwanted current intensity changes caused by side reactions can overlap with the target current signal and become difficult to distinguish, thus affecting the accuracy and reliability of neurochemical sensing. While existing sensing technologies enhance specificity through membrane confinement effects and charge repulsion, in-situ polymerization modification of conductive polymers can easily mask catalytically active sites, leading to sensitivity degradation. Therefore, there is an urgent need to develop a new microelectrode design approach to synergistically optimize sensitivity and anti-interference capabilities.

[0004] In the prior art, patent CN113786853B discloses a single-atom catalyst and its preparation method, as well as a microelectrode and its preparation method and application. The microelectrode includes an electrode, a composite layer coated on the electrode surface, and a polymer layer covering the composite layer. The composite layer contains a single-atom catalyst or a mixture of a single-atom catalyst and carbon material. The single-atom catalyst includes graphitic carbon nitride as a support and copper single atoms supported on the support as an active component; the atomic percentage of the active component in the single-atom catalyst is 0.07-0.2%; the graphitic carbon nitride is mpg-C3N4. This microelectrode uses a highly selective single-atom catalyst as an electrocatalyst, achieving accurate detection of hydrogen peroxide on this electrode, while remaining unaffected by oxygen and most other substances in the brain.

[0005] Single-atom catalysts (SACs) have shown groundbreaking potential in the field of sensing due to their maximized metal atom utilization, unique coordination, and electronic structure. However, in applications involving complex real-world samples, the long-term stability, sensitivity, safety, and resistance to background interference of SAC-based microelectrodes require further improvement and validation. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a microelectrode that uses asymmetrically coordinated PtN1C3 with high selectivity and high sensitivity as a catalyst to support in-situ dynamic monitoring of hydrogen peroxide in cells and living brains.

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

[0008] A microelectrode, the microelectrode comprising an electrode, a composite layer, and a polymer layer;

[0009] The composite layer is modified on the electrode surface, and the polymer layer is modified on the electrode surface of the modified composite layer;

[0010] The composite layer includes a PtN1C3 single-atom catalyst, which comprises a support and a metal active component; the support is a nitrogen-doped multi-walled carbon nanotube; the metal active component is Pt; the metal active component is dispersed on the support in the form of isolated single atoms; the Pt is coordinated with 3 carbon atoms and 1 nitrogen atom to form a PtN1C3 coordination configuration.

[0011] Furthermore, the microelectrode is an implantable microelectrode.

[0012] Furthermore, the electrode is a carbon fiber electrode; the polymer layer is PEDOT.

[0013] Furthermore, the PtN1C3 single-atom catalyst contains a single N atom dopant, which causes the electronic structure of the central metal atom to become asymmetric.

[0014] Furthermore, the Pt loading in the PtN1C3 single-atom catalyst is 0.5-2.0 wt%, preferably 1.0-1.5 wt%, and most preferably 1.26 wt%.

[0015] Furthermore, the d-band center of the central Pt atom in the PtN1C3 single-atom catalyst is -2.662 eV.

[0016] This invention reveals that the designability of SACs (Self-Coordination Asymmetric Electrodes) promises to achieve high selectivity without sacrificing sensitivity. Specifically, by employing different strategies to modulate the electron cloud structure of the SAC, the d-band center is shifted in the desired direction, thereby achieving optimal adsorption strength for the target substance while preventing other cross-reactions. Current manipulation techniques, such as defect site construction, axial ligand introduction, and heteroatom doping, all influence the electron cloud configuration by introducing asymmetric factors into periodic mirror structures. Further research shows that these systems containing asymmetric factors often exhibit superior intrinsic activity and selectivity. Therefore, asymmetric coordination engineered SACs developed based on symmetry-breaking strategies may provide a new approach for developing implantable microelectrodes that combine both sensitivity and selectivity.

[0017] This invention further extends this theoretical advantage to the field of neurochemical sensing, and reports the atomic-level electronic structure reconstruction of Pt metal active sites through a symmetry destruction strategy to create PtN1C3SAC that combines high sensitivity and high selectivity, and uses it as a catalytic material for sensing hydrogen peroxide (H2O2) in the brain. This invention uses nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) fully impregnated with K2PtCl4 solution as a precursor. By controlling the ratio of dopant to precursor and performing high-temperature annealing, Pt-N-MWCNTs with asymmetric N1C3 coordination and typical N4 coordination in the first shell were successfully prepared. Through theoretical calculations and simulations, we found that the asymmetry of the electronic structure of the central metal atom caused by single N atom doping shifts the d-band center of the Pt atom (-2.662 eV) upward compared to PtN4 (-2.725 eV), bringing it closer to the Fermi level, making it more sensitive than other PtN... x C 4-x The (x=0,2-4) configuration better balances the adsorption and desorption processes, thus exhibiting a lower rate-determining step (RDS) energy barrier (0.173 eV). Furthermore, in reactions with common neurochemicals, the RDS barriers of each interfering agent are significantly higher than the 0.173 eV RDS in the hydrogen peroxide reduction reaction (HPRR), indicating that thermodynamically, the interfering agents are virtually impossible to react on the PtN1C3 surface.

[0018] This invention develops a novel PEDOT / PtN1C3 / CFE based on PtN1C3 and systematically evaluates its electrochemical performance in vitro. Subsequently, the PEDOT / PtN1C3 / CFE was used to monitor H2O2 kinetic changes in the living brain induced by resorcinol (Res) and rosmarinic acid (RA) to verify its application in real-world complex environments. Results show that the PEDOT / PtN1C3 / CFE can effectively monitor H2O2 kinetic changes in the living brain induced by resorcinol and rosmarinic acid, exhibiting high sensitivity and selectivity. This microelectrode design provides new insights for developing higher-performance SAC microelectrode platforms based on symmetry disruption strategies, enabling high-performance sensing of neurochemical substances in the living brain.

[0019] Furthermore, the PtN1C3 single-atom catalyst is prepared using the following method:

[0020] 1) K2PtCl4 precursor was mixed with N-MWCNT and reacted to prepare K2PtCl4 / N-MWCNT;

[0021] 2) The K2PtCl4 / N-MWCNT obtained in step 1) was mixed and ground with urea, and then thermally annealed to obtain a PtN1C3 single-atom catalyst;

[0022] The mass ratio of K2PtCl4 / N-MWCNT to urea is 1:3-6.

[0023] Preferably, the mass ratio of the K2PtCl4 precursor to the N-MWCNT is 1 to 2:1, and more preferably 9:8.

[0024] Preferably, the reaction conditions for step 1) are: magnetic stirring; the magnetic stirring speed is 1200-2000 rpm, and the time is 3-6 hours.

[0025] Preferably, the magnetic stirring conditions are vigorous stirring at 1500 rpm for 4 hours.

[0026] Preferably, the solution obtained from the reaction in step (1) is rapidly frozen and then freeze-dried under vacuum to obtain K2PtCl4 / N-MWCNT powder.

[0027] More preferably, the freezing temperature is -70 to -90°C, and even more preferably -80°C.

[0028] As a preferred option, step (1) is as follows:

[0029] Step A: Dissolve the K2PtCl4 precursor in ultrapure water to prepare a Pt stock solution;

[0030] Step B: Disperse the N-MWCNTs in ultrapure water and sonicate to ensure complete dispersion, thereby obtaining an N-MWCNT suspension;

[0031] 3) The Pt stock solution obtained in step A is added dropwise to the N-MWCNT suspension obtained in step B. Pt is loaded onto carbon nanotubes by magnetic stirring. After rapid freezing, vacuum freeze-drying is performed to obtain K2PtCl4 / N-MWCNT powder.

[0032] Preferably, in step A, the concentration of the Pt stock solution is 2-5 mg / mL, more preferably 3 mg / mL.

[0033] Preferably, in step B, the ultrasonic treatment time is 20-60 minutes, more preferably 30 minutes.

[0034] Preferably, in step 2), the mass ratio of K2PtCl4 / N-MWCNT to urea is 1:4.

[0035] Furthermore, in step 2), thermal annealing is performed under a N2 atmosphere.

[0036] Preferably, hot annealing is carried out in a tube furnace.

[0037] Further, in step 2), the thermal annealing includes: heating to 700-1000°C at a rate of 8-20°C per minute, holding the reaction at that temperature for 40-120 minutes, and then cooling to room temperature to obtain a PtN1C3 single-atom catalyst.

[0038] Preferably, in step 2), the thermal annealing includes: heating to 750-850°C at a rate of 8-15°C per minute, holding the reaction at that temperature for 50-70 minutes, and then cooling to room temperature to obtain a PtN1C3 single-atom catalyst.

[0039] More preferably, in step 2), the thermal annealing includes: heating to 800°C at a rate of 10°C per minute, holding the reaction at that temperature for 1 hour, and then cooling to room temperature to obtain a PtN1C3 single-atom catalyst.

[0040] The second objective of this invention is to provide a method for preparing the aforementioned microelectrode.

[0041] To achieve the above objectives, the present invention adopts the following technical solution:

[0042] The fabrication method of microelectrodes includes the following steps:

[0043] (1) PtN1C3 single-atom catalyst was modified onto the surface of bare CFE by electrochemical deposition to obtain PtN1C3 / CFE;

[0044] (2) The PtN1C3 / CFE obtained in step (1) is deposited with PEDOT:TFB to modify the surface of the PtN1C3 / CFE with PEDOT to obtain PEDOT / PtN1C3 / CFE.

[0045] Further, in step (1), the electrochemical deposition uses bare CFE as the working electrode, Pt wire as the counter electrode, and Ag / AgCl as the reference electrode; the electrochemical deposition is performed by electrodeposition at a potential of 1.0-2.0V for 4500-6500 seconds using the amperometric method; more preferably, it is performed by electrodeposition at a potential of 1.5V for 5400 seconds.

[0046] Furthermore, in step (1), the deposition solution is a mixture of PtN1C3 and acetonitrile.

[0047] Preferably, the concentration of the deposition solution is 0.5-2.0 mg / mL, more preferably 1 mg / mL.

[0048] Preferably, PtN1C3 is ultrasonically dispersed in acetonitrile to form a 1 mg / mL deposition solution.

[0049] Furthermore, in step (2), the electrolyte is an acetonitrile solution containing 0.005-0.03M EDOT and 0.05-0.3M TFB; the PEDOT:TFB deposition is performed at 1.0-1.5V for 15-60 seconds.

