All-hydrogel neural electrode and preparation method and application thereof

Abstract

The invention provides a full hydrogel neural electrode which is composed of four layers of hydrogel, namely a PSFD hydrogel layer, an LPPDMEAs layer, a PSFD hydrogel layer and an MHD layer. The invention further provides a preparation method and application of the full-hydrogel neural electrode. According to the invention, a PEDOT: PSS hydrogel conductive path with ultrahigh conductivity is provided, so that the recording capability of the PEDOT: PSS hydrogel conductive path is achieved compared with that of a metal electrode; according to the full-hydrogel neural electrode disclosed by the invention, due to tight interlayer combination, excellent interface compliance is ensured by introducing dopamine into each layer of the full-hydrogel neural electrode, so that the full-hydrogel neural electrode can be attached to a complex geometrical shape of a soft nervous tissue, and interface mismatching among a conductive layer, a packaging layer, a shielding layer and a tissue-electrode interface is avoided; the natural polymer packaging layer improves the compatibility, and the electromagnetic shielding layer is used for improving the signal-to-noise ratio. The full-hydrogel neural electrode can provide artificial electrical stimulation for the cerebral cortex of a rat during epileptic seizure, and the prepared full-hydrogel neural electrode has the potential to become a valuable tool for better understanding the development and functions of brain diseases and neurodegenerative diseases and treating the neurodegenerative diseases.

Description

Technical Field

[0001] The invention relates to a full-hydrogel neural electrode and a preparation method and application thereof. Background Art

[0002] Brain diseases and neurodegenerative diseases, such as epilepsy, Alzheimer's disease, and mental illness, seriously affect the quality of life of patients and bring heavy socioeconomic burdens. In the brain, a large number of neurons generate and transmit electrophysiological signals in a systematic manner 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 accurately detect and even modulate neural activity signals is very helpful for predicting the onset of patients and preparing artificial stimulation in time to treat the disease.

[0003] Over 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 aggregation caused by foreign body reactions between metal electrodes and tissues can lead to decreased sensing performance. All-hydrogel neural electrodes have attracted much attention due to their natural mechanical compliance. Since they match the modulus of brain tissue, they avoid subsequent problems caused by modulus mismatch.

[0004] Another prerequisite for all-hydrogel neural electrodes is the ability to record electrophysiological signals with high precision, which depends on the electrical conductivity or ionic conductivity of the conductive hydrogel layer. Among all hydrogel-based conductive materials, poly(3,4-ethyldioxythiophene):poly(styrene sulfonic acid) (PEDOT:PSS) has superior softness, biocompatibility, and electrochemical stability, making it competitive in flexible electrode design (Fan et al. 2019; Zhou et al. 2023). However, considering the requirement to improve electrode performance to capture weak neural electrical signals, and the fact that the conductivity of untreated PEDOT:PSS is usually much lower than that of metal (~0.1s / cm), it limits its performance in certain electrical applications. Excessive PSS (polystyrene sulfonate) is one of the reasons for the low conductivity. In addition, PEDOT:PSS is acidic and may react with electrode materials, leading to electrode corrosion. PEDOT:PSS is sensitive to water and oxygen, which may affect the stability and life of the device.

[0005] An important step to improve the conductivity is to achieve PEDOT and PSS doping or post-treatment with chemical reagents, including but not limited to polar solvents, dopamine, acids, and ionic liquids, which can reduce the Coulomb force between PEDOT and PSS, thereby promoting the PSS chains to move away from the PEDOT chains. In addition, physical treatments, such as electric fields, heating, ultraviolet light, and lasers, have been shown to effectively induce phase separation of PEDOT:PSS.

[0006] The existing PEDOT:PSS hydrogel has toxic dopants, and the finished product faces complicated water washing and detoxification steps; physical means: heat treatment, electric field, UV and other effects are usually low, and excessive laser intensity will cause carbonization of PEDOT, destroying its conjugated conductive structure, and the degree of phase separation is limited. For example, in 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.), there is a lack of stable interlayer interface adhesion and insufficient conductivity of the conductive path; Laser-induced wet stability and adhesion of pure conducting polymer hydrogels (Won, Daeyeon, et al. Laser-induced wet stability and adhesion of pure conducting polymer hydrogels. Nature Electronics, 2024, 1-12.), PET is used as the substrate and PDMS is used as the encapsulation method. These chemical polymers are not as biocompatible as natural polymers, and their modulus is relatively high, which has the risk of inducing immune response 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 interface adhesion and insufficient conductivity of the conductive pathway, it is difficult to obtain high-precision patterning capabilities in the template method for circuit preparation using sodium alginate with conductive fillers. Summary of the invention

