A full hydrogel neural electrode and its preparation method and use

Abstract

The application provides a full hydrogel nerve electrode which is composed of four layers of hydrogel, namely a PSFD hydrogel layer, an LPPDMEA layer, a PSFD hydrogel layer and an MHD layer; the application also provides a preparation method and application of the full hydrogel nerve electrode. The application provides a PEDOT:PSS hydrogel conductive path with ultrahigh conductivity, which achieves a record capacity compared with a metal electrode; the introduction of dopamine in each layer of the full hydrogel nerve electrode ensures excellent interface compliance, so that the full hydrogel nerve electrode can be attached to the complex geometry of soft neural tissue and avoid interface mismatch between the conductive layer, the packaging layer, the shielding layer and the tissue-electrode interface; the natural polymer packaging layer improves compatibility, and the electromagnetic shielding layer is used to improve the signal-to-noise ratio. The full hydrogel nerve electrode can provide artificial electrical stimulation to the cerebral cortex of a rat during a seizure, and the prepared full hydrogel nerve electrode has the potential to become a valuable tool for better understanding the development, function and treatment of brain diseases and neurodegenerative diseases and neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to a fully hydrogel neural electrode, its preparation method, and its uses. Background Technology

[0002] Brain diseases and neurodegenerative disorders, such as epilepsy, Alzheimer's disease, and mental illnesses, severely impact patients' quality of life and impose a heavy socioeconomic burden. In the brain, numerous neurons systematically generate and transmit electrophysiological signals to communicate with other neurons and other areas of the brain. The occurrence of brain diseases, characterized by permanent or temporary dysfunction of the central nervous system, often leads to paradoxical discharges in neurons. Based on this electrophysiological phenomenon, the application of neural electrodes to precisely detect and even modulate neural activity signals is extremely helpful in predicting the onset of disease and preparing timely artificial stimulation for treatment.

[0003] For the past decade, stereotactically placed single metal electrodes (such as silver, gold, and platinum) have been the gold standard for brain recording and artificial stimulation applications. However, the inherent rigidity of metal electrodes (up to 200 GPa) has been shown to cause tissue damage, and glial cell aggregation caused by foreign body reactions between the metal electrode and tissue can lead to decreased sensing performance. All-hydrogel neural electrodes have attracted considerable attention due to their natural mechanical compliance. Their modulus-match to brain tissue avoids subsequent problems caused by modulus mismatch.

[0004] Another prerequisite for all-hydrogel neural electrodes is the ability to record high-precision electrophysiological signals, which depends on the conductivity or ionic conductivity of the conductive hydrogel layer. Among all hydrogel-based conductive materials, poly(3,4-ethyldioxothiabenzene):poly(styrene sulfonate) (PEDOT:PSS) exhibits superior flexibility, biocompatibility, and electrochemical stability, making it competitive in flexible electrode designs (Fan et al. 2019; Zhou et al. 2023). However, the need to improve electrode performance for capturing weak neural electrical signals, and the fact that untreated PEDOT:PSS typically has a much lower conductivity than metals (~0.1 S / cm), limits its performance in certain electrical applications. Excessive PSS (polystyrene sulfonate) is one of the reasons for the low conductivity. Furthermore, PEDOT:PSS is acidic and may react with electrode materials, leading to electrode corrosion. PEDOT:PSS is sensitive to water and oxygen, which can affect device stability and lifespan.

[0005] A key step in improving conductivity is to dope PEDOT and PSS with chemical reagents or post-treatment, including but not limited to polar solvents, dopamine, acids, and ionic liquids, which can reduce the Coulomb forces between PEDOT and PSS, thereby promoting the separation of PSS chains from PEDOT chains. Furthermore, physical treatments such as electric fields, heating, ultraviolet light, and lasers have been shown to effectively induce phase separation of PEDOT:PSS.

[0006] Existing PEDOT:PSS hydrogels suffer from dopant toxicity, and the finished product faces a complex water washing and detoxification process. Physical methods such as heat treatment, electric fields, and UV radiation are generally ineffective, and excessively high-intensity lasers can cause carbonization of PEDOT, destroying its conjugated conductive structure and limiting the degree of phase separation. For example, the literature "Soft and elastic hydrogel-based microelectronics for localized low-voltage neuromodulation" (LIU, Yuxin, et al. Soft and elastic hydrogel-based microelectronics for localized low-voltage neuromodulation. Nature biomedical engineering, 2019, 3.1: 58-68.) lacks stable interlayer adhesion and has insufficient conductivity of conductive pathways; "Laser-induced wet stability and adhesion of pure conducting polymer hydrogels" (Won, Daeyeon, et al. Laser-induced wetstability and adhesion of pure conducting polymer hydrogels. Nature Electronics, 2024, 1-12.) uses PET as a substrate and PDMS encapsulation. These chemical polymers are not as biocompatible as natural polymers, and their high modulus poses a risk of inducing immune responses and glial layer formation for long-term implantation; "Viscoelastic surface electrode arrays to interface with viscoelastic tissues" (Tringides, Christina M., et al. Viscoelastic surface electrode arrays to interface with viscoelastic tissues. Nature nanotechnology, 2021, 16.9: 1019-1029. Due to the lack of stable interlayer adhesion and insufficient conductivity of conductive pathways, the fabrication of circuits with sodium alginate and conductive fillers is difficult to achieve high-precision patterning capabilities in the template method. Summary of the Invention

[0007] This invention provides a dopamine (DA) integrated all-hydrogel neural electrode for capturing weak neural signals.

[0008] This invention provides an all-hydrogel neural electrode, which is composed of four hydrogel layers: PSFD hydrogel layer, LPPDMEAs layer, PSFD hydrogel layer, and MHD layer.

[0009] The PSFD hydrogel layer serves as an insulating encapsulation layer.

[0010] The LPPDMEAs layer is a conductive layer;

[0011] The MHD layer is a shielding layer;

[0012] in,

[0013] The PSFD hydrogel is prepared by mixing dopamine (DA) modified degummed fibrous protein (SF) with polyethylene glycol diglycidyl ether (PEGDE) with an average Mn of 500.

[0014] The LPPDMEAs layer is a multi-electrode array formed by depositing PPD aqueous solution onto the PSFD hydrogel layer and then laser etching it.

[0015] The PPD aqueous solution is a PEDOT:PSS solution doped with dopamine (DA);

[0016] The MHD layer is composed of Mxene and dopamine (DA) modified hyaluronic acid.

[0017] The method for preparing the PPD aqueous solution is as follows:

[0018] DA·HCl was added to a PEDOT:PSS aqueous solution (10 mL, 10 mg / mL) for synthesis. After stirring at room temperature for 30 minutes, PPD aqueous solution was obtained.

[0019] The preparation method of the LPPD hydrogel is as follows:

[0020] A PPD aqueous solution was deposited on a PSFD hydrogel layer to form a film, which was then annealed at 60°C. The PPD film was then laser etched under the following conditions: laser power 0.185W and scanning speed 20mm / s.

[0021] The preparation method of the MHD hydrogel is as follows:

[0022] EDC and NHS were added to a 10 wt% hyaluronic acid (HA) solution to activate the carboxyl groups, and then DA·HCl was added. The reaction solution was stirred overnight at room temperature under nitrogen protection. Then, the solution was dialyzed in deionized water for 3 days using a 10000 Da dialysis membrane, and the pure product HAD was obtained by freeze drying.

[0023] Then, 2.5 wt% HAD solution was mixed with MXene at concentrations of 0, 1, 1.5 and 2 mg / mL, respectively. Then, 30 μL of 1 mg / mL HRP solution and 12.5 μL of 0.5 mol / L H2O2 were added. After stirring evenly, the mixture was placed at 40℃ for 10 minutes to obtain MHD hydrogel.