[0050] Preferably, in step (2), the electrolyte is an acetonitrile solution containing 0.01M EDOT and 0.1M TFB.

[0051] Preferably, in step (2), the PEDOT:TFB deposition is performed at 1.3V for 20 seconds.

[0052] As a preferred embodiment, the method for preparing the microelectrode specifically includes the following steps:

[0053] (1) PtN1C3 was ultrasonically dispersed in acetonitrile to form a deposition solution; bare CFE was used as the working electrode, Pt wire as the counter electrode, and Ag / AgCl as the reference electrode; electrodeposition was performed at a potential of 1.5V for 5400 seconds by the Ampere method; after deposition, the electrode was removed, washed three times with ultrapure water, and dried to obtain PtN1C3 / CFE;

[0054] (2) PEDOT:TFB deposition: Using an acetonitrile solution containing 0.01MEDOT and 0.1M TFB as the electrolyte, a voltage of 1.3V was applied for 20 seconds to deposit PEDOT on the electrode surface to obtain PEDOT / PtN1C3 / CFE.

[0055] As a preferred option, acetonitrile should be used before the day of use. The molecular sieve is kept dry.

[0056] Preferably, the monomer solution is bubbled with N2 before electrodeposition.

[0057] As a preferred embodiment, the carbon fiber electrode is prepared using the following method:

[0058] S1: Using a micropipette puller, glass capillaries are drawn into two conical glass-coated carbon fiber tip electrodes. The fine tip of each tip is broken into 10-15 μm diameters. The drawn capillaries are used as sheaths for CFEs.

[0059] S2: The carbon fiber was ultrasonically treated in acetone, HNO3, KOH and ultrapure water in sequence for 5 minutes each; then it was vacuum dried.

[0060] S3: Connect a single carbon fiber on a clean glass plate to a copper wire that is slightly longer than the glass capillary tube using silver conductive adhesive. After a period of time, carefully insert the carbon fiber with the copper wire attached into the capillary tube, so that the carbon fiber is exposed at the narrow opening end of the capillary tube and the copper wire is exposed at the other end of the capillary tube, thus creating CFEs.

[0061] S4: Immerse the end of the glass capillary vertically into the molten paraffin bath, being careful not to let the paraffin come into contact with the carbon fiber tip. Then place it in room temperature air to allow the paraffin to solidify. Finally, under a microscope, use a scalpel to cut the exposed CFEs to about 200 μm to 250 μm.

[0062] Preferably, in S1, the inner diameter of the glass capillary is 1.2-2 mm and the length is 90-150 mm.

[0063] As a more preferred embodiment, in S1, the inner diameter of the glass capillary is 1.5 mm and the length is 100 mm.

[0064] Preferably, in S2, the diameter of the carbon fiber is 6-8 μm, more preferably 7 μm.

[0065] Preferably, in S2, the concentration of HNO3 is 2-5M, more preferably 3M; and the concentration of KOH is 0.5-2M, more preferably 1M.

[0066] Preferably, in S2, each ultrasonic treatment lasts for 4-10 minutes, more preferably for 5 minutes.

[0067] Preferably, in S2, the vacuum drying temperature is 50-70°C and the time is 5-8 hours; more preferably, the vacuum drying is carried out at 60°C for 6 hours.

[0068] Preferably, in S3, the diameter of the copper wire is 150-250 μm, more preferably 200 μm.

[0069] Preferably, in step S3, the placement is carried out at 70-90°C for 0.5-2 hours, and more preferably at 80°C for 1 hour.

[0070] The third objective of this invention is to provide an application of the aforementioned microelectrode in in-situ dynamic monitoring of H2O2 levels.

[0071] The fourth objective of this invention is to provide an application of the aforementioned microelectrode in monitoring the kinetic changes of H2O2 in cells and / or the living brain.

[0072] The fifth objective of this invention is to provide an application of the aforementioned microelectrode in monitoring the dynamic changes of H2O2 in cells and / or living brains induced by resorcinol and rosmarinic acid.

[0073] The beneficial effects of this invention are as follows:

[0074] 1. This invention develops a novel microelectrode (PEDOT / PtN1C3 / CFE) based on PtN1C3. This microelectrode can sensitively and specifically monitor the dynamic changes of hydrogen peroxide in the brain of live rats induced by resorcinol and rosmarinic acid. This invention provides new insights into the design of electrochemical sensing platforms using asymmetric coordination-engineered single-atom catalysts, and also offers a new option for high-performance sensing of neurochemical substances in the live brain. This is of great significance for studying the kinetic changes of hydrogen peroxide in the live brain and its related physiological and pathological processes.

[0075] 2. The microelectrode of this invention uses asymmetrically coordinated PtN1C3 as a catalyst, successfully achieving in-situ dynamic monitoring of hydrogen peroxide in cells and the living brain. Furthermore, this microelectrode exhibits good electrocatalytic performance and mechanical stability in the reduction of H2O2.

[0076] 3. The microelectrode of the present invention has the characteristic of high selectivity. Common interfering substances in the brain, such as ascorbic acid, dopamine, DOPAC, glucose, 5-hydroxytryptamine, and adrenaline, cannot interfere with the sensor's detection of H2O2.

[0077] 4. The microelectrode of this invention has the characteristic of high sensitivity. Its sensitivity is 692.1 μA·mM. -1 ·cm -2 The detection limit is as low as 0.845 μM.

[0078] 5. The microelectrode of the present invention has excellent antifouling properties and can effectively resist non-specific protein adsorption, making it very suitable for continuous selective monitoring of H2O2 in vivo.

[0079] 6. The microelectrode of the present invention can achieve long-term stable monitoring of H2O2, is non-cytotoxic, and has excellent biocompatibility, further ensuring the safety of live implantation. Attached Figure Description

[0080] Figure 1 Figure 1 shows the experimental results of the synthesis and characterization of PtN1C3 and PtN4; where, Figure 1 -a is a schematic diagram of the synthesis process of Pt-N-MWCNTs; Figure 1 -b is a TEM image of PtN1C3; Figure 1 -c is the HAADF-STEM image (10nm) of PtN1C3; Figure 1 -d is a HAADF-STEM image (5nm) of PtN1C3, with the insertion mark indicating the Pt content measured by inductively coupled plasma mass spectrometry. Figure 1 -e is a HAADF-STEM image (2nm) of PtN1C3, where red circles indicate the presence of single metal atoms; Figure 1 -f is the EDS element mapping image (overlay) corresponding to PtN1C3; Figure 1 -g is a distribution image of C, N, O and Pt elements in PtN1C3; Figure 1 -h is the EDS energy spectrum of N element in PtN1C3; Figure 1 -i represents the EDS energy spectrum of Pt element in PtN1C3; Figure 1 -j represents the XRD patterns of PtN1C3, PtN4, and N-MWCNT;

[0081] Figure 2 TEM image of PtN4;

[0082] Figure 3 This is an annular dark-field scanning transmission electron microscope (AC HAADF STEM) image of PtN4, where red circles indicate the presence of single metal atoms;

[0083] Figure 4 The full EDS spectrum of PtN1C3;

[0084] Figure 5 The figures show the atomic structure analysis results of PtN1C3 and PtN4 using X-ray absorption spectroscopy; where, Figure 5 -a is the XPS full spectrum of PtN1C3; Figure 5 -b is the N1s high-resolution XPS spectrum of PtN1C3; Figure 5 -c is the high-resolution XPS spectrum of Pt4f for PtN1C3; Figure 5 -d is the XPS full spectrum of PtN4; Figure 5 -e is the N1s high-resolution XPS spectrum of PtN4; Figure 5 -f represents the high-resolution XPS spectrum of Pt4f for PtN4; Figure 5 -g represents the Pt L3 edge X-ray absorption near-edge structure (XANES) spectrum of PtN1C3, Pt foil, and PtO2; Figure 5 -h represents the Fourier transform extended X-ray absorption fine structure (FT-EXAFS) spectra of PtN1C3, Pt foil, and PtO2; Figure 5 -i represents the EXAFS fitting curve of PtN1C3 in R space; Figure 5 -j represents the XANES spectra of PtN4, Pt foil, and PtO2 in PtL3; Figure 5 -k represents the EXAFS spectra of PtN4, Pt foil, and PtO2; Figure 5 -l is the EXAFS fitting curve of PtN4 in R space; Figure 5 -m represents the k of the Pt foil (PtN1C3 reference sample). 2 Wavelet transform (WT) plot of weighted EXAFS signal; Figure 5 -n is the k of PtO2 (PtN1C3 reference sample). 2 Wavelet transform diagram of weighted EXAFS signal; Figure 5 -o is the k of PtN1C3 2 Wavelet transform diagram of weighted EXAFS signal; Figure 5 -p represents the k of the Pt foil (PtN4 reference sample). 2 Wavelet transform diagram of weighted EXAFS signal; Figure 5 -q represents the k of PtO2 (PtN4 reference sample). 2 Wavelet transform diagram of weighted EXAFS signal; Figure 5 -r is the k of PtN4 2 Wavelet transform diagram of weighted EXAFS signal;

[0085] Figure 6 High-resolution XPS spectra of C1s and O1s of PtN1C3; among which, Figure 6 -a is the high-resolution XPS spectrum of PtN1C3's C1s; Figure 6 -b is the O1s high-resolution XPS spectrum of PtN1C3;

[0086] Figure 7 High-resolution XPS spectra of C1s and O1s of PtN4; among which, Figure 7 -a is the high-resolution XPS spectrum of PtN4 in C1s; Figure 7 -b is the O1s high-resolution XPS spectrum of PtN4;

[0087] Figure 8 The image shows the EXAFS spectral fits of the Pt foil (PtN1C3 reference sample) in k-space and R-space; where, Figure 8 -a is the EXAFS spectral fitting diagram of Pt foil (PtN1C3 reference sample) in k-space; Figure 8 -b is the EXAFS spectrum fitting diagram of Pt foil (PtN1C3 reference sample) in R space;

[0088] Figure 9 The image shows the EXAFS spectral fits of the Pt foil (PtN4 reference sample) in k-space and R-space; where, Figure 9 -a is the EXAFS spectral fitting diagram of Pt foil (PtN4 reference sample) in k-space; Figure 9 -b is the EXAFS spectral fit plot of Pt foil (PtN4 reference sample) in R space;