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

[0008] The present invention provides a full hydrogel neural electrode, which is composed of four layers of hydrogel, namely: PSFD hydrogel layer-LPPDMEAs layer-PSFD hydrogel layer-MHD layer;

[0009] The PSFD hydrogel layer is 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 a hydrogel prepared by mixing degummed silk fibroin SF modified with dopamine DA and polyethylene glycol diglycidyl ether PEGDE with an average Mn of 500;

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

[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] Wherein, the preparation method of the PPD aqueous solution is:

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

[0019] Wherein, the preparation method of the LPPD hydrogel is:

[0020] The PPD aqueous solution was deposited on the PSFD hydrogel layer to form a film, and annealed at 60°C. The PPD film was then processed by laser etching. The laser etching conditions were: laser power 0.185 W; scanning speed 20 mm / s.

[0021] Wherein, the preparation method of the MHD hydrogel is:

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

[0023] Then, 2.5 wt% HAD solution was mixed with MXene with concentrations of 0, 1, 1.5 and 2 mg / mL, respectively, and 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 °C for 10 minutes to obtain MHD hydrogel.

[0024] Wherein, the preparation method of the PSFD hydrogel is:

[0025] 8 wt% degummed silk fibroin SF solution was placed in an ice bath, followed by the addition of EDC and NHS, and after stirring for 30 minutes, DA·HCl was added to the solution; after one hour of reaction, the mixture was dialyzed with deionized water using a 14000Da dialysis bag, and pure SFD was obtained by freeze drying;

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

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

[0028] a. Deposit the PPD film on the PSFD hydrogel layer and then perform laser etching to form a 16-channel pattern;

[0029] b. Wash with water to remove the untreated part, and the remaining LPPD membrane absorbs water to form an LPPDMEAs layer;

[0030] c. Place another insulating PSFD hydrogel layer on top of the LPPD MEAs hydrogel layer, followed by secondary laser etching to expose 16 electrode sites;

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

[0032] Among them, the conditions of laser etching described in step a are: laser power 0.185W; scanning speed 20mm / s.

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

[0034] The present invention also provides the use of the all-hydrogel neural electrode in preparing a device for recording neural activity, treating or curing neurodegenerative diseases.

[0035] Beneficial effects of the present invention:

[0036] 1. Provide ultra-high conductivity PEDOT:PSS hydrogel conductive pathway (high phase separation, ultra-high conductivity of 4173S / CM), which has achieved the record capacity compared with metal electrodes;

[0037] 2. Tight interlayer bonding: Among them, polyphenol chemistry has various interactions such as hydrogen bonding, electrostatic interaction, thiol reduction and π-π / cation-π interaction. The introduction of dopamine in each layer of the all-hydrogel neural electrode ensures excellent interface compliance, enabling it to fit the complex geometry of soft neural tissue and avoid interface mismatch between the conductive layer-encapsulation layer-shielding layer and the tissue-electrode interface. And the problem of material failure caused by physiological stress and reduced recording ability;

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

[0039] The all-hydrogel neural electrode of the present invention is capable of providing 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. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0043] Figure 4 COMSOL was used to simulate the thermal decomposition process of surface PSS during laser etching;

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

[0045] Figure 6.Images of PEDOT:PSS treated with 350 (optimal power) and 400 (excess power) lasers. A) PEDOT:PSS treated with 350 lasers exhibits a bluish-black color similar to the original PEDOT:PSS. B) PEDOT:PSS treated with 400 lasers 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 PSS:PEDOT by laser treatment and water washing (bottom). C) Schematic diagram of laser-triggered photothermal effect, leading to thermal decomposition of separated PSS parts. D) Laser-induced ionization of air to form plasma, and the luminescent electric field formed by the plasma. E) Finite element modeling of laser-triggered photogenerated electric field. F) Focus 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 illustrate the carbonization process within PEDOT:PSS at 400 excess laser intensity. (K) Schematic diagram of excessive laser intensity-induced carbonization effect within PEDOT:PSS;

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

[0048] Fig. 9 .This image shows the obvious aggregation phenomenon in the PEDOT:PSS solution after adding excess dopamine (15mg / mL). Excess dopamine destroys the ionic environment of the PEDOT:PSS solution, resulting in obvious micellar aggregation of PEDOT:PSS molecules;

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

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

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

[0052] Fig.13.Electrochemical properties of PEDOT:PSS and PPDs. A) Cyclic voltammetry (CV) curves of PEDOT:PSS, PPD2.5, PPD5 and PPD10. B) Electrochemical impedance spectroscopy (EIS, from 1Hz to 10MHz) of PEDOT:PSS, PPD2.5, PPD5 and PPD10. C) Nyquist plots of PEDOT:PSS, PPD2.5, PPD5 and PPD10. D) Phase angles of PEDOT:PSS, PPD2.5, PPD5 and PPD10;