[0024] The preparation method of the PSFD hydrogel is as follows:

[0025] An 8 wt% degummed silk fibroin SF solution was placed in an ice bath, followed by the addition of EDC and NHS. After stirring for 30 minutes, DA·HCl was added to the solution. After reacting for one hour, the mixture was dialyzed with deionized water using a 14000 Da dialysis bag and then freeze-dried to obtain pure SFD.

[0026] Polyethylene glycol diglycidyl ether (PEGDE) with an average Mn of 500 was added to a 5 wt% SF-DA solution and then dynamically stirred at 60°C. The solution was poured into a substrate, and a thin film was formed after the moisture evaporated in the ambient air. After absorbing water, a PSFD hydrogel was obtained.

[0027] This invention provides a method for preparing the all-hydrogel neural electrode, which includes the following steps:

[0028] a. A PPD film is deposited on a PSFD hydrogel layer and then laser etched to form a 16-channel pattern;

[0029] b. Wash with water to remove the untreated parts. The remaining LPPD membrane absorbs water and forms an LPPDMEAs layer.

[0030] c. Place another insulating PSFD hydrogel layer on top of the LPPD MEAs hydrogel layer, and then perform a second laser etching to expose 16 electrode points;

[0031] d. Add an MHD shielding layer on top and link it through a DA multi-interaction PSFD layer to form a four-layer all-hydrogel neural electrode.

[0032] The laser etching conditions described in step a are: laser power 0.185W; scanning speed 20mm / s.

[0033] The laser etching described in step c involves selectively etching away the second layer of PSFD after the second layer of PSFD is encapsulated, exposing the electrode points for contact with tissue.

[0034] The present invention also provides the use of the described all-hydrogel neural electrode in the preparation of devices for recording neural activity, treating or treating neurodegenerative diseases.

[0035] The beneficial effects of this invention are:

[0036] 1. Provides a PEDOT:PSS hydrogel conductive pathway with ultra-high conductivity (high phase separation, ultra-high conductivity of 4173 S / CM), achieving recording capabilities comparable to metal electrodes;

[0037] 2. Tight interlayer bonding: Polyphenol chemistry involves various interactions such as hydrogen bonding, electrostatic interactions, thiol reduction, and π-π / cation-π interactions. The introduction of dopamine into each layer of the all-hydrogel neural electrode ensures excellent interfacial compliance, allowing it to conform to the complex geometry of soft nerve tissue and avoiding interfacial mismatch between the conductive layer-encapsulation layer-shielding layer and the tissue-electrode interface. This also avoids material failure and decreased recording capacity caused by physiological stress.

[0038] 3. Reasonable structural design and material selection: The natural polymer encapsulation layer improves compatibility, and the electromagnetic shielding layer is used to improve the signal-to-noise ratio.

[0039] The all-hydrogel neural electrode of this invention can provide artificial electrical stimulation to the cerebral cortex of rats during epileptic seizures. The prepared all-hydrogel neural electrode has the potential to become a valuable tool for better understanding the development, function, and treatment of brain diseases and neurodegenerative diseases. Attached Figure Description

[0040] Figure 1 Flowchart of the preparation method of the all-hydrogel neural electrode of the present invention;

[0041] Figure 2 The effect of laser scanning speed on the resolution of patterned PEDOT:PSS hydrogels. (A) Images of laser-processed PEDOT:PSS films (top, before washing) at different scanning speeds and hydrogels formed with different pattern resolutions (bottom, after washing). Scale bar: 50 μm. (B) Statistical analysis of the resolution of patterned PEDOT:PSS hydrogels processed at different laser scanning speeds;

[0042] Figure 3 Laser-etched cat pattern PEDOT:PSS hydrogel;

[0043] Figure 4 COMSOL was used to simulate the pyrolysis process of surface PSS during laser etching.

[0044] Figure 5 XPS S2p spectrum of PEDOT:PSS at 400 laser intensity;

[0045] Figure 6Images of PEDOT:PSS treated with 350 (optimal power) and 400 (excess power) lasers. A) PEDOT:PSS treated with a 350 laser exhibits a bluish-black color similar to the original PEDOT:PSS. B) PEDOT:PSS treated with a 400 laser exhibits a yellow color due to the formation of carbides.

[0046] Figure 7 Laser-induced phase separation of PEDOT:PSS. A) Schematic diagram of femtosecond laser treatment in PEDOT:PSS film. B) Preparation steps of cat-patterned PEDOT:PSS hydrogel (top) and molecular changes of PEDOT after laser treatment and water washing (bottom). C) Schematic diagram of laser-triggered photothermal effect leading to pyrolysis of separated PSS portions. D) Laser-induced air ionization to form plasma, and the luminescent electric field generated by the plasma. E) Finite element modeling of the laser-triggered photoelectric field. F) Focal temperature of PEDOT:PSS surface at different laser intensities. G) XPS S2p and H) Raman spectra of PEDOT:PSS at different laser intensities. I) XPS C1s and J) Raman spectra illustrating the carbonization process within PEDOT:PSS at an excess laser intensity of 400. (K) Schematic diagram of carbonization effect caused by excessive laser intensity within PEDOT:PSS.

[0047] Figure 8 1H NMR spectra of PEDOT:PSS and PPDs at different dopamine (DA) concentrations;

[0048] Figure 9 This image shows the obvious aggregation phenomenon in the PEDOT:PSS solution after the addition of excess dopamine (15 mg / mL). Excess dopamine disrupts the ionic environment of the PEDOT:PSS solution, leading to significant micellar aggregation of PEDOT:PSS molecules.

[0049] Figure 10 XPS spectra of PEDOT:PSS and PPDs at different dopamine concentrations;

[0050] Figure 11 Raman spectra of PEDOT:PSS and PPDs at different dopamine concentrations;

[0051] Figure 12 Thermogravimetric curves of PEDOT:PSS and PPDs at different dopamine concentrations;

[0052] Figure 13Electrochemical performance of PSDs and PPDs in PEDOT. A) Cyclic voltammetry (CV) curves of PSDs, PPD2.5, PPD5, and PPD10 in PEDOT. B) Electrochemical impedance spectroscopy (EIS, from 1 Hz to 10 MHz) of PSDs, PPD2.5, PPD5, and PPD10 in PEDOT. C) Nyquist curves of PSDs, PPD2.5, PPD5, and PPD10 in PEDOT. D) Phase angles of PSDs, PPD2.5, PPD5, and PPD10 in PEDOT.

[0053] Figure 14 AFM phase images of PPDs and LPPDs at different dopamine concentrations. PEDOT enrichment regions are shown in purple, and PSS enrichment regions are shown in yellow and green.

[0054] Figure 15 Thermogravimetric analysis (TGA) curves of LPPDs at different dopamine concentrations;

[0055] Figure 16 XPS spectra of PPL and LPPD hydrogels with different dopamine concentrations;

[0056] Figure 17 Raman spectra of PPL and LPPD hydrogels with different dopamine concentrations;

[0057] Figure 18A) A schematic diagram of the internal structure of untreated PEDOT:PSS transformed into PEDOT:PSS (LPPD) induced by DA and laser synergy. B) A schematic diagram showing the conformational transformation of PEDOT mediated by the dual strategy: from the random coil structure of PEDOT:PSS to the regular linear structure of LPPD, accompanied by the enhancement of PEDOT-enriched structural domains. C) AFM phase images of PEDOT:PSS, LPP, PPD10, and LPPD10 (left), where PEDOT-enriched domains are purple, and PSS-enriched domains are yellow and green; the right side shows the area change of PEDOT-enriched domains calculated using ImageJ software. D) Micro-region mechanical distribution of PEDOT:PSS, LPP, PPD10, and LPPD10 in nanoindentation experiments. E) Thermogravimetric analysis (TGA) curves of PEDOT:PSS, LPP, PPD10, and LPPD10 (left), and the corresponding decomposition temperatures of each PEDOT group (right). F) XPS S2p spectra of PEDOT:PSS, LPP, PPD10, and LPPD10. G) PEDOT / PSS ratios of the four materials based on XPS results. H) Raman spectra of PEDOT:PSS, LPP, PPD10, and LPPD10. I) Quinone / benzoyl ratios of the four materials calculated using Raman spectroscopy results.