[0089] Figure 10 The EXAFS fit plot of PtN1C3 in k-space;

[0090] Figure 11 The EXAFS fit plot of PtN4 in k-space;

[0091] Figure 12 The amperometric responses and corresponding fitted curves of PtN1C3 / GCE and PtN4 / GCE with continuous addition of 0.5 mM H2O2 are shown; among them, Figure 12 -a is the amperometric response of PtN1C3 / GCE and PtN4 / GCE with continuous addition of 0.5mM H2O2; Figure 12 -b is Figure 12 -a The corresponding fitted line plot;

[0092] Figure 13 To determine the different PtN values ​​in a neutral environment with an electrode potential of -0.3V. x C 4-x Calculated free energy diagram of HPRR configuration;

[0093] Figure 14 The optimized structures of H2O2 adsorbed on PtC4, PtN1C3, PtN2C2-1, PtN2C2-2, PtN3C1, and PtN4, and the calculated charge density differences among PtC4, PtN1C3, PtN2C2-1, PtN2C2-2, PtN3C1, and PtN4 are plotted; among them, Figure 14 -a is the optimized structural diagram of H2O2 adsorbed on PtC4; Figure 14 -b is the optimized structural diagram of H2O2 adsorbed on PtN1C3; Figure 14 -c is the optimized structural diagram of H2O2 adsorbed on PtN2C2-1; Figure 14 -d is the optimized structural diagram of H2O2 adsorbed on PtN2C2-2; Figure 14-e represents the optimized structural diagram of H2O2 adsorbed on PtN3C1; Figure 14 -f is the optimized structural diagram of H2O2 adsorbed on PtN4; Figure 14 -g is the calculated charge density difference diagram for PtC4; Figure 14 -h is the calculated charge density difference diagram for PtN1C3; Figure 14 -i is the calculated charge density difference plot for PtN2C2-1; Figure 14 -j is the calculated charge density difference plot of PtN2C2-2; Figure 14 -k is the calculated charge density difference plot of PtN3C1; Figure 14 -l is the calculated charge density difference diagram for PtN4;

[0094] Figure 15 For different PtN x C 4-x The calculated band structure and projected density of states (PDOS) of the Pt d orbitals are shown in the figure; among them, Figure 15 -a is the calculated band structure result of PtC4; Figure 15 -b is the calculated Pt d orbital density of states of PtC4; Figure 15 -c is the calculated band structure result of PtN1C3; Figure 15 -d is the calculated Pt d orbital projected density of states diagram for PtN1C3; Figure 15 -e is the calculated band structure result of PtN2C2-1; Figure 15 -f is the plot of the calculated Pt d orbital density of states for PtN2C2-1; Figure 15 -g is the calculated band structure diagram of PtN2C2-2; Figure 15 -h is the plot of the calculated Pt d orbital projected density of states for PtN2C2-2; Figure 15 -i is the calculated band structure result of PtN3C1; Figure 15 -j is the plot of the calculated Pt d orbital projected density of states of PtN3C1; Figure 15 -k is the calculated band structure diagram of PtN4; Figure 15 -l is the calculated Pt d orbital density of states of PtN4;

[0095] Figure 16 The graph shows the selective experimental results for PtN1C3; where, Figure 16 -a is a schematic diagram of PtN1C3 selectivity; Figure 16 -b is the amperometric response curve of PtN1C3 / GCE in 0.1M PBS to 100μM H2O2, 3,4-dihydroxyphenylacetic acid, glucose, adrenaline, dopamine, uric acid, ascorbic acid, 5-hydroxytryptamine, and the addition of 100μM H2O2 again. Figure 16 -c is Figure 16 -b corresponding statistical bar chart (n=3); Figure 16 -d represents the free energy diagram of the reaction of H2O2 on PtN1C3, with the inset showing the corresponding catalytic mechanism schematic diagram; Figure 16 -e represents the free energy diagram of the reaction of AA on PtN1C3, where the inset is a schematic diagram of the corresponding catalytic mechanism; Figure 16 -f is the free energy diagram of the reaction of 5-HT on PtN1C3, where the inset is a schematic diagram of the corresponding catalytic mechanism; Figure 16 -g represents the free energy diagram of the reaction of DA on PtN1C3, with the inset showing the corresponding catalytic mechanism schematic diagram; Figure 16 -h is the free energy diagram of the reaction of Glu on PtN1C3, where the inset is a schematic diagram of the corresponding catalytic mechanism; Figure 16 -i represents the free energy diagram of the reaction of UA on PtN1C3, where the inset is a schematic diagram of the corresponding catalytic mechanism; Figure 16 -j is the Pt d orbital PDOS calculation result diagram when H2O2 reacts on PtN1C3; Figure 16 -k is the plot of the calculated projection density of states of the Pt d orbitals and Op orbitals when AA reacts on PtN1C3; Figure 16 -l is the calculated Pt d orbital projected density of states for the reaction of 5-HT on PtN1C3; Figure 16 -m is the plot of the calculated projection density of states of the Pt d orbitals and Op orbitals when DA reacts on PtN1C3; Figure 16 -n is the calculated density of states of the Ptd and Op orbitals of Glu during the reaction on PtN1C3; Figure 16 -o is the calculated Pt d orbital density of states for the reaction of UA on PtN1C3;

[0096] Figure 17 The graph shows the calculated Hamiltonian population (COHP) of the crystal orbitals of dopamine.

[0097] Figure 18 The graph shows the COHP calculation results of the Pt-O bond in the ascorbic acid reaction;

[0098] Figure 19 The graph shows the COHP calculation results for Pt-O bonds in the glucose reaction;

[0099] Figure 20 Figures show the experimental results related to the construction and electrochemical performance of PEDOT / PtN1C3 / CFE; among them, Figure 20 -a is a schematic diagram of the PEDOT / PtN1C3 / CFE preparation process; Figure 20 -b is the SEM image of the bare CFE; Figure 20-c is the SEM image of PEDOT / PtN1C3 / CFE; Figure 20 -d represents bare CFE, PtN1C3 / CFE, and PEDOT / PtN1C3 / CFE in 5 mM [Fe(CN)6] containing 0.1 M KCl. 3- / 4- EIS diagram in solution; Figure 20 -e is a graph showing the response of PEDOT / PtN1C3 / CFE to different interfering substances; Figure 20 -f is the cyclic voltammetry (CV) curve of PEDOT / PtN1C3 / CFE in 2mM H2O2 solution at different scan rates (ν); Figure 20 -g is used for Figure 20 -f Corresponding current versus scan rate graph; Figure 20 -h is a typical cyclic voltammetry curve of PEDOT / PtN1C3 / CFE in 0.1M PBS solution (pH 7.40) with 0mM, 2mM and 4mM H2O2 added, with a scan rate ν = 100mV / s; Figure 20 -i is the Ampere current response of PEDOT / PtN1C3 / CFE to the continuous addition of 5μM to 100μM (5, 10, 20, 50, 100μM) H2O2; Figure 20 -k is the Ampere current response of PEDOT / PtN1C3 / CFE to the continuous addition of 200μM to 5mM (0.2, 0.5, 1, 2, 5mM) H2O2; Figure 20 -l is an image of PEDOT / PtN1C3 / CFE before bending; Figure 20 -m is the image after the 50th bend of PEDOT / PtN1C3 / CFE; Figure 20 -n is the CV curve of PEDOT / PtN1C3 / CFE under different bending times in 0.1M PBS solution (pH 7.40) containing 4mM H2O2; Figure 20 -o is the Ampere current response diagram of PEDOT / PtN1C3 / CFE and bare CFE to 1mM H2O2, where I0 and I represent the current values ​​at the initial time and the specified time, respectively; Figure 20 -p is Figure 20 -o corresponding statistical bar chart (n=3); Figure 20 -q is a graph showing the cell viability of PC12 cells after 24 hours of co-incubation with PEDOT / PtN1C3 / CFE or naked CFE (n=3); Figure 20 -r is a bright-field and fluorescence image of PC12 cells stained with calcein-AM (green) and propidium iodide (PI, red); Figure 20-s are fluorescence microscopy images of PEDOT / PtN1C3 / CFE and bare CFE treated with 5 mg / ml FITC-BSA; Figure 20 -t is the contact angle measurement result of bare CC (n=3); Figure 20 -u is a graph showing the contact angle measurement results of PEDOT / PtN1C3 / CC (n=3); Figure 20 -v is the amperometric response (n=3) of PEDOT / PtN1C3 / CFE and naked CFE to 1mM H2O2 in 0.1M PBS solution (pH 7.40) containing 10mg / ml BSA at a potential of -0.3V vs. Ag / AgCl. Figure 20 -w is Figure 20 -v Corresponding statistical bar chart (n=3);

[0100] Figure 21 Cyclic voltammetry curves of bare CFE, PtN1C3 / CFE, and PEDOT / PtN1C3 / CFE in 0.1M PBS solution (pH 7.40) without H2O2;

[0101] Figure 22 Ampere current curves and corresponding statistical results are shown for PEDOT / PtN1C3 / CFE at different potentials when 1 mM H2O2 is continuously added to stirred 0.1 M PBS (pH = 7.40); among them, Figure 22 -a is the Ampere current curve; Figure 22 -b is Figure 22 Statistical results for -a;

[0102] Figure 23 The amperometric response curves and corresponding statistical bar charts (n=3) of PEDOT / PtN1C3 / CFE in 0.1M PBS to 100μM H2O2, DOPAC, Glu, E, DA, UA, AA, 5-HT, and after the re-addition of 100μM H2O2 are shown. Figure 23 -a is the Ampere response curve; Figure 23 -b is Figure 23 -a Corresponding statistical bar chart (n=3);

[0103] Figure 24 The image shows the differential pulse voltammetry (DPV) plots of PEDOT / PtN1C3 / CFE in PBS containing 1 mM H2O2, 1 mM 5-HT, 1 mM MAA, 1 mM DA, 1 mM DOPAC, 1 mM ME, 1 mM Glu, and 1 mM UA; where, Figure 24 -a is the DPV diagram of PEDOT / PtN1C3 / CFE in PBS containing 1mM H2O2; Figure 24-b is the DPV diagram of PEDOT / PtN1C3 / CFE in PBS containing 1mM 5-HT; Figure 24 -c is the DPV plot of PEDOT / PtN1C3 / CFE in PBS containing 1mMAA; Figure 24 -d is the DPV plot of PEDOT / PtN1C3 / CFE in PBS containing 1mMDA; Figure 24 -e is the DPV diagram of PEDOT / PtN1C3 / CFE in PBS containing 1mM DOPAC; Figure 24 -f is the DPV plot of PEDOT / PtN1C3 / CFE in PBS containing 1 mM E; Figure 24 -g is the DPV diagram of PEDOT / PtN1C3 / CFE in PBS containing 1mM Glu; Figure 24 -h is the DPV plot of PEDOT / PtN1C3 / CFE in PBS containing 1 mA;

[0104] Figure 25 Cyclic voltammetry curves of bare CFE, PtN1C3 / CFE, and PEDOT / PtN1C3 / CFE in 0.1M PBS solution (pH 7.40) containing 2mM H2O2;

[0105] Figure 26 The graph shows the typical amperometric current response of PEDOT / PtN1C3 / CFE after continuous addition of H2O2 (500 μM each time) to PBS before and after immersion in cell culture medium for 2 hours, and the pre-calibration and post-calibration curves obtained under these conditions; where, Figure 26 -a is a typical Ampere current response diagram; Figure 26 -b means in Figure 26 The curves obtained under condition -a are shown in blue before calibration and purple after calibration.