[0053] Fig.14 .AFM phase images of PPDs and LPPDs at different dopamine concentrations. PEDOT-enriched domains are shown in purple, and PSS-enriched domains are shown in yellow and green;

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

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

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

[0057] Fig.18: Dual strategy for DA and laser synergistically induced phase separation of PEDOT:PSS. A) Schematic diagram of the internal structure of untreated PEDOT:PSS transformed into PEDOT:PSS (LPPD) synergistically treated with DA and laser. B) Schematic diagram showing the conformational transition 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 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; on the right is the area change of PEDOT-enriched domains calculated using ImageJ software. D) Micro-regional mechanical distribution of PEDOT:PSS, LPP, PPD10, and LPPD10 during nanoindentation tests. E) Thermogravimetric analysis (TGA) curves of PEDOT:PSS, LPP, PPD10, and LPPD10 (left), and the corresponding decomposition temperatures of each group of PEDOT (right). F) XPS S2p spectra of PEDOT:PSS, LPP, PPD10 and LPPD10. G) PEDOT / PSS ratios of the four groups of materials based on XPS results. H) Raman spectra of PEDOT:PSS, LPP, PPD10 and LPPD10. I) Quinone / benzoyl ratios of the four groups of materials calculated using Raman spectra results;

[0058] Fig.19 Preparation of highly conductive LPPD hydrogels by DA / laser co-treatment. (A) Schematic diagram showing the molecular structural transformation induced by DA doping in PEDOT:PSS solution. (B) Schematic diagram showing the femtosecond laser-induced pyrolysis of PSS and the formation of PEDOT semicrystalline domains in LPPD films. (C) AFM images of PEDOT:PSS and (D) ESR spectra of PEDOT:PSS and LPPD. (E) (F) XPS S2p spectra 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] Fig. 20 . FTIR spectra of SF and PSF hydrogels treated with different PEGDE weight ratios;

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

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

[0062] Fig.23 Electrical conductivity of MHD hydrogels with different MXene concentrations;

[0063] Fig.24 .X-band electromagnetic interference shielding effectiveness (EMI SE) of MHD2 hydrogel before and after 500 bends;

[0064] Fig.25 .Electromagnetic interference shielding effectiveness (EMI SE) of MHD2 hydrogel in X-band (8.212.4GHz), Ku-band (12.414GHz), K-band (1418GHz) and Ka-band (1840GHz);

[0065] Fig.26 Preparation and characterization of encapsulation layer and shielding layer. (A) Schematic diagram of the preparation of encapsulation layer (PSFD hydrogel). (B) HNMR spectra of 1SFD and SF. (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 PSFD 10% hydrogel. (F) Schematic diagram of the preparation of EMI shielding layer (MHD hydrogel). (G) Schematic diagram of the absorption-dominated EMI shielding mechanism of MHD hydrogel. (H) HNMR spectra of 1HAD and HA; (A) TEM image of MXene. (J) SEM 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 SETMHD2 hydrogels in different states;

[0066] Fig. 27 .16-channel microelectrode array (MEAs) model parameters;

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

[0068] Fig.29Fabrication and characterization of all-hydrogel neural electrodes. (A) Flowchart showing the fabrication of a 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 16 individual electrodes and electrode exposure after laser etching (bottom). (D) Tight interlayer bonding initiated 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 conformity 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. Images depicting the neural electrode after being (J) rubbed and (K) bent while maintaining its intact structure with no signs of delamination. (L) SEM image showing the cross-sectional morphology of the LPPD hydrogel. (M) Optical and fluorescence images showing adhesion and growth of pheochromocytoma (PC12) cells on LPPD MEAs. (N) Resistance changes of 16-channel electrodes when subjected to stretching, twisting, and bending. (O) SEM 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 LPPMEAs (without DA, right) under 15% strain.

[0069] Fig.30 .The implantation location of the prepared all-hydrogel neural electrode in the rat brain;

[0070] Fig.31 .Representative multi-channel (channels 9-16) local field potential (LFP) signals recorded in resting state of epileptic rats (top) and their corresponding time-frequency spectrogram (bottom);

[0071] Fig.32 .Representative multi-channel (channels 9-16) local field potential (LFP) signals recorded in the early epileptic state of epileptic rats (top) and their corresponding time-frequency spectrograms (bottom);

[0072] Fig.33 .Representative multi-channel (channels 9-16) local field potential (LFP) signals recorded in late epileptic state of epileptic rats (top) and their corresponding time-frequency spectrograms (bottom);

[0073] Fig.34 .Representative multi-channel (channels 9-16) local field potential (LFP) signals recorded in the post-recovery state of epileptic rats (top) and their corresponding time-frequency spectrograms (bottom);