[0058] Figure 19 Highly conductive LPPD hydrogels were prepared by DA / laser co-processing. (A) Schematic diagram showing the molecular structure transformation induced by DA doping in PEDOT:PSS solution. (B) Schematic diagram showing the formation of PEDOT semi-crystalline domains in LPPD films induced by femtosecond laser pyrolysis of PSS. (C) AFM image of PEDOT: PSS and (D) ESR spectra of PPD and LPPD. (E) XPS S2p and Raman spectra of PEDOT: PSS, PPD, LPP and LPPD. Calculated (G) PEDOT / PSS ratio and (H) quinone / benzoyl ratio. Schematic diagram of charge transfer between PEDOT and DA. (J) CV curves and (K) conductivity of PEDOT: PSS, PPD, LPP and LPPD;

[0059] Figure 20 FTIR spectra of SF and PSF hydrogels treated with different PEGDE weight ratios;

[0060] Figure 21 FTIR spectra of PSF and PSFD hydrogels treated with PEGDE at different weight ratios;

[0061] Figure 22XRD spectra of MXene and MHD hydrogels with different concentrations of MXene;

[0062] Figure 23 Conductivity of MHD hydrogels with different MXene concentrations;

[0063] Figure 24 X-band electromagnetic interference shielding effectiveness (EMI SE) of MHD2 hydrogel before and after 500 bending cycles;

[0064] Figure 25 Electromagnetic interference shielding effectiveness (EMI SE) of MHD2 hydrogel in X-band (8.21-2.4 GHz), Ku-band (12.414 GHz), K-band (1418 GHz) and Ka-band (1840 GHz);

[0065] Figure 26 Preparation and characterization of encapsulation and shielding layers. (A) Schematic diagram of encapsulation layer (PSFD hydrogel) preparation. (B) 1SFD and SF HNMR spectra. (C) FTIR spectra of PSFD hydrogels treated with different weight ratios of PEGDE. (D) Stress-strain curves of PSFD, PSF and hydrogels in tensile mode. (E) Optical transparency diagram of 10% PSFD hydrogel. (F) Schematic diagram of EMI shielding layer (MHD hydrogel) preparation. (G) Schematic diagram of EMI shielding mechanism dominated by absorption of MHD hydrogel. (H) 1HAD and HA HNMR spectra; (I) TEM image of MXene. (J) Scanning electron microscope image of MHD hydrogel. (K) X-band EMISE, (L) SER, SEA and SET MHD hydrogels containing different MXene contents. (M) Schematic diagram of the transition from MHD hydrogel to MHD foam. (N) Curves of SEA, SER and SET MHD2 hydrogels in different states;

[0066] Figure 27 Parameters of a 16-channel microelectrode array (MEAs) mode;

[0067] Figure 28 Live / dead staining of PC12 cells co-cultured with different layers and the entire four-layer neural electrode system. The PSFD group represents PC12 cells co-cultured with the PSFD layer; the LPPD group represents PC12 cells co-cultured with the LPPD layer; the LPP group represents PC12 cells co-cultured with the LPP layer; the MHD group represents PC12 cells co-cultured with the MHD layer; and the PSFD-MHD-LPPD group represents PC12 cells co-cultured with the four-layer neural electrode system consisting of the MHD, PSFD, LPPD, and PSFD layers. Scale bar: 250 μm;

[0068] Figure 29Fabrication and characterization of all-hydrogel neural electrodes. (A) Flowchart showing the fabrication process of the 16-channel neural electrode. (B) Schematic diagram of the proposed 4-layer neural electrode. Encapsulation layer: PSFD hydrogel, conductive layer: LPPD hydrogel MEAs, shielding layer: MHD hydrogel. (C) Image showing the prepared neural electrode attached to an FPC (top). Microscopic view of the arrangement of the 16 individual electrodes and the exposure of the electrodes after laser etching (bottom). (D) Tight interlayer bonding induced by polyphenol chemistry. (E) Schematic diagram of neural electrode-tissue interface interaction. (F) Adhesion strength of LPPD MEAs encapsulated by PSF or PSFD layers. (G) Image showing the conformation of the neural electrode to the surface of the pig brain. (H) Image of LPPD MEAs. (I) Image of LPPD MEAs wrapped around a glass rod. The image depicts the neural electrode after being rubbed (J) and bent (K) while maintaining its intact structure without signs of delamination. (L) Scanning electron microscopy image showing the cross-sectional morphology of the LPPD hydrogel. (M) Optical and fluorescence images showing the adhesion and growth of pheochromocytoma (PC12) cells on LPPD MEAs. (N) Resistance changes of the 16-channel electrode under stretching, twisting, and bending. (O) Scanning electron microscopy images of the interface between the conductive layer and PSFD encapsulation layer of LPPD MEAs (left) or the conductive layer and PSF encapsulation layer of LPPD MEAs (right) without DA at 15% strain;

[0069] Figure 30 The implantation site of this prepared all-hydrogel neural electrode in the rat brain;

[0070] Figure 31 Representative multichannel (channels 9-16) local field potential (LFP) signals (top) and their corresponding time-frequency spectra (bottom) recorded in epileptic rats at rest;

[0071] Figure 32 Representative multichannel (channels 9-16) local field potential (LFP) signals (top) and their corresponding time-frequency spectra (bottom) recorded in early epileptic states of rats;

[0072] Figure 33 Representative multichannel (channels 9-16) local field potential (LFP) signals (top) and their corresponding time-frequency spectra (bottom) recorded in late-stage epileptic rats;

[0073] Figure 34 Representative multichannel (channels 9-16) local field potential (LFP) signals (top) and their corresponding time-frequency spectra (bottom) recorded in epileptic rats after recovery;

[0074] Figure 35The effect of electromagnetic shielding on background noise reduction. Local field potentials (LFPs) recorded by neural electrodes with (top) and without (bottom) electromagnetic shielding. After removing the electromagnetic shielding, the recorded LFP background noise increased significantly;

[0075] Figure 36 LFP recorded from the surface of the cerebral cortex. (A) LFP signal recorded on day 1 post-surgery; (B) LFP signal recorded 2 weeks post-surgery. (C) Signal-to-noise ratio (SNR) of LFP signals obtained from the 16-channel all-hydrogel neural electrode on day 1 and day 7 post-surgery. (D) An image showing a freely moving rat equipped with the 16-channel all-hydrogel neural electrode. The tip of the device was fixed with dental cement after implantation.