[0106] Figure 27 Figure 1 shows the experimental results of H2O2 sensing related to PEDOT / PtN1C3 / CFE in PBS solution containing PC12 cells and in vivo; among them, Figure 27 -a is the H2O2 release current response diagram recorded by PEDOT / PtN1C3 / CFE when PBS containing PC12 cells is added with PBS (NC), 100mM resorcinol (Res), 500U catalase (CAT) + 100mM Res, or 100μM rosmarinic acid (RA) + 100mM Res. Figure 27 -b is Figure 27 -a Statistical results of H2O2 release (n=3) Figure 27 -c shows fluorescence imaging of intracellular reactive oxygen species (ROS) levels in PC12 cells under different treatment conditions; Figure 27 -d is a graph showing the quantitative analysis results of ROS generation (n=3); Figure 27 -e is a schematic diagram of PEDOT / PtN1C3 / CFE used for in vivo H2O2 monitoring; Figure 27 -f is a diagram of the catalytic mechanism of HPRR on PtN1C3; Figure 27 -g represents the local microinjection (200 nL·s) into the cerebral cortex recorded by PEDOT / PtN1C3 / CFE. -1 H2O2 release current response diagram at PBS (NC), 100mM Res, 500U CAT+100mM Res, and 100μM RA+100mM Res (lasting 5 seconds); Figure 27 -h is Figure 27 -g corresponding H2O2 release statistics (n=3); data points represent the average of multiple samples, and all error bars are standard deviations; statistical analysis was performed using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0107] Figure 28 The diagram shows the pre-calibration curve and linearity graph for H2O2 detection in vivo; among them, Figure 28 -a is the amperometric current response of PEDOT / PtN1C3 / CFE to PBS (pH 7.4) with the continuous addition of 500 μM H2O2; Figure 28 -b is a graph showing the functional relationship between current intensity and H2O2 concentration. Detailed Implementation

[0108] 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.

[0109] Unless otherwise specified, all reagents involved in this invention can be purchased through conventional commercial channels. For example, in the embodiments of this invention, nitrogen-doped multi-walled carbon nanotubes (N-MWCNTs) were purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd., China; 3,4-ethylenedioxythiophene (EDOT) was purchased from Beijing Cool Chemical Technology Co., Ltd., China; tetrabutylammonium tetrafluoroborate (TFB) and rosmarinic acid (RA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., China; hydrogen peroxide (AR, 30wt%), acetone, nitric acid, urea, and potassium hydroxide were purchased from Chongqing Chuandong Chemical Group Co., Ltd., China; potassium tetrachloroplatinate (K2PtCl4), resorcinol (Res), dopamine (DA), uric acid (UA), ascorbic acid (AA), 3,4-dihydroxyphenylacetic acid (DOPAC), adrenaline (E), and serotonin (5-HT) were purchased from... Alfa Esa (Shanghai Titan Technology Co., Ltd., China); glucose (Glu) was purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd.; acetonitrile was purchased from Chengdu Chron Chemical Co., Ltd., China; phosphate buffered saline (PBS) was prepared by mixing 0.1M Na₂HPO₄·12H₂O and 0.1M NaH₂PO₄·2H₂O in a ratio of 81:19, and the pH was adjusted to 7.40 using a pH meter with either 0.1M Na₂HPO₄·12H₂O or 0.1M NaH₂PO₄·2H₂O; potassium ferrocyanide trihydrate (K₄[Fe(CN)₆]) and potassium ferricyanide (K₃[Fe(CN)₆]) were purchased from Shanghai Aladdin Reagent Co., Ltd.; potassium ferricyanide solution ([Fe(CN)₆]) was also purchased. 3- / 4- The assay was prepared using PBS containing 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6] and 0.1 M KCl; CCK-8 and calcein / propidium iodide cell viability / cytotoxicity assay kits were purchased from Beyotime Biotechnology Co., Ltd.; RPMI 1640 basal medium was purchased from Gibco; fetal bovine serum (FBS) was provided by Lonsera; ultrapure water (18.2 MΩ) used in the experiment was prepared by an ultrapure water system (AOSIDE INSTRUMENT); lyophilized catalase (A001847-0002) was purchased from Shanghai Sangon Biotech Co., Ltd.

[0110] In this embodiment of the invention, the morphological characteristics of the modified carbon fiber electrode (CFE) were captured by scanning electron microscopy (SEM) using a Hitachi S-8010 field emission gun scanning electron microscope (Hitachi, Tokyo, Japan). Transmission electron microscopy (TEM) images were recorded on a JEM-2100F (Nippon Electron Instruments Co., Ltd.) operating at 200 kV. X-ray diffraction (XRD) patterns of Pt-N-MWCNTs were obtained using an X-ray diffractometer (XRD-6100, Shimadzu). X-ray photoelectron spectroscopy (XPS) measurements were performed using a photoelectron spectrometer (K-Alpha, Thermo Fisher Scientific), with the following settings: radiation source: Al Kα source; test energy: 1486.8 eV; test spot area: 400 μm; test tube voltage: 15 kV; tube current: 10 mA; and background vacuum in the analysis chamber: 2 × 10⁻⁶. -9 Ar ion etching was performed at mbar, with an energy of 2000 V and a typical depth of 3-5 nm. Inductively coupled plasma mass spectrometry (ICP-MS) was performed on an Agilent 7800 ICP-MS spectrometer. Dark-field scanning transmission electron microscopy (STEM) characterization was performed using a FEI-Themis Z (FEI / Thermo Fisher Scientific, USA) at 200 kV. X-ray absorption spectra (XAS) of the sample at the Pt K-edge (7709 eV), including X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS), were collected at the Singapore Synchrotron Radiation Facility (SSLS) center, where a monochromator was used with a pair of channels to cut the Si(111) crystal. Pt K-edge XANES data were recorded in transmission mode. Pt foil and PtO2 were used as references. The storage ring was operated at an energy of 2.5 GeV with an average electron flux of less than 200 mA. The acquired EXAFS data were extracted and processed using the ATHENA module in the FEFIT software package according to standard procedures. By applying the Bessel window function, in Obtain x(k) within the range of k 3 The result of the weighted Fourier transform (FT) in R space.

[0111] In this embodiment of the invention, the electrochemical measurement method is as follows:

[0112] All electrochemical measurements were performed using an electrochemical workstation (CHI 660E, Shanghai, China). The glassy carbon electrode (GCE) was cleaned by physical polishing and chemical cleaning, and then subjected to Fe(CN)6… 3- / Fe(CN)6 4-The reversible electrode reaction was examined. Then, for sensitivity measurements, 4 μL of 10 mg / mL PtN1C3 or PtN4 was dropped onto a clean GCE and dried at room temperature. For selectivity measurements, 8 μL of 10 mg / mL PtN1C3 or PtN4 was added and dried. A classic three-electrode system was constructed with Pt wire as the CE, Ag / AgCl as the RE, and PtN1C3 / GCE, PtN4 / GCE, or PEDOT / PtN1C3 / CFE as the WE. The modified electrodes were characterized by EIS in a 5 mM potassium ferricyanide solution containing 0.1 M KCl. The EIS spectral frequency range was 1,000,000 Hz to 0.08 Hz. It, CV, and DPV were performed in 0.1 M PBS solution (pH = 7.40). The optimal operating voltage was -0.3 V. The CV characterization conditions were: potential range -1.0V to 1.0V, scan rate 100mV / s. The DPV voltage range was -1.0V to 1.0V, pulse amplitude 50mV, and pulse period 0.5s.

[0113] In this embodiment of the invention, the method for quantitative testing of cytotoxicity is as follows:

[0114] PC12 cells (rat adrenal pheochromocytoma cells) were purchased from Shanghai Aicell Biotechnology Co., Ltd. PC12 cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin (5% CO2, 37℃). CC cells with the same modification were cut into appropriate sizes and sterilized by exposure to ultraviolet (UV). After digestion and resuspending, PC12 cells were seeded in 96-well plates and cultured for 24 hours. Then, small pieces of CC were added to the wells. After 24 hours of incubation, the medium was removed, and 100 μL of fresh medium containing CCK-8 (10 μL) was added to each well, followed by incubation for 2 hours. The absorbance at 450 nm was recorded when the solution turned orange. Relative cell viability was calculated using the following formula: (ODtest / ODControl) × 100%.

[0115] In this embodiment of the invention, the fluorescence staining and cell imaging methods are as follows:

[0116] PC12 cells were cultured on identically modified CCs (1cm × 1cm) to assess electrode biocompatibility. After a period of time, the culture medium was aspirated, the cells were washed once with PBS, and then stained with the fluorescent live / dead cell dyes calcein acetoxymethyl (AM) and propidium iodide (PI). Finally, the stained cells were incubated at 37°C for 30 minutes in a cell culture incubator and observed and imaged using a fluorescence microscope.