[0076] Figure 37 4. Schematic diagram of the 16-channel neural electrode electron transfer interface for ECoG signal recording of LFP (A) in vivo multichannel monitoring. (B) Schematic diagram of LFP signal recording and electrical stimulation treatment in 4-AP-induced epileptic rats. (C) LFP signals recorded at rest, epileptic state, and recovery state after electrical stimulation by CH-1. Representative multichannel recorded LFP signals (top) and corresponding time-spectrum diagrams (bottom) of (D) rest state, (E) early epilepsy, (F) late epilepsy, and (G) recovery state. (H) Power spectral density (PSD) of LFP signals in different states. (I) LFP signals recorded by Ag electrodes (CH-1 to CH-8), and this work used LPPD hydrogel electrodes (CH-9 to CH-16). (J) Signal-to-noise ratio of LFP signals recorded by Ag electrodes, this work used LPPD hydrogel electrodes;

[0077] Figure 38 Immunofluorescence images of a flexible 16-channel all-hydrogel neural electrode and a rigid silver neural electrode two weeks after insertion into a brain slice. iNOS: inducible nitric oxide synthase, a marker of immune activation and inflammation. Arg1: arginase-1, a marker of macrophage polarization. Scale bar: 100 μm. The lack of interaction between the silver electrode and the substrate led to direct electrode detachment after implantation, as well as more intense macrophage aggregation and tissue damage.

[0078] Figure 39 Peak signals recorded in 16 channels 2 weeks after implantation;

[0079] Figure 40Multichannel in vivo monitoring of action potentials. (A) Schematic diagram showing appropriate contact between recording electrodes and neurons. (B) Time-stabilized and (C) peak waveforms of action potentials recorded in a resting state. (D) Two spike waveforms from different neurons and (E) their principal component analysis (PCA). (F) and (G) Autocorrelation plots of the two recorded peaks. (H) Schematic diagram of peaks recorded before and after epileptic induction. (I) Eight action potentials recorded within a very small time window. (J) The spike waveform of the third group. (K) The superimposed voltage trajectory of the spikes. (L) Array layout of the average peak time delay calculated from 50 action potentials. (M) Total number of peaks recorded per second. (N) Total number of peaks recorded per channel within 60 seconds. (O) Immunofluorescence images of rat brain slices 2 weeks after insertion of all-hydrogel neural electrodes and rigid Ag neural electrodes. Brain slices without implanted electrodes served as controls (sham-operated group). NF: Neurofilaments, markers of neuronal development. GFAP: Glial cell fibrillary acidic protein, a product of glial cells. Detailed Implementation

[0080] Experimental materials:

[0081] PEDOT: PSS was purchased from Clevios (PH1000, Germany). Dopamine hydrochloride (DA·HCl), 1-(3-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from A-Latin Ltd. (Shanghai, China). Hyaluronic acid (HA, Mw = 700k-800k Da) was obtained from Bloomberg Biotechnology Co., Ltd. HRP (activity: 248 units / mg) was from TCI (Japan). Fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Sigma-Aldrich (USA). MXene was obtained from Beike (China). Unless otherwise stated, all other chemicals were obtained from Chengdu Kelon Chemical Co., Ltd.

[0082] Example 1: Preparation method of the all-hydrogel neural electrode of the present invention

[0083] a. A PPD film is deposited on a PSFD hydrogel layer and then laser etched to form a 16-channel pattern;

[0084] b. Wash with water to remove the untreated parts. The remaining LPPD membrane absorbs water and forms an LPPDMEAs layer.

[0085] c. Place another insulating PSFD hydrogel layer on top of the LPPD MEAs hydrogel layer, and then selectively perform secondary laser etching to expose 16 electrode points for contact with the target tissue.

[0086] d. Fix an MHD shielding layer on top to form a four-layer all-hydrogel neural electrode.

[0087] Selective etching refers to selectively etching away the second layer of PSFD after encapsulation, focusing only on the electrode points (the circular areas) to expose the electrode points for contact with tissue. Other areas are left untreated to ensure insulation between the treated areas and the tissue.

[0088] The detailed flowchart is as follows: Figure 1 As shown:

[0089] The preparation methods for each raw material are as follows:

[0090] 1) Preparation of PPD (DA-doped PEDOT:PSS)

[0091] PPD was synthesized by directly adding DA·HCl (10 mg) to a PEDOT:PSS aqueous solution (10 mL, 10 mg / mL). After stirring at room temperature (RT) for 30 minutes, the product was obtained and stored at 4°C for subsequent use. The successful synthesis of PPD was subsequently verified by 1H NMR spectroscopy (600 MHz, Bruker, USA).

[0092] 2) Preparation of LPP (laser-treated PEDOT:PSS) and LPPD (laser and DA dual-strategy treated PEDOT:PSS)

[0093] A femtosecond laser system (INNO Laser, China) emitting a continuous-wave laser (532 nm) was used to selectively induce phase separation in PEDOT:PSS-based materials. The untreated portions were then removed by water washing. The laser power was set to 250, 300, 350, and 400 kW (these are the power values ​​displayed by the instrument, corresponding to 0.067 W, 0.114 W, 0.185 W, and 0.280 W respectively). Laser scanning speeds of 10, 20, 30, 40, and 50 mm / s were used to select the optimal speed.

[0094] 3) Synthesis of HD (HA grafted with DA) and preparation of MHD (HD hydrogel mixed with MXENE) hydrogel

[0095] EDC (440 mg) and NHS (288 mg) were added to a 10 wt% HA solution to activate the carboxyl groups, followed by the addition of 0.47 g DA·HCl. The reaction solution was stirred overnight at room temperature (RT) under nitrogen protection. Then, the solution was dialyzed in a DIW for 3 days using a 10,000 Da dialysis membrane, and the purified HD product was obtained by freeze-drying. To form a series of MHD hydrogels, a 2.5 wt% HD solution was mixed with different concentrations of MXene (0, 1, 1.5, and 2 mg / mL, respectively), followed by the addition of 30 μL of HRP solution (1 mg / mL) and 12.5 μL of H2O2 (0.5 mol / L). After stirring at room temperature for 5 minutes, the mixture was incubated at 40 °C for 10 minutes to form an MHD hydrogel.

[0096] 4) Preparation of debonded SF (silk fibroin for encapsulation layer)

[0097] The degummed SF was prepared according to the literature (Cui, Yajing, et al. A stretchable and transparent electrode based on PEGylated silk fibroin for in vivo dual-modal neural-vascular activity probing. Advanced Materials, 2021, 33.34:2100221.). Specifically, 5 grams of silkworm cocoons were soaked in 2 liters of 0.02 M Na₂CO₃ solution and then boiled at 100 °C for half an hour. The product was rinsed with deionized water (DIW) to remove residual Na₂CO₃ solution. This process was repeated three times to obtain a crude product. 2 grams of the obtained product were added to 8 mL of 9.3 M LiBr solution and stirred at 60 °C for 4 hours. Subsequently, the solution was dialyzed with DIW using an 8000 Da dialysis bag, and then the degummed SF was obtained by freeze-drying.

[0098] 5) Preparation of SFD (SF grafted with DA)

[0099] Five mL of 8 wt% degummed SF solution was placed in an ice bath, followed by the addition of 37.5 mg EDC and 45 mg NHS. After stirring for 30 minutes, 216 mg DA·HCl was added to the solution. After reacting for one hour, the mixture was dialyzed against a DIW for 3 days using a dialysis bag (14000 Da), and pure SFD was obtained by freeze-drying. The successful synthesis of the product was then verified by 1H NMR spectroscopy (600 MHz, Bruker, USA).

[0100] 6) Preparation of SF and PSFD (PEGDE-initiated SFD hydrogels)

[0101] PEGDE (average Mn 500) was added to a 5 wt% SF or SF-DA solution, and then stirred at 60 °C for 15 minutes. The solution was poured into a substrate and air-dried under RT to remove residual water and form a film. Subsequently, Fourier transform infrared spectroscopy (FT-IR) was performed (Nicolet IS5, ThermoFisher Scientific, USA) to characterize the changes in the SFD molecular structure after PEGDE-induced film formation.

[0102] Example 2: Experiment on the selection of laser etching conditions during the fabrication of the all-hydrogel neural electrode of the present invention.

[0103] A femtosecond laser system emitting a continuous-wave laser (532 nm) (INNO Laser, China) is used to selectively induce phase separation based on PEDOT:PSS materials. The "selectivity" refers to the selective and patterned processing of PEDOT:PSS by controlling the laser etching path according to a programmed pattern.

[0104] Power screening:

[0105] 1. Purpose:

[0106] a. Screening power parameters to achieve stable hydrogel construction.

[0107] b. Control the power to obtain the group of materials with the highest degree of phase separation;

[0108] c. And to seek the parameters that enable the laser thermal effect to fully lead to the pyrolysis of PSS;

[0109] d. Find the parameters that cause PEDOT carbonization due to excessive heat effect.

[0110] 2. The results showed that 250 was the minimum parameter that could induce hydrogel construction, while 400 would lead to PEDOT carbonization. Therefore, after screening between 250 and 400, 350 was found to be the most suitable parameter, which ensured hydrogel construction, high degree of phase separation, PSS pyrolysis, and PEDOT retention.

[0111] Scan speed filtering:

[0112] 1. Purpose:

[0113] a. Neurons are tiny, and only electrode recording sites smaller than 100 micrometers can be used to record action potentials. Different scanning speeds will affect the thickness of the electrode circuit and the size of the electrode points.

[0114] b. Bonding to the substrate: Excessively fine and tiny dimensions result in a small bonding area between the electrode and the PSFD substrate, leading to unstable bonding.

[0115] 2. The results show that a velocity of 20 mm / s is optimal, providing stable binding to the basal layer and a size that is well-suited to the neuron. (See [link to study]). Figure 2 .

[0116] 3. PEDOT: Optimization of laser energy in PSS phase separation

[0117] Because lasers offer extremely high energy injection and fine processing precision, they can simultaneously meet the requirements for designing micropatterns and adjusting the content of conductive / insulating phases in the flexible conductive polymer PEDOT:PSS. By programming and controlling the scanning path (the pattern being a cat), laser-etched PEDOT:PSS(LPP) films can be obtained. Figure 3 , Figure 7 A, Figure 7 B).

[0118] During laser etching, the resulting thermal effect reduces the electrostatic force between PEDOT and PSS, causing some of the PEDOT phase to separate from the PSS phase. Figure 7 C). Simultaneously, its thermal effect leads to the pyrolysis of the insulating PSS phase, which has a lower decomposition temperature. High-energy lasers also excite the ionization of the surrounding air, forming an electric field composed of plasma. Figure 7 D). The two partially separated phases are in this electric field, and because they carry opposite charges, further phase separation will occur. Induced by the laser photothermal / photogenerated electric field, the π-π stacking of the PEDOT domains and the interchain entanglement in the PSS chains are regulated and reach equilibrium. Upon contact with water, the regulated PEDOT stable domains form a gel framework, and due to the water enrichment of the residual hydrophilic PSS phase, the laser-scanned portion transforms into a cat-shaped hydrogel.

[0119] Then, in the COMSOL simulation system, laser intensities of 250, 300, 350, and 400 (specific energy values ​​of the instrument parameters, 0.067W, 0.114W, 0.185W, and 0.280W, respectively) were selected, see [link to COMSOL simulation]. Figure 4 .

[0120] The results show that 350 is the optimal laser intensity for inducing the pyrolysis of non-conductive PSS. Figure 7 F). Subsequently, X-ray photoelectron spectroscopy (XPS) S2p spectroscopy verified the successful phase separation of PEDOT:PSS, with an increase in the peak value of PEDOT between 167 and 162 eV and a decrease in the peak value of PSS between 172 and 164 eV. Figure 7 G, Figure 5 ). Figure 7The Raman spectrum in H showed a redshift of the adsorption peak with increasing laser intensity, indicating a conformational transformation of PEDOT after phase separation. Excessive laser intensity of 400 nm may lead to carbonization of PEDOT:PSS. Figure 7 IK and Figure 6 Therefore, in the following experiments, the laser intensity for optimal phase separation was fixed at 350.

[0121] 4. Dual-strategy therapy for inducing phase separation in PEDOT: PSS

[0122] Laser treatment can induce phase separation in PEDOT:PSS, but this single treatment method is limited and it is difficult to further improve the conductivity. Combining chemical doping with laser treatment can synergistically enhance phase separation, achieving broad modulation of the conductivity of PEDOT:PSS. Considering the biocompatibility of flexible bioelectronic devices, non-toxic DA is used as a chemical dopant to further induce phase separation in PEDOT:PSS.

[0123] First, the effect of using DA alone on the phase separation of PEDOT:PSS was evaluated. PEDOT:PSS doped with DA was named PPD. After several screenings and characterizations, the DA doping concentration was determined to be 10 mg / ml (the doped product was named PPD10) for subsequent laser processing. Figures 8-13 ).like Figure 18 As shown in Figure A, in principle, the electrostatic contact between the primary amine group of DA and the sulfonic acid ion of PSS weakens the Coulomb force between PEDOT and PSS, thereby synergistically enhancing the laser-induced phase separation in PEDOT:PSS. Subsequently, the separated PEDOT chains further aggregate and recrystallize under π-π conjugation to form PEDOT-enriched domains. This process is accompanied by a conformational transformation of PEDOT from benzene (random helical shape) to quinone (regular linear shape). Figure 18 B). It was further confirmed that a laser intensity of 350 nm was also the optimal parameter for inducing phase separation of PEDOT and PSS within the PPD10 group, showing the largest PEDOT peak in the XPS S2p spectrum and the most significant redshift of the PEDOT peak in the Raman spectrum. Therefore, for PEDOT:PSS DA / laser dual-strategy treatment, with a DA concentration of 10 mg / mL and a laser intensity of 350 nm, the product after dual-strategy treatment is termed LPPD10.

[0124] Phase separation regulation and electrochemical properties of PPD hydrogels were studied, showing adjustable dopamine concentration, such as... Figure 8As shown, the introduction of dopamine (DA) led to phase separation of PEDOT and PSS. In the 1H NMR spectrum, a peak of the catechol ring appeared in the DA-doped PEDOT:PSS group (PPDs). The degree of phase separation in PPDs increased with increasing DA concentration, while excessive DA concentration (15 mg / mL) may lead to significant aggregation of PEDOT:PSS. Figure 9 Subsequently, XPS analysis was performed to investigate the phase separation between PEDOT and PSS in the system under different DA doping concentrations. The results showed that the degree of phase separation gradually increased with increasing DA concentration. Figure 10 As phase separation intensifies, the increased concentration of free PEDOT molecular chains forms denser crystalline domains through π-π conjugation interactions. This transformation manifests as a conformational change in PEDOT from a benzene to a quinone structure, a change confirmed by Raman analysis. Figure 11 This strongly confirms the point. Furthermore, the increased number of crystalline domains resulting from enhanced phase separation leads to a higher decomposition temperature for PEDOT. Figure 12 ).

[0125] Subsequently, PPDs with different DA concentrations were electrochemically characterized. Cyclic voltammetry (CV) curves showed that DA doping into PEDOT:PSS initiated a quasi-reversible redox reaction, manifested as a new oxidation peak at approximately 0.3 V. Furthermore, the area enclosed by the CV curves was positively correlated with the increase of DA content, indicating superior charge storage capacity. Figure 13 A). For example Figure 13 As shown in B and C, compared to untreated PEDOT:PSS, PPDs exhibit reduced impedance and a smaller Nyquist plot radius in the 1–10 MHz frequency range. Furthermore, the observed reduction in phase angle indicates that DA doping effectively enhances the synchronicity of current-voltage changes. Figure 13 D). These changes indicate that a DA concentration of 10 mg / ml is the optimal parameter for DA-doped PEDOT:PSS and does not induce aggregation.