[0117] In this embodiment of the invention, the method for determining intracellular reactive oxygen species levels is as follows:

[0118] To verify the amount of reactive oxygen species induced by different treatment conditions, PC12 cells were seeded in culture dishes and cultured at 37°C, 5% CO2, and in a humid environment for 24 hours. They were then incubated at 37°C with DCFH-DA (Beyotime Biotechnology) for 30 minutes, followed by the addition of freshly prepared Res solution to a final concentration of 100 μM and fresh Res solution containing 100 μM RA, and incubated for 10 minutes. Imaging and analysis were performed using an inverted fluorescence microscope (ECLIPSE Ti2, Nikon).

[0119] In this embodiment of the invention, the density functional theory calculation method is as follows:

[0120] Density functional theory (DFT) calculations were performed using the Vienna ab initio simulation software package (VASP). The interaction between core and valence electrons was determined using a projected enhanced wave (PAW) pseudopotential. The exchange-correlation functional was performed using the Perdew-Burke-Ernzerhof (PBE) functional under the generalized gradient approximation. Van der Waals interactions were calculated using the DFT-D3 method with Grimme semi-empirical dispersion correction.

[0121] The cutoff energy of the plane-wave basis set was set to 450 eV. During structural optimization, a (3×2×1) k-point grid generated using the Gamma scheme was used to sample the Brillouin zone. The total energy convergence threshold for the ground-state electron density was set to 10. -5 eV, when the maximum force on any ion is less than eV, At that time, the structural optimization is considered to have converged. Approximately [value missing] is set along the z-direction. A vacuum layer is used to avoid interaction between the periodic substrate and its repeating pattern. For the N-doped system, a (4×4×1) graphene supercell is used, where Pt atoms are embedded in double-vacancy sites (denoted as PtNX). Molecular calculations are performed in (15×15×15) supercells. The sampling was performed in a box using the Gamma grid scheme with a cutoff energy of 450 eV.

[0122] The HPRR pathway includes the following steps:

[0123] H₂O₂ + * → H₂O₂

[0124] H2O2*→2OH*

[0125] 2OH*+H + +e - →H₂O + OH⁻

[0126] OH*+H + +e - →H2O+*

[0127] Under standard conditions (298 K, pH 2 = 1 bar, Ph = 0), when the reference potential is set to the potential of the standard hydrogen electrode, (H + +e - The chemical potential of H₂ (i.e., the free energy per H₂) is equal to 1 / 2 the chemical potential of H₂. The change in reaction free energy (ΔG) is further estimated using the following function:

[0128] ΔG=ΔH-TΔS-qU+kBTln10 xpH+

[0129] Wherein, ΔH: enthalpy of reaction in each elementary step, calculated using reaction energy (ΔE) corrected for zero point energy (ZPE); ΔS: entropy change at temperature T; U: applied potential; q: charge transferred in each elementary step; kB: Boltzmann constant.

[0130] We constructed a series of surface structure models of PtNXC4-X (X = 0-4) using Vesta. We investigated the effect of different N atom coordination numbers on HPRR using Pt as the active site as a single-atom catalyst. From thermodynamic and electronic structural perspectives, we theoretically explained the high electrocatalytic activity of the PtN1C3 configuration for HPRR compared to the typical PtN4 configuration. Finally, we continued to construct other small molecule adsorption and catalytic models to theoretically explain the selectivity of PtN1C3 for H2O2.

[0131] In this embodiment of the invention, the in-situ detection method for H2O2 secreted by cells is as follows:

[0132] PC12 cells were purchased from Shanghai Aicell Biotechnology Co., Ltd. They were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin at 5% CO2 and 37°C. Cells were seeded in cell culture dishes for 24 hours, and the amperometric current response was measured at the optimal applied potential (-0.3V) using sterile PEDOT / PtN1C3 / CFE obtained after UV irradiation. During the measurement, after signal stabilization, H2O2 released by the cells was detected using 100 μM Res, 100 μM Res + 100 μM RA, and 100 μM Res + 500 UCAT as stimuli.

[0133] In this embodiment of the invention, the in vivo experiment is as follows:

[0134] Adult male SD rats (300-350g) were anesthetized with isoflurane (4% induction, 2% maintenance) using a gas pump R520 (Reward Life Sciences Co., Ltd., Shenzhen, China) and fixed in a stereotactic frame for craniotomy. According to the stereotactic procedure, PEDOT / PtN1C3 / CFE was inserted into the right cortex (AP = 3mm, L = 2mm lateral to the anterior fontanelle, V = 1mm from the skull surface). A prepared micron-sized Ag / AgCl (saturated KCl) reference electrode and a platinum wire counter electrode were placed within the dura mater. Exogenous PBS containing 100 mmol Res, 100 μmol RA, or 500 U / mL CAT was microinjected into the localized area of ​​the intracranial microelectrode via a quartz capillary (4 cm long, 50 μm inner diameter, 375 μm outer diameter) implanted parallel to the PEDOT / PtN1C3 / CFE in the right cortex. This capillary was also referred to as a single-atom catalyst-based microsensor. These solutions were delivered from a gas-impermeable syringe via a microinjection pump R480 (Shenzhen Ruiwode Life Sciences Co., Ltd., China). All local microinjections were administered into the rat cerebral cortex at 200 nL / s. -1 The injection rate was [not specified]. The local microinjection volume of Res was 1000 nL. In inhibitor treatment, 2000 nL of RA and CAT were injected, respectively, before Res injection, and incubated for 15 min and 1 min, respectively. PEDOT / PtN1C3 / CFE was polarized at -0.3 V for amperometric measurement of H2O2 in the rat cerebral cortex.

[0135] In this embodiment of the invention, the statistical analysis method is as follows:

[0136] Statistical analyses were performed using Excel and GraphPadPrism 9. The sample size for each statistical analysis was greater than or equal to 3 (n≥3). Statistical comparisons between two groups were performed using two-tailed t-tests and one-way ANOVA. Significance was expressed as *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001. ns indicates no statistically significant difference.

[0137] In this embodiment of the invention, the carbon fiber electrodes (CFEs, 7 μm in diameter) are prepared using the following method:

[0138] Using a micropipette drawer (MP-500, RWD, Shenzhen, China), glass capillaries (1.5 mm inner diameter, 100 mm length) were drawn into two conical glass-coated carbon fiber tip electrodes, each tip being broken into 10-15 μm diameter sections. The drawn capillaries served as sheaths for the CFEs. Then, carbon fibers (7 μm diameter, Shenzhen Tony Electronics Co., Ltd., Shenzhen, China) were sequentially sonicated in acetone, 3M HNO3, 1M KOH, and ultrapure water (18.2 MΩ) for 5 minutes each. They were then dried in a vacuum drying oven at 60°C for 6 hours. Single carbon fibers on a clean glass plate were attached to copper wires (200 μm diameter) slightly longer than the glass capillaries using silver conductive adhesive and placed at 80°C for 1 hour. The CFEs were then fabricated by carefully inserting the carbon fibers with the copper wires into the capillaries, exposing the carbon fibers at the narrow opening end and the copper wires at the other end. Next, the end of the glass capillary was vertically immersed in a pool of molten paraffin, taking care not to let the paraffin touch the carbon fibers at the tip. It was then placed in room temperature air to allow the paraffin to solidify. Finally, under a microscope, the exposed CFEs were cut with a scalpel to approximately 200–250 μm.

[0139] Example 1. Preparation of PtN1C3 and PtN4

[0140] (1) Preparation of K2PtCl4 / N-MWCNT: The K2PtCl4 precursor was dissolved in ultrapure water to form a 3 mg / mL stock solution. 0.16 g of N-MWCNTs were dispersed in 100 mL of ultrapure water and sonicated for 30 minutes to ensure thorough dispersion and form a suspension. 600 μL of the Pt stock solution was added dropwise to the N-MWCNT suspension, which was then vigorously stirred at a maximum speed of 1500 rpm for 4 hours on a magnetic stirrer to load Pt onto the carbon nanotubes. The suspension was then rapidly frozen at -80 °C and transferred to a vacuum freeze dryer until a black powder K2PtCl4 / N-MWCNT was obtained.

[0141] (2) Preparation of PtN1C3: The K2PtCl4 / N-MWCNT powder prepared in step (1) was ground with urea at a mass ratio of 1:4. Then, it was heated to 800°C in a tube furnace under N2 flow at a rate of 10°C per minute and held at 800°C for 1 hour. Then, it was cooled to room temperature to obtain PtN1C3.

[0142] (3) Preparation of PtN4: The K2PtCl4 / N-MWCNT powder prepared in step (1) was ground with urea at a mass ratio of 1:10. Then, it was heated to 800°C in a tube furnace under N2 flow at a rate of 10°C per minute and held at 800°C for 1 hour. Then, it was cooled to room temperature to obtain PtN4.

[0143] Example 2. Characterization of PtN1C3 and PtN4

[0144] By anchoring Pt single atoms onto N-MWCNTs, materials with large surface areas and good electrical conductivity can be synthesized. For example... Figure 1 As shown in -a, the synthesis of Pt-N-MWCNTs with different coordination environments involves several steps. In short, a small amount of Pt cations is first dispersed in ultrapure water containing N-MWCNTs under vigorous stirring, followed by freeze-drying and thermal annealing with specific dopant precursors in different proportions. With a constant Pt loading of 1.26 wt%, no metal clusters or nanoparticles were observed in the bright-field transmission electron microscopy (TEM) images of PtN1C3. See details... Figure 1 -b. We also observed the same result in the TEM images of PtN4, see details. Figure 2 . Figure 1 -c~ Figure 1 -e shows the aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) characterization of PtN1C3, which retains the structure of N-MWCNTs, with clearly discernible edges of the multilayered rolled graphene layers, while isolated Pt single atoms are identified as bright spots due to their higher Z contrast compared to adjacent C / N sites. Similarly, Figure 3 This proves that the Pt in PtN4 is also atomically dispersed. Figure 1 As indicated in -d, the Pt content of PtN1C3, measured by inductively coupled plasma mass spectrometry (ICP-MS), is 1.26 wt%. Figure 1 -f and Figure 1 -g shows that carbon (C), nitrogen (N), and platinum (Pt) elements are uniformly distributed throughout the structure, as revealed by energy-dispersive X-ray spectroscopy (EDS) elemental mapping image. Furthermore, Figure 1 -h、 Figure 1 -i and Figure 4 The EDS spectrum clearly shows the characteristic peaks of Pt and N elements, suggesting the possible formation of Pt-N. X Species. Figure 1 The high-resolution X-ray diffraction (XRD) pattern shown in -j indicates that neither PtN1C3 nor PtN4 contains any long-range ordered Pt crystal structures or nanoparticles.