[0126] AFM phase images show that unprocessed PEDOT:PSS exhibits large PSS-rich regions (yellow or green) enclosing small PEDOT-rich regions (purple), while only doped PPD or laser-treated PEDOT:PSS (LPP) shows an increase in purple PEDOT-rich regions. Figure 18 C and Figure 14Furthermore, the dual-strategy treated LPPD10 showed the most significant increase in the area of ​​conductive PEDOT-enriched regions, demonstrating the significant improvement of PEDOT:PSS phase separation by the dual strategy. The increase in PEDOT aggregation domains also contributed to the increase in modulus, and an appropriate modulus is crucial for maintaining structural integrity under cyclic compressive stress (see...). Figure 18 D). Thermogravimetric analysis (TGA) results show that LPPD10 exhibits the highest decomposition temperature (see D). Figure 18 E). During the pyrolysis of PSS, unbonded PEDOT chains with high decomposition temperatures recrystallize under π-π interactions, forming a wider range of PEDOT-enriched structural domains. This change leads to a gradual increase in the decomposition temperature of PEDOT:PSS materials from untreated to treated alone, and then to dual-strategy treatment. The increasing trend in decomposition temperature becomes more pronounced with increasing DA doping concentration. Figure 15 XPS detection further validated that the dual-strategy processed LPPD10 has a higher PEDOT / PSS ratio than PPD10 and LPP (see [link]). Figure 18 F and G). Raman spectroscopy analysis also confirmed a similar trend through the conformational shift of the PEDOT domain. Notably, LPPD10 exhibited the highest quinone / benzene ratio, clearly indicating the efficient conversion of benzene structures to quinone structures under the dual-strategy treatment (see F and G). Figure 18 H and I). Furthermore, by adjusting the concentration of DA in the dual-treatment LPPD group, the PEDOT / PSS ratio and quinone / benzoyl ratio can be modulated to some extent, thereby achieving fine-tuning of the PEDOT and PSS phase separation (see [link to relevant documentation]). Figure 16 Based on the above results, it is concluded that the synergistic effect of DA doping significantly enhances the laser-induced phase separation of PEDOT:PSS, providing a possibility for constructing more differentiated gradient conductive structures. This method foreshadows great potential in fabricating neural electrodes with higher recording accuracy.

[0127] Example 3: Performance testing of the all-hydrogel neural electrode of the present invention

[0128] 1. Characterization

[0129] Phase separation in PEDOT:PSS was rigorously characterized using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Raman spectroscopy, and electron spin resonance (ESR). Furthermore, the material was analyzed using scanning electron microscopy (SEM). Dynamic mechanical analysis (DMA) was also used to evaluate the mechanical properties.

[0130] 2. Statistical Analysis

[0131] Unless otherwise stated, all data are expressed as mean ± standard deviation (SD). Statistical analysis among multiple groups was performed using one-way ANOVA. **p<0.05, ***p<0.01, ***p<0.001, and ****p<0.0001 were considered statistically significant.

[0132] 3. Results and Discussion

[0133] 3.1. Dual-strategy construction of PEDOT: PSS-based conductive hydrogel layer

[0134] To prepare a highly conductive hydrogel layer, preliminary phase separation was first achieved using PPD. Figure 19 A demonstrates the interaction between the positively charged primary amine group on DA and the negatively charged sulfonic acid group on PSS. This interaction weakens the Coulomb force between PEDOT and PSS, allowing the PEDOT chain to break free from the entanglement of PSS and undergo a transition from a coiled to a linear conformation. Subsequently, the PPD solution was dried on a substrate to form a thin film, and further deep phase separation was performed using a high-energy laser to obtain LPPD treated with a DA / laser dual-strategy. Figure 19 (B) During this process, laser-induced photon vibrations and thermal effects disrupt the interaction between PEDOT and PSS, while simultaneously promoting the pyrolysis of PSS. Subsequently, the released PEDOT chains form semi-crystalline domains through π-π coupling, constructing a water-resistant LPPD hydrogel composed of a hydrophilic PSS residual matrix and a hydrophobic PEDOT-rich semi-crystalline domain, with its pattern design consistent with the laser scanning path.

[0135] Considering the pattern resolution of MEAs, the effect of laser scanning speed on the pattern resolution of LPPD hydrogels was evaluated, and 20 mm / s was selected as the optimal scanning speed.

[0136] Atomic force microscopy (AFM) results showed that PEDOT:PSS underwent a significant transformation from a helical structure to a linear structure of PPD. Figure 19 C) confirmed the effect of DA doping. Electron spin resonance (ESR) analysis showed a significant decrease in the ESR signal within the LPPD, attributed to the increased bipolaron content after laser treatment, which is closely related to the improved crystallinity of PEDOT and the pyrolysis process of PSS. Figure 19 D).

[0137] X-ray photoelectron spectroscopy (XPS) S2p spectral analysis showed that the absorption peak of PEDOT in LPP and LPPD was significantly enhanced, while the absorption peak of PSS was correspondingly weakened, directly reflecting the promoting effect of laser-induced thermal effect on the pyrolysis of PSS. Figure 19 E).

[0138] Raman spectroscopy further confirmed the molecular conformational transformation and crystallization process of the PEDOT chains after phase separation. Figure 19 F). PEDOT:PSS is dominated by benzene ring structures, while the quinone structure significantly increases after phase separation, indicating improved crystallinity. The PEDOT / PSS ratio and quinone / benzoyl ratio calculated using XPS and Raman data show that LPPD has the highest PEDOT / PSS ratio (0.81 / 0.19) and quinone / benzoyl ratio (0.67 / 0.33), indicating that DA / laser co-processing achieves efficient phase separation. Figure 19 G, Figure 19 H).

[0139] PEDOT-based materials, as hybrid ion-electron conductive polymers, exhibit electron transfer pathways through the migration of free-delocalized electrons within the o-quinone structure of DA. Cyclic voltammetry (CV) analysis confirmed the electron transfer process between PEDOT and DA, with LPPD exhibiting higher charge storage capacity and conductivity. Figure 19 I, Figure 19 J, Figure 19 In particular, LPPD exhibits a high conductivity of 4176 S / cm, approximately 14 times that of pure PEDOT:PSS, demonstrating excellent conductivity.

[0140] 3.2. Preparation of encapsulating hydrogel layer and shielding hydrogel layer

[0141] SF, a naturally derived polymer, is renowned for its excellent biocompatibility and is widely used in encapsulation materials for neural electrodes. To ensure tight integration of the layers in the neural electrode, this study modified SF with DA and selected PEGDE as a crosslinking agent, successfully preparing a PSFD hydrogel encapsulation layer. Figure 26 A). Figure 26 The 1H NMR spectrum in sample B shows characteristic peaks for the catechol ring and the protons of the -CH group, confirming the successful synthesis of SFD. Simultaneously, the peak at 1055 cm⁻¹ in the FTIR spectrum indicates successful cross-linking of PSF (PEGDE-crosslinked SF) with the PSFD hydrogel. Figure 20 ).

[0142] Tensile test results showed that PSFD 10% (containing 10wt% PEGDE) hydrogel exhibited good tensile properties and a mechanical modulus matching the tissue. Figure 21 In addition, the PSFD 10% hydrogel also possesses excellent optical transparency. Figure 26 E), which is of great significance for the development of new visual wearable devices. Unless otherwise stated, PSFD will be referred to as PSFD 10% group in subsequent sections.

[0143] To improve the accuracy of neural signal recording, this study introduced a high-performance electromagnetic interference (EMI) shielding layer to create an effective electromagnetic environment. Figure 26 F demonstrates the process of preparing MHD2 hydrogel by crosslinking Mxene and HAD using HRP / H2O2 multiple bonds. Figure 26 G elucidated the shielding mechanism of MHD, whose open pore structure allows electromagnetic waves to penetrate, and then disperse and reflect through a three-dimensional network, extending the electromagnetic wave path. At the same time, the polarization loss caused by interface mismatch further enhances the shielding effect (Li et al. 2018; Iqbal et al. 2020).

[0144] ^1H NMR results confirmed the successful synthesis of HAD, and TEM images showed the morphology of sheet-like Mxene. Figure 26 I), while the XRD pattern confirmed the successful introduction of Mxene into the MHD hydrogel. Figure 22 SEM images revealed the porous structure of the MHD hydrogel, supporting the hypothesis that electromagnetic waves can penetrate the shielding layer.

[0145] The EMI shielding performance of MHD2 hydrogel was evaluated by fixing the Mxene concentration at 2 mg / mL. In the X-band (8.2–12.4 GHz), the average shielding effectiveness (SE) of MHD2 reached 43.2 dB, indicating that more than 96.64% of the incident electromagnetic waves were effectively blocked, far exceeding the commercial SE threshold of 20 dB (Zhu et al. 2021). Figure 26 L shows the contributions of total SE (SET), absorption-induced SE (SEA), and reflection-induced SE (SER), with SET mainly composed of SEA, reflecting the high conductivity (1.62 S / m) of the MHD2 hydrogel. Figure 23 It promotes the development of mobile carriers, interface polarization, and dipole polarization.

[0146] By comparing the surface area (SE) of wet MHD2 hydrogels with that of dehydrated MHD2 foam materials, it was found that the water surface area within the hydrogel helps to enhance polarization loss and dielectric loss. Figure 26 M). The EMI shielding performance of wet MHD2 was significantly higher than that of dehydrated foam material (43.2 dB vs 22.5 dB), indicating that a humid environment is more conducive to EMI shielding performance. Figure 26 N). Furthermore, the MHD2 hydrogel maintains stable shielding performance even after 500 stretches. Figure 24 It exhibits excellent EMI shielding capabilities within the GHz frequency range (including X, Ku, K, and Ka bands). Figure 25 This has potential application value in reducing environmental noise and improving the signal-to-noise ratio of signal acquisition. In subsequent chapters, MHD will be referred to as MHD2 group.

[0147] 3.3. Assembly of a four-layer all-hydrogel flexible neural electrode

[0148] The fabrication process of the all-hydrogel neural electrode is as follows: Figure 29 As shown in Figure A, a PPD film is first deposited on the PSFD hydrogel layer, and a 16-channel pattern is formed by laser etching. Figure 27 Subsequently, the untreated portion was removed, and the remaining LPPD membrane absorbed water to form a conductive MEAs layer. Next, an insulating PSFD hydrogel layer was placed on top of the LPPD MEAs layer, and laser etching was performed again to expose 16 electrode points. Finally, the MHD shielding layer was fixed, forming a four-layer all-hydrogel neural electrode. Figure 29 B). This electrode outputs an electrical signal via an FPC connector. Figure 29 C).

[0149] Throughout the electrode structure, the introduction of DA ensures tight integration and flexible interface compliance between layers through multiple interactions, which is crucial for the reliability and stability of neural signal acquisition. Figure 29 D). Furthermore, the electrode exhibits tight adhesion to the target tissue, improving the signal-to-noise ratio and reducing motion artifacts. Figure 29 EF). Overlap shear tests demonstrated that the PSFD hydrogel exhibited higher adhesion strength compared to the undoped DA PSF hydrogel. Figure 29 E). This property allows the all-hydrogel neuroelectrode to adhere firmly to brain tissue. Figure 29 G), and independent LPPD MEAs also exhibit excellent shape retention on complex surfaces such as fingers. Figure 29 H).

[0150] The multiple dynamic bonds of DA significantly enhance the flexibility and bonding stability of the electrode, maintaining elasticity and structural integrity even after deformations such as stretching and torsion. Figure 29 This flexibility is partly due to the porous structure of the LPPD hydrogel, which not only promotes stress dissipation (IK). Figure 29 L), and also meets the needs of cell growth, ensuring the high biocompatibility of the overall device ( Figure 29 M, Figure 28 ).

[0151] Given that the electrochemical performance and interface integration of flexible electronic devices are susceptible to mechanical stimulation, the mechanical stability of the all-hydrogel neural electrode was evaluated. At 1 kHz, by applying deformations such as stretching, twisting, and bending, it was found that the electrode incorporating DA exhibited smaller impedance changes and maintained good electrochemical performance and interface integration under different external pressures, verifying its superiority and reliability in neural recording. Figure 29 NO).

[0152] 3.4. In vivo multichannel monitoring of local field potentials (LFP)

[0153] Electrocorticography (ECoG) recording is one of the key techniques for studying neural activity (Buzsaki et al. 2012). Figure 36 A elucidates the generation and recording mechanism of ECoG: the release of neurotransmitters in the synaptic cleft, accompanied by the activation of voltage-gated calcium channels, converts chemical signals into electrical signals (action potentials) between neurons (Bean, 2007). Conductive channels constructed using microelectrode arrays (MEAs) provide an efficient electronic transport interface for ECoG recording (Rivnay et al. 2017). Local field potentials (LFPs) are generated by the coordinated oscillations of neuronal action potentials and reflect a wide range of ECoG activity (Buzsáki et al. 2012).

[0154] To verify the LFP capture capability of this neural electrode, the electrode was implanted on the surface of the rat cortex. Figure 30 The study recorded signal changes in a 4-aminopyridine (4-AP)-induced seizure model, covering the resting state, epileptic state, and recovery phase after electrical stimulation therapy. Figure 37 B). Figure 37 C shows representative LFP signals of channel 1 in different states: the amplitude is low at rest; during epileptic seizures, the amplitude fluctuates rapidly from early to late stages, presenting typical sharp epileptic waves; after electrical stimulation, the amplitude stabilizes and basically returns to normal levels.

[0155] Figure 37 DG and Figures 31-34 The time-frequency spectra of multi-channel (channels 1 to 8) LFP signals are presented, revealing the enhanced spectral power caused by abnormal neuronal discharge during epileptic seizures and the energy reduction corresponding to voltage-gated ion channel activity after electroneuromodulation (Zhang et al. 2023). Simultaneously, LFP power spectral density (PSD) analysis indicates that the energy released by abnormal neuronal discharges is reduced during epileptic states. Figure 37 H).

[0156] To compare the performance of LPPD hydrogel electrodes with conventional Ag electrodes, some channels were replaced with Ag electrodes for ECoG recording. Figure 37 The results showed that the two electrodes were similar in signal amplitude and waveform, but the LPPD hydrogel electrode had significantly lower background noise, with a signal-to-noise ratio almost 10 times that of the Ag electrode. Figure 37 J). Furthermore, the electromagnetic shielding effect of the MHD hydrogel further improves the signal-to-noise ratio (SNR). Figure 35 It is worth noting that, two weeks after implantation, the signal-to-noise ratio and amplitude of the LFP signal remained at a high level. Figure 36 ).