[0145] The composition and related Pt valence states of PtN1C3 and PtN4 were further investigated using in-situ X-ray photoelectron spectroscopy (XPS). See details in [link to article]. Figure 5 -a and Figure 5-d. The XPS spectra depict the compositional information, with approximately 0.15 at% Pt content observed in the sample. The XPS spectra of the PtN1C3, N 1s region are shown below. Figure 5 -b, after core layer decomposition, identified three distinct coordination structures: pyridine nitrogen (398.5 eV), graphitic nitrogen (401.2 eV), and Pt-N (399.9 eV). Furthermore, pyrrole nitrogen (400.6 eV) was also observed in the N1s region of PtN4, which can be attributed to the introduction of a higher precursor ratio; see [link to relevant documentation] for details. Figure 5 -e. Figure 5 The -c command plotted the XPS spectrum of the core layer in the Pt4f region, where Pt4f... 7 / 2 The binding energy is 72.1 eV. Notably, this value is higher than that of metallic platinum (71.2 eV), indicating that partial charge transfer occurred from the central Pt atom to the adjacent metallic impurity dopant. A similar finding can be observed in the Pt 4f region of PtN4, see [link to details]. Figure 5 -f. For more information on C1s and O1s, see [link / description]. Figure 6 and Figure 7 Furthermore, we used Pt L3-edge XAS spectroscopy to reveal the chemical states and coordination environments of PtN1C3 and PtN4 at the atomic level. The aforementioned characteristic of partial depletion of free electrons in the Pt valence band was demonstrated through… Figure 5 -d and Figure 5 The X-ray near-edge absorption structure (XANES) spectrum shown in -g was further confirmed, in which the intensity of the white line of the prepared catalyst was higher than that of the platinum foil, indicating the stable presence of slightly positively charged Pt on the N-MWCNTs support. δ+ Species. Furthermore... Figure 5 -e and Figure 5 -h displays Fourier transform extended X-ray absorption fine structure spectra (FT-EXAFS). The Pt foil at approximately... The area exhibits typical first-shell Pt-Pt pairing, see details. Figure 8 and Figure 9 The Pt-O interaction is located approximately in PtO2. Neither of these two conditions was observed in either of the prepared catalysts. For PtN1C3 and PtN4, the dominant R-space features are approximately... and This may be due to the characteristic bonding of Pt-N and the difference in coordination number. To further investigate the distribution of Pt atoms in PtN1C3, we performed wavelet transform (WT) analysis on the PtL3-edge using EXAFS. Figure 5 -j~ Figure 5 As shown in Figure -l, only one Pt-N coordination-related feature was observed in the WT spectrum of PtN1C3. The maximum intensity at this location differs from the WT spectrum of the Pt foil, which is at... There is a maximum intensity value corresponding to the Pt-Pt contribution, indicating that there are no Pt-Pt bonds in PtN1C3, but rather isolated Pt atoms. Similarly, only one maximum intensity value related to Pt-N coordination was observed in the WT of PtN4, see [link to WT]. Figure 5 -m~ Figure 5 -r. Based on the fitted curves obtained in k and R spaces ( Figure 5 -f、 Figure 5 -i、 Figure 10 and Figure 11 Based on Tables 1 and 2, the quantitative structural parameters of Pt in PtN1C3 and PtN4 were obtained using least-squares EXAFS fitting. Analysis showed that the coordination numbers of PtN1C3 and PtN4 are approximately 1 (PtN1C3) and 4 (PtN4), respectively, and the average bond lengths of Pt-N are... (PtN1C3) and (PtN4). However, since the precursor used was chloride, some Cl was not completely removed, resulting in some Pt-Cl coordination, which is normal. In PtN1C3, the second shell of Pt coordinates with C in CN, with an N bridge. In summary, given the similar manufacturing methods and processing procedures of the two samples, we believe that reducing the introduced urea ratio before thermal annealing directly contributed to the reduction of N doping content, disrupting the symmetric structure of PtN4, thus obtaining PtN1C3 with an asymmetric coordination structure.

[0146] Table 1. EXAFS Fitting Parameters for Pt L3 Edge (S0) 2 =0.89)

[0147]

[0148] In Table 1, CN represents the coordination number; R represents the bond length; and σ represents the σ value. 2 Debye-Waller factor; ΔE: internal potential correction; R factor: goodness of fit. *Fit with fixed parameters.

[0149] Table 2. EXAFS Fitting Parameters for Pt L3 Edge (S0) 2 =0.70 (from Pt foil)

[0150]

[0151] In Table 2, CN represents the coordination number; R represents the bond length; and σ represents the σ value. 2 Debye-Waller factor; ΔE: internal potential correction; R factor: goodness of fit.

[0152] Example 3. Electrocatalytic performance of PtN1C3

[0153] First, the electrocatalytic performance of PtN1C3 and PtN4 in phosphate buffered saline (PBS) for HPRR was investigated by amperometric method (it). Figure 12 As shown, with the continuous addition of H2O2, the current signals of both PtN1C3 / GCE and PtN4 / GCE exhibited a stepwise response, while the reduction current signal of PtN1C3 / GCE was significantly greater than that of PtN4 / GCE, with a sensitivity increase of approximately 4.9 times. This indicates that the electrocatalytic performance of asymmetrically coordinated PtN1C3 is significantly improved compared to that of classically symmetrically coordinated PtN4. To further explore the reasons for the high HPRR electrocatalytic activity of PtN1C3, we used DFT calculations to investigate the different configurations of Pt-N, including PtN1C3 and PtN4. x -C 4-x (x = 0–4) Catalytic mechanism of HPRR (see SI for more calculation details). We constructed Pt-N with different configurations. x -C 4-x The calculation model at -0.3V, i.e., PtC4, PtN1C3, PtN2C2-1 (cis configuration, two N atoms on the same side), PtN2C2-2 (trans configuration, two N atoms on opposite sides), PtN3C1, and PtN4 with different C and N positions, is presented. The Gibbs free energy (ΔG) and adsorption structure for each basic step are also described. Figure 13 , Figure 14 -a~ Figure 14 As shown in -f, the basic reactions of the HPRR reaction include the adsorption of H2O2 molecules, the homolytic cleavage of H2O2 into two hydroxyl groups adsorbed on Pt atoms, and two proton transfers that cause the adsorbed hydroxyl groups to generate water. Figure 13 The free energy diagram of HPRR shown indicates that Pt-N x -C 4-x The rate-determining step for the reaction (x=0-3) is the adsorption of H2O2, while for PtN4 the rate-determining step is the homolytic cleavage of H2O2. The reactivity decreases in the order of PtN1C3 > PtN3C1 > PtN2C2-1 > PtN2C2-2 > PtC4 > PtN4. This means that among all C and N atom coordination configurations, PtN1C3 exhibits the lowest reaction energy barrier (0.173 eV) for HPRR, making it the optimal N-doped coordination configuration for HPRR catalysis. Therefore, PtN1C3 is one of the most ideal HPRR catalyst choices. This is likely attributed to the asymmetry of the central metal atom's electronic structure caused by single N atom doping, which can be explained by… Figure 14 -g~ Figure 14It can be intuitively obtained from the differential charge density map shown in -l, which will cause the shift of the d-band center of Pt atoms. The Sabatier principle states that for a good catalyst with a determined active center structure, it should bind atoms or molecules with moderate strength, neither too weakly to make the reactants difficult to activate nor too strongly to make the products difficult to desorb. The d-band center theory believes that the adsorption strength of the catalytic reaction on the transition metal surface is closely related to the position of the d-band center of the active metal center. Judging the adsorption strength through the d-band center of the central site Pt atoms can provide a conceptual framework for us to explore the optimal catalyst. For this purpose, we calculated the band structures of different coordination systems and focused on the contribution of the d-orbital PDOS of the transition metal Pt to the total DOS and the value of its d-band center. See Figure 15 . PtC4 (-3.426 eV) < PtN3C1 (-2.769 eV) < PtN4 (-2.725 eV) < PtN1C3 (-2.662 eV) < PtN2C2-2 (-2.505 eV) < PtN2C2-1 (-2.244 eV). The d-band center (-2.662 eV) of the central Pt atom in the PtN1C3 center is slightly shifted upward compared to PtN4 (-2.725 eV), closer to the Fermi level, making it better balance the adsorption-desorption process. In addition, among the other configurations calculated, PtC4 and PtN3C1 have difficulty in adsorption due to the obvious deviation of the d-band center. Although the d-band centers of PtN2C2-1 and PtN2C2-2 are closer to the Fermi level, the adsorption barrier of H2O2 may be higher because of their lower DOS near the Fermi level. In summary, the symmetry-broken PtN1C3 is closer to the optimal adsorption-desorption position than other Pt-N x -C 4-x (x = 0, 2, 3, 4), theoretically explaining the high HPRR catalytic activity of PtN1C3.

[0154] Example 4. Selectivity of PtN1C3

[0155] Example 3 elaborated on the high HPRR electrocatalytic activity of PtN1C3 prepared by the symmetry-breaking strategy and its possible mechanism. This example further explores its high selectivity for H2O2, as shown in Figure 16 -a. For this purpose, we directly modified PtN1C3 on a glassy carbon electrode (GCE) again and measured its responses to other neurochemical substances in the brain, AA (ascorbic acid), DA (dopamine), DOPAC (3,4-dihydroxyphenylacetic acid), Glucose (glucose), 5-HT (5-hydroxytryptamine), E (epinephrine) and H2O2 with the same concentration by the i-t method. The results are shown in Figure 16 -b to Figure 16As shown in -c. Compared to H2O2, the amperometric responses produced by other neurochemicals are extremely weak, almost negligible. The above experimental results indicate that PtN1C3 only exhibits a significant response to H2O2, while showing no response to other neurochemicals in the brain, demonstrating its high selectivity for H2O2. To further explore the possible mechanism of PtN1C3's high selectivity for H2O2, we performed DFT simulations to study the possible reaction mechanisms of some interfering substances in PtN1C3 at an applied potential of -0.3V, and further investigated the thermodynamic possibility of these substances undergoing redox reactions in PtN1C3. For example... Figure 16 -d and Figure 16 As shown in -j, during the reduction of H2O2, only the first step of H2O2 adsorption is an endothermic reaction, which is the rate-determining step (RDS) of the reaction, ΔG = 0.173 eV. The projected density of states of the d orbitals of Pt atoms has relatively abundant electronic states near the Fermi level, indicating that Pt atoms have relatively active d orbital valence electrons. This may be one of the reasons why Pt-N1C3 can effectively adsorb H2O2 molecules. The adsorbed H2O2 spontaneously homolytically splits into two *OH groups and adsorbs onto Pt atoms. This step does not involve electron transfer, and the reaction intermediate is in a relatively high-energy unstable state. The subsequent two steps of OH* desorption involve proton transfer steps. Due to its good adsorption capacity with hydroxyl groups, PtN1C3 can both stabilize the hydroxyl groups to complete the proton transfer step and effectively desorb the products to avoid side reactions caused by the long-term occupation of active sites.

[0156] When DA, AA, and Glu undergo oxidation reactions, the first adsorption step is always an exothermic reaction, such as... Figure 16 -e、 Figure 16 -g、 Figure 16 -h、 Figure 16 -k、 Figure 16 -m and Figure 16 As shown in Figure -n, in the DA oxidation reaction, the projected densities of states of the d orbitals of Pt atoms and the p orbitals of the O atoms in the ligand do not overlap well, indicating that there is almost no hybridization between the Pt atoms and the O atoms in the DA ligand. The Hamiltonian population integral (ICOHP) of the crystal orbitals below the Fermi level is -0.044 eV, meaning that DA tends to be physically adsorbed on the surface of the catalyst. See details. Figure 17 In the oxidation reaction of AA and Glu, the valence electrons in the d orbitals of Pt atoms and the electrons in the p orbitals of O atoms exhibit a high degree of overlap, with their ICOH values ​​being -1.355 eV and -1.138 eV, respectively. (See details...) Figure 18 and Figure 19The study demonstrates that Pt atoms, acting as active sites, can undergo orbital hybridization with O atoms of AA and Glu ligands, indicating a strong covalent bond between Pt and O. This excessively strong adsorption may be one reason why PtN1C3 does not exhibit catalytic activity. The adsorption of the three substances mentioned above is exothermic, which explains their easy adsorption onto the electrode surface. However, the subsequent dehydrogenation and oxidation are endothermic, with RDS ΔG values ​​of 1.282 eV, 0.763 eV, and 1.985 eV, respectively. This indicates that thermodynamically, it is almost impossible for PtN1C3 to catalyze these reactions, a conclusion that also applies to the oxidation of 5-HT and UA. Figure 16 -f、 Figure 16 -i、 Figure 16 -l and Figure 16 As shown in Figure -o, the RDS of the reactions of 5-HT and UA on PtN1C3 are both the final step, namely the desorption of *H2O and oxidation products (5-HT-H, UA-H) from PtN1C3, with maximum Gibbs free energy changes of 1.922 eV and 1.753 eV, respectively. The maximum ΔG of the reactions of the above interfering substances on PtN1C3 is much higher than the maximum Gibbs free energy change of 0.337 eV of the H2O2 reduction reaction, indicating that the tendency for HPRR reactions to occur on PtN1C3 is much greater than that of other common electrochemically active substances in the brain. These results not only thermodynamically explain the excellent selectivity of PtN1C3, but also highly agree with the experimental results.

[0157] Example 5. Preparation of PEDOT / PtN1C3 / CFE

[0158] (1) Preparation of PtN1C3 / CFE

[0159] PtN1C3 was ultrasonically dispersed in acetonitrile to form a deposition solution of 1 mg / mL. A classic three-electrode system was constructed, using bare CFE as the working electrode (WE), Pt wire as the counter electrode (CE), and Ag / AgCl as the reference electrode (RE). Electrodeposition was then performed at 1.5 V for 5400 seconds using an amperometric method. After deposition, the electrode was removed, washed three times with ultrapure water, and dried to obtain PtN1C3 / CFE.

[0160] (2) Preparation of PEDOT / PtN1C3 / CFE

[0161] The PtN1C3 / CFE obtained in step (1) was modified onto the surface of the PtN1C3 / CFE by PEDOT:TFB deposition, resulting in PEDOT / PtN1C3 / CFE. Specifically, the PEDOT:TFB deposition was performed in an acetonitrile solution containing 0.01M EDOT and 0.1M TFB at 1.3V for 20 seconds to prepare PEDOT / PtN1C3 / CFE. Acetonitrile was used before the day of use. The molecular sieve is kept dry, and the monomer solution is bubbled with N2 before electrodeposition.

[0162] Example 6. Electrochemical performance of PEDOT / PtN1C3 / CFE

[0163] Based on the excellent electrocatalytic performance of PtN1C3 in HPRR, we constructed a PEDOT / PtN1C3 / CFE sensing platform for in vivo brain H2O2 monitoring via electrochemical deposition, and performed a series of physical and chemical characterizations to verify the successful construction of the sensor. The preparation process of PEDOT / PtN1C3 / CFE is as follows: Figure 20 -a is shown. Figure 20 -b and Figure 20 The SEM image shown in -c confirms that PtN1C3 has been successfully modified onto the carbon fiber surface. Electrochemical impedance spectroscopy (EIS) provides information on charge transfer resistance (Rct), which is represented by the size of the leading semicircle of the Nyquist plot. (See details...) Figure 20 -d. Compared to bare CFE, the impedance of PtN1C3 / CFE is significantly reduced. This is because the modification of PtN1C3 increases the electrode conductivity and electron transfer rate, which is beneficial for the electrocatalytic reduction of H2O2. However, the impedance of PEDOT / PtN1C3 / CFE increases significantly. This may be because even though PEDOT is a polymer film with excellent conductivity, it is still inferior to completely carbon-based materials. Meanwhile, Figure 21 Different cyclic voltammetric (CV) plots of bare CFE, PtN1C3 / CFE, and PEDOT / PtN1C3 / CFE were shown, demonstrating the surface modification of the electrodes during the layer-by-layer modification process. These results all demonstrate the successful modification of CFE by PtN1C3 and PEDOT.

[0164] Before practical application, we evaluated the sensor's H2O2 detection performance using an in vitro system. First, we tested its selectivity. For example... Figure 23As shown, the current response of the PEDOT / PtN1C3 / CFE interfering substances is extremely small, indicating that their presence cannot interfere with the sensor's detection of H2O2. Meanwhile, differential pulse voltammetry (DPV) results show that H2O2 undergoes a strong reduction reaction at the optimal application potential of -0.3V, while other common interfering substances in the brain hardly undergo redox reactions. (See details...) Figure 20 -e and Figure 24 These results are consistent with Figure 16 -b and Figure 16 The results in -c are consistent, indicating that PEDOT / PtN1C3 / CFE can achieve direct and excellent selectivity at the level of catalytic materials.

[0165] Subsequently, we used CV to evaluate the electrocatalytic performance of PEDOT / PtN1C3 / CFE. For example... Figure 25 As shown, compared to bare CFE, the reduction current of PtN1C3 / CFE and PEDOT / PtN1C3 / CFE for 2mM H2O2 increases from 0V to higher potentials. To further investigate the electrocatalytic reaction kinetics, we studied the CV response of PEDOT / PtN1C3 / CFE to 500μM H2O2 at different scan rates (ν). Figure 20 -f and Figure 20 As shown in -g, the peak current exhibits a linear correlation with ν, indicating that the electrochemical surface interaction is adsorption-controlled. Furthermore, as... Figure 20 As shown in -h, the current also increases with increasing H2O2 concentration gradient (0, 2, 4 mM), demonstrating that PEDOT / PtN1C3 / CFE has good electrocatalytic performance for H2O2 reduction. Figure 20 -i、 Figure 20 -j and Figure 20 As shown in Figure -k, when the concentration of H2O2 in PBS increases continuously, the reduction current rapidly rises to a stable value. PEDOT / PtN1C3 / CFE exhibits a strong linear correlation with H2O2 levels from 5 μM to 5 mM. The equation for the linear relationship between H2O2 concentration and current is I / nA = 0.6921C. H2O2 / μM -25.38(R 2 =0.9970), the signal-to-noise ratio (S / N=3) obtained from the current response of 50 μM H2O2 is used to estimate the detection limit (LOD) as (0.845 μM), which is the same as or lower than other H2O2 sensors reported in the literature, and the linear range exceeds that of most previously reported H2O2 sensors, as detailed in Table 3. Figure 20 -l、 Figure 20 -m and Figure 20-n shows the microscopic images and CV curves of the electrode before and after 50 bending cycles. The CV curves before and after bending almost overlap, indicating that mechanical deformation has no significant effect on its electrocatalytic activity and that it has good mechanical stability.

[0166] Subsequently, in order to evaluate the stability of PEDOT / PtN1C3 / CFE, we conducted long-term in vitro monitoring experiments. Figure 20 -o shows that the bare CFE has been on a downward trend, while PEDOT / PtN1C3 / CFE has only decreased slightly. Figure 20 Statistical data from -p showed that the current response of bare CFE decreased by 45%, while the current response of PEDOT / PtN1C3 / CFE decreased by only about 7%, demonstrating that PEDOT / PtN1C3 / CFE can achieve long-term stable monitoring of H2O2. This may be attributed to the inherent stability of SAC and the use of a smaller tetrafluoroborate (TFB) anion during electrodeposition, which, compared to the commonly used PSS, prevents the PEDOT coating from absorbing water, swelling, and peeling off, thus improving interfacial stability. To ensure the safety of in vivo implantation, we first determined the cytotoxicity of PEDOT / PtN1C3 using CCK-8 assays. Figure 20 As shown in -q, compared with bare carbon cloth (CC), the PEDOT / PtN1C3 modified CC did not significantly inhibit cell viability, indicating that it is non-toxic to cells. Next, we demonstrated the excellent biocompatibility of the PEDOT / PtN1C3 modified CFE through live / dead cell staining. Figure 20 As shown in the figure, after 24 hours, compared with naked CC, PC12 proliferated well on PEDOT / PtN1C3 / CC and maintained high viability with a very low number of dead cells.