[0157] 3.5. In vivo multichannel monitoring of action potentials within a single neuron

[0158] Action potential monitoring is crucial for accurately capturing neural activity down to the level of a single neuron (Bean, 2007). Utilizing the high precision of laser processing, the recording electrodes (~15 μm in diameter) fabricated in this study are matched to the size of neurons (≤100 μm) (Hong & Lieber, 2019), laying the foundation for single-neuron action potential monitoring. Figure 40 A shows the layout of the recording electrodes. Figure 40 B represents the time series of action potentials recorded over 100 seconds. The waveforms of each potential are similar, but the amplitude fluctuates between 35 and 78 μV due to differences in the relative position or orientation of the electrodes and neurons. Figure 40 C and 40D showed that when the electrode points contacted multiple neurons, action potentials with different peak values ​​could be recorded simultaneously, and PCA analysis ( Figure 40 E) and autocorrelation plot ( Figure 40 F,G) further confirmed that these signals originated from different neurons, indicating that the 16-channel neural electrode has the ability to record signals from single neurons or multiple neurons.

[0159] In a 4-AP-induced rat epilepsy model, thresholding was used to analyze neuronal spike activity. Results showed that multichannel recordings revealed nearly synchronized specific action potentials, reflecting the synchronous firing of abnormal neurons. Figure 40 H,I). By extracting peak time ( Figure 40 J) and compared action potentials recorded by different channels, finding a time lag in epileptic states (J) Figure 40 K). Calculate the time delay of 50 spikes ( Figure 40 L) revealed its propagation characteristics in the cortical region, consistent with the neuronal firing patterns in status epilepticus. Electroneuromodulation effectively suppressed epileptiform discharge activity ( Figure 40 M, 40N).

[0160] Long-term biocompatibility studies have shown that, two weeks after implantation of the all-hydrogel neural electrode under the skull of rats, no significant tissue damage or inflammatory response was induced, while the rigid Ag electrode control group showed significant damage. Figure 40 O, Figure 38 Furthermore, the electrode maintained a stable action potential capture capability even two weeks after implantation. Figure 39 This suggests its long-term application potential in the diagnosis, monitoring and treatment of clinical neurological diseases.

[0161] 4. Conclusion

[0162] Here, a four-layer all-hydrogel neural electrode was successfully fabricated, which combines PEDOT:PSS phase separation technology with the adhesion-enhancing properties of polyphenols (DA). Through the synergistic effect of laser and DA, the conductive layer achieved an ultra-high conductivity of 4176 S / cm and a fine resolution of 15 μm, laying a solid foundation for recording weak neural signals. Simultaneously, an integrated DA-optimized insulating encapsulation layer ensures the independence and effective isolation of each recording channel. Furthermore, the introduction of an EMI shielding layer composed of HAD and MXene significantly improves the electrode's resistance to electromagnetic interference (EMI).

[0163] DA not only promotes phase separation within the conductive layer, but also strengthens the tight bonding between layers and the tight integration of the electrode with the tissue interface, thereby endowing the all-hydrogel neuroe with excellent electrochemical stability, which can maintain stable performance even under various deformation conditions such as stretching, curling, and bending.

[0164] In in vivo experiments, this neural electrode demonstrated the ability to accurately record local field potential (LFP) signals in epileptic rats, with a significantly improved signal-to-noise ratio compared to commercial silver electrodes. Further analysis showed that the electrode could distinguish and record spike signals from two different neurons, highlighting its great potential for capturing neural signals from small clusters of neurons.

[0165] Of particular importance is that this all-hydrogel neural electrode can also apply artificial electrical stimulation to the rat cerebral cortex during epileptic seizures, demonstrating its potential application in therapeutic intervention. In summary, the all-hydrogel neural electrode prepared in this invention is expected to become a powerful tool for exploring the development mechanisms, functional characteristics, and treatment strategies of brain diseases and neurodegenerative diseases.

Claims

1. A fully hydrogel neural electrode, characterized in that: It consists of four hydrogel layers: PSFD hydrogel layer - LPPDMEAs layer - PSFD hydrogel layer - MHD layer; The PSFD hydrogel layer serves as an insulating encapsulation layer. The LPPDMEAs layer is a conductive layer; The MHD layer is a shielding layer; in, The PSFD hydrogel is prepared by mixing dopamine (DA) modified degummed fibrous protein (SF) with polyethylene glycol diglycidyl ether (PEGDE) with an average Mn of 500. The LPPDMEAs layer is a multi-electrode array formed by depositing PPD aqueous solution onto the PSFD hydrogel layer and then laser etching it. The PPD aqueous solution is a PEDOT:PSS solution doped with dopamine (DA); The MHD layer is composed of Mxene and dopamine (DA) modified hyaluronic acid.

2. The all-hydrogel neural electrode according to claim 1, characterized in that: The method for preparing the PPD aqueous solution is as follows: The PPD aqueous solution was synthesized by adding DA·HCl to PEDOT:PSS aqueous solution and stirring at room temperature for 30 minutes.

3. The all-hydrogel neural electrode according to claim 1, characterized in that: The preparation method of the LPPD hydrogel is as follows: A PPD aqueous solution was deposited on a PSFD hydrogel layer to form a film, which was then annealed at 60°C. The PPD film was then laser etched under the following conditions: laser power 0.185W and scanning speed 20mm / s.

4. The all-hydrogel neural electrode according to claim 1, characterized in that: The preparation method of the MHD hydrogel is as follows: EDC and NHS were added to a 10 wt% hyaluronic acid (HA) solution to activate the carboxyl groups, and then DA·HCl was added. The reaction solution was stirred overnight at room temperature under nitrogen protection. Then, the solution was dialyzed in deionized water for 3 days using a 10000 Da dialysis membrane, and the pure product HAD was obtained by freeze drying. Then, 2.5 wt% HAD solution was mixed with MXene at concentrations of 0, 1, 1.5 and 2 mg / mL, respectively. Then, 30 μL of 1 mg / mL HRP solution and 12.5 μL of 0.5 mol / L H2O2 were added. After stirring evenly, the mixture was placed in a 40°C environment for 10 minutes to obtain MHD hydrogel.

5. The all-hydrogel neural electrode according to claim 1, characterized in that: The preparation method of the PSFD hydrogel is as follows: An 8 wt% degummed silk fibroin SF solution was placed in an ice bath, followed by the addition of EDC and NHS. After stirring for 30 minutes, DA·HCl was added to the solution. After reacting for one hour, the mixture was dialyzed with deionized water using a 14000 Da dialysis bag and then freeze-dried to obtain pure SFD. Polyethylene glycol diglycidyl ether (PEGDE) with an average Mn of 500 was added to a 5 wt% SF-DA solution and then dynamically stirred at 60°C. The solution was poured into a substrate, and a thin film was formed after the moisture evaporated in the ambient air. After absorbing water, a PSFD hydrogel was obtained.

6. A method for preparing the all-hydrogel neural electrode according to any one of claims 1-5, characterized in that: It includes the following steps: a. A PPD film is deposited on a PSFD hydrogel layer and then laser etched to form a 16-channel pattern; b. Wash with water to remove the untreated parts. The remaining LPPD membrane absorbs water and forms an LPPDMEAs layer. c. Place another insulating PSFD hydrogel layer on top of the LPPD MEAs hydrogel layer, and then perform a second laser etching to expose 16 electrode points; d. Add an MHD shielding layer on top and link it through a DA multi-interaction PSFD layer to form a four-layer all-hydrogel neural electrode.

7. The method for preparing the all-hydrogel neural electrode according to claim 6, characterized in that: The laser etching conditions described in step a are: laser power 0.185W; scanning speed 20mm / s; The laser etching described in step c involves selectively etching away the second layer of PSFD after the second layer of PSFD is encapsulated, exposing the electrode points for contact with tissue.

8. Use of the all-hydrogel neural electrode according to any one of claims 1-5 in the preparation of a device for recording neural activity, treating or treating neurodegenerative diseases.

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