[0167] Table 3. Performance comparison of the sensor presented in this work with previously reported H2O2 detection sensors.

[0168]

[0169]

[0170] The references in Table 3 are as follows:

[0171] [1]Li Z.Ordered-vacancy-induced cation intercalation into layereddouble hydroxides:a general approach for high-performancesupercapacitors.Chem.2018;4(9):2168-2176.doi:10.1016 / j.chempr.2018.06.007.

[0172] [2]Karam P,Halaoui LI.Sensing of H2O2 at Low Surface DensityAssemblies of Pt Nanoparticles in Polyelectrolyte.AnalyticalChemistry.2008Jun 11;80(14):5441–8.

[0173] [3]Zhang Y,Yang W,Wang Y,Jia J,Wang J.Nonenzymatic hydrogen peroxidesensor based on a glassy carbon electrode modified with electrospun PdO-NiOcomposite nanofibers.Microchimica Acta.2013 Jun 28;180(11-12):1085–91.

[0174] [4]Zhang J D,Oyama M.Gold nanoparticle-attached ITO as abiocompatible matrix for myoglobin immobilization:direct electrochemistry andcatalysis to hydrogen peroxide.Journal of Electroanalytical Chemistry.Volume577,Issue 2,1April 2005,Pages 273-279.

[0175] [5]Hua M et al.The intrinsic redox reactions of polyamic acidderivatives and their application in hydrogen peroxidesensor.Biomaterials.Volume 32,Issue 21,July 2011,Pages 4885-4895.

[0176] [6]Jiang F,Yue R,Du Y,Xu J,Yang P.A one-pot'green'synthesis ofPd-decorated PEDOT nanospheres for nonenzymatic hydrogen peroxidesensing.Biosens Bioelectron.2013;44:127-131.doi:10.1016 / j.bios.2013.01.003.

[0177] [7]Du X,Chen Y,Dong W,Han B,Liu M,Chen Q,et al.A nanocomposite-basedelectrochemical sensor for non-enzymatic detection ofhydrogenperoxide.Oncotarget.2016 Dec 27;8(8).

[0178] [8]Li,Yancai,et al."Carbon quantum dots / octahedral Cu2Onanocomposites for non-enzymatic glucose and hydrogen peroxide amperometricsensor."Sensors andActuators B:Chemical 206(2015):735-743.

[0179] [9]Jin,E.,et al."Fabrication of graphene / prussian blue compositenanosheets and their electrocatalytic reduction of H2O2."ElectrochimicaActa55.24(2010):7230-7234.

[0180]

[10] Bian X, Lu

[0181]

[11] Kurowska, E., et al. "Silver nanowire array sensor for sensitive andrapid detection of H2O2." Electrochimica Acta 104 (2013): 439-447.

[0182] Antifouling resistance is one of the biggest challenges facing implantable electrodes. Small biomolecules and non-specific proteins can occupy the active sites of the electrode, leading to decreased sensitivity. Therefore, we investigated the antifouling resistance of PEDOT / PtN1C3 / CFE at multiple levels. First, we immersed naked CFE and PEDOT / PtN1C3 / CFE in a FITC-labeled bovine serum albumin (BSA, 5 mg / mL) solution for 4 hours, and then captured microscopic images using a fluorescence microscope. Figure 20 The -s data showed that the adsorption of BSA on the PEDOT / PtN1C3 / CFE surface was significantly less than that on bare CFE, indicating its ability to resist non-specific protein adsorption. Subsequently, we compared the adsorption of BSA on bare CC and PEDOT / PtN1C3 / CFE surfaces. 3 / Water contact angle tests were conducted on CC, and the results showed that the water contact angle of PEDOT / PtN1C3 / CC was significantly reduced, indicating a higher anti-protein adsorption capacity. See details... Figure 20 -t and Figure 20 -u. Simultaneously, we immersed PEDOT / PtN1C3 / CFE in discarded culture medium from PC12 cells and evaluated its current response to H2O2 before and after immersion. See details... Figure 26The current response after immersion was 91.09% of that before immersion, with only a slight decrease in sensitivity, demonstrating that PEDOT / PtN1C3 / CFE can reliably monitor in complex environments. Finally, we evaluated the instantaneous kinetics of electrode contamination by directly adding 10 mg / ml BSA to PBS containing 100 μM H2O2. Figure 20 -v and Figure 20 As shown in the figure, the current of the bare CFE continuously decreased after the addition of BSA, while the PEDOT / PtN1C3 / CFE maintained a stable response to H2O2, with the current value decreasing by only about 5%, demonstrating that PEDOT / PtN1C3 / CFE has excellent antifouling properties. These results collectively indicate that PEDOT / PtN1C3 / CFE is very suitable for continuous selective monitoring of H2O2 in vivo.

[0183] Example 7. Application of PEDOT / PtN1C3 / CFE in cells and in vivo

[0184] Finally, to verify the high sensitivity and selectivity of PEDOT / PtN1C3 / CFE in real-world biological environments, we applied it to quantitatively analyze the biochemical processes related to resorcinol (Res)-induced H2O2 release in vivo. Many industrial chemicals induce neurotoxicity by producing H2O2 in the brain. Res is a widely used industrial chemical, but there is currently no direct evidence that Res can induce H2O2 production. First, we explored the role of Res at the cellular level. Res was rapidly injected into culture dishes containing PC12 cells, and the H2O2 level was tracked in real time using PEDOT / PtN1C3 / CFE. Figure 27 -a and Figure 27 -b shows the ampere response and corresponding statistics obtained by the sensor under different treatment conditions. The addition of 100 μM Res produced a significant current response, which may be related to the large amount of H2O2 released from PC12 stimulated by Res. In contrast, when Res was co-added with 100 μM rosmarinic acid (RA), the current response was significantly reduced, suggesting a decrease in H2O2 production, which may be related to the scavenging effect of RA on H2O2. To clarify that the sensor's response originated from the generated H2O2, we mixed Res with catalase (CAT) and added it to the above system, observing a significant reduction in the ampere response. Next, we used a reactive oxygen species (ROS) detection kit to investigate the ROS levels within PC12 after different treatments. Figure 27 -c and Figure 27As shown in Figure -d, ROS levels in the control group were very low. However, intracellular ROS levels in Res-treated cells were significantly increased, suggesting that Res can stimulate PC12 to produce high levels of ROS. In contrast, intracellular ROS levels in cells co-treated with Res and RA were significantly decreased, indicating that RA can reduce the ROS content generated by Res stimulation. These in vitro results preliminarily demonstrate that Res can induce H2O2 production. Finally, we investigated this at the in vivo level. Res was rapidly injected locally into the rat cerebral cortex, and PEDOT / PtN1C3 / CFE was implanted near the injection site to track the H2O2 signal generated by stimulation. See details below. Figure 27 -e and Figure 27 -f. For example... Figure 27 -g and Figure 27 As shown in the h-axis, consistent with cell experiments, microinjection of 91 mmol Res (1000 nmol) into the cortex produced a significant current response, while pre-injection of 100 μM RA (2000 nmol) 15 min prior to Res significantly reduced the current response. Furthermore, we quantified the release of H2O2 based on pre-calibration, such as... Figure 28 As shown, quantitatively, microinjection of 91 mmol Res (1000 nmol) increased H2O2 by 115 μM, while pre-injection of 100 μM RA (2000 nmol) reduced H2O2 production to 46.7 μM after Res injection. In conclusion, PEDOT / PtN1C3 / CFE can quantitatively monitor H2O2 release at both cellular and in vivo levels, providing not only novel direct evidence for Res-induced H2O2 production but also a reliable tool for future exploration and application of more mechanisms.

Claims

1. A microelectrode, characterized in that, The microelectrode comprises an electrode, a composite layer, and a polymer layer; The composite layer is modified on the electrode surface, and the polymer layer is modified on the electrode surface of the modified composite layer; The composite layer includes a PtN1C3 single-atom catalyst, which comprises a support and a metal active component; the support is a nitrogen-doped multi-walled carbon nanotube; the metal active component is Pt; the metal active component is dispersed on the support in the form of isolated single atoms; the Pt is coordinated with 3 carbon atoms and 1 nitrogen atom to form a PtN1C3 coordination configuration.

2. The microelectrode according to claim 1, characterized in that, The electrode is a carbon fiber electrode; the polymer layer is PEDOT.

3. The microelectrode according to claim 1, characterized in that, The PtN1C3 single-atom catalyst was prepared using the following method: 1) K2PtCl4 precursor was mixed with N-MWCNT and reacted to prepare K2PtCl4 / N-MWCNT; 2) The K2PtCl4 / N-MWCNT obtained in step 1) was mixed and ground with urea, and then thermally annealed to obtain a PtN1C3 single-atom catalyst; The mass ratio of K2PtCl4 / N-MWCNT to urea is 1:3-6.

4. The method for preparing the microelectrode according to any one of claims 1 to 3, characterized in that, Includes the following steps: (1) PtN1C3 single-atom catalyst was modified onto the surface of bare CFE by electrochemical deposition to obtain PtN1C3 / CFE; (2) The PtN1C3 / CFE obtained in step (1) is deposited with PEDOT:TFB to modify the surface of the PtN1C3 / CFE with PEDOT to obtain PEDOT / PtN1C3 / CFE.

5. The preparation method according to claim 4, characterized in that, In step (1), the electrochemical deposition uses bare CFE as the working electrode, Pt wire as the counter electrode, and Ag / AgCl as the reference electrode; the electrochemical deposition is performed by the Ampere method at a potential of 1.0-2.0V for 4500-6500 seconds.

6. The preparation method according to claim 4, characterized in that, In step (1), the deposition solution is a mixture of PtN1C3 and acetonitrile.

7. The preparation method according to claim 4, characterized in that, In step (2), the electrolyte is an acetonitrile solution containing 0.005-0.03M EDOT and 0.05-0.3M TFB; the PEDOT:TFB deposition is carried out at 1.0-1.5V for 15-60 seconds.

8. The application of the microelectrode according to claim 1 in in-situ dynamic monitoring of H2O2 levels.

9. The application of the microelectrode of claim 1 in monitoring the kinetic changes of H2O2 in cells and / or living brain.

10. The application of the microelectrode of claim 1 in monitoring H2O2 kinetic changes in cells and / or living brains induced by resorcinol and rosmarinic acid.

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