[0074] Fig.35.The effect of the electromagnetic shield on reducing background noise. Local field potentials (LFPs) recorded from neural electrodes with (above) and without (below) the electromagnetic shield. After removing the electromagnetic shield, the background noise of the recorded LFP increased significantly;

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

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

[0077] Fig.38 . Immunofluorescence images of soft 16-channel all-hydrogel neural electrodes and rigid silver neural electrodes 2 weeks after insertion into brain slices. 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 absence of any interaction between the silver electrode and the substrate resulted in direct detachment of the electrode after implantation, as well as more intense macrophage aggregation and tissue damage;

[0078] Fig.39 Spike signals recorded from 16 channels 2 weeks after implantation;

[0079] Fig.40. In vivo multichannel monitoring of action potentials. (A) Schematic diagram showing the appropriate contact between recording electrodes and neurons. (B) Temporal tamping and (C) spike waveforms of action potentials recorded in the resting state. (D) Two spike waveforms from different neurons and (E) their principal component analysis (PCA). (F) and (G) Autocorrelograms of two recorded spikes. (H) Schematic diagram of spikes recorded before and after seizure induction. (I) Eight groups of action potentials recorded in a very small time window. (J) Spike waveforms of the third group. (K) Superimposed voltage traces of spikes. (L) Array layout of the average spike time delay calculated from 50 groups of action potentials. (M) Total number of spikes recorded per second. (N) Total number of spikes recorded in 60 seconds for each channel. (O) Immunofluorescence images of rat brain slices 2 weeks after the insertion of all-hydrogel neural electrodes and rigid Ag neural electrodes. Brain slices without electrode implantation were used as controls (sham group). NF: neurofilament, a marker in neuronal development. GFAP: Glial fibrillary acidic protein, a product produced by glial cells. DETAILED DESCRIPTION

[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 Co., Ltd. (Shanghai, China). Hyaluronic acid (HA, Mw = 700k-800k Da) was obtained from Blumchi Biotech. HRP (activity: 248 units / mg) was from TCI (Japan). Fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Sigma-Aldrich (USA). MXene was sourced from Beike (China). All other chemicals were obtained from Chengdu Cologne Chemicals Co., Ltd. unless otherwise stated.

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

[0083] a. Deposit the PPD film on the PSFD hydrogel layer and then perform laser etching to form a 16-channel pattern;

[0084] b. Wash with water to remove the untreated part, and the remaining LPPD membrane absorbs water to form 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 sites to contact the target tissue;

[0086] d. An MHD shielding layer is fixed on the top to form a four-layer all-hydrogel neural electrode.

[0087] Selectivity: After the second layer of PSFD is encapsulated, laser etching is performed on the electrode point (the circular area) separately, and the second layer of PSFD is selectively etched away to expose the electrode point for contact with the tissue. No laser treatment is performed on other places to ensure insulation between other places and tissues.

[0088] The specific flow chart is as follows Figure 1 As shown:

[0089] Wherein, the preparation method of each raw material is as follows:

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

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

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

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

[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 group, followed by the addition of 0.47 g DA·HCl. The reaction solution was stirred at room temperature (RT) overnight under nitrogen protection. Then, it was dialyzed in DIW for 3 days using a dialysis membrane (10,000 Da), and the pure product of HD was obtained by freeze drying. In order 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 min, the MHD hydrogel was formed after being placed at 40 °C for 10 min.

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

[0097] The preparation method of degummed SF can be 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 cocoons were soaked in 2 liters of 0.02M Na2CO3 solution and then boiled at 100°C for half an hour. The product was rinsed with deionized water (DIW) to wash away the residual Na2CO3 solution. This process was repeated 3 times to obtain a crude product. 2 grams of the resulting product was added to 8 milliliters of 9.3MLiBr solution and stirred at 60°C for 4 hours. Subsequently, the solution was dialyzed with DIW using an 8000Da dialysis bag, and then the degummed SF was obtained by freeze drying.

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

[0099] 5 ml of 8 wt% degummed SF solution was placed in an ice bath, followed by the addition of 37.5 mg of EDC and 45 mg of NHS. After stirring for 30 minutes, 216 mg of DA·HCl was added to the solution. After one hour of reaction, the mixture was dialyzed in DIW for 3 days using a dialysis bag (14000 Da), and pure SFD was obtained by freeze drying. Afterwards, the successful synthesis of the product was verified using 1H NMR spectroscopy (600 MHz, Bruker, USA).

[0100] 6) Preparation of SF and PSFD (SFD hydrogel initiated by PEGDE)

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

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

[0103] A femtosecond laser system (INNO Laser, China) emitting continuous wave laser (532 nm) was used to selectively induce phase separation of PEDOT:PSS-based materials. The “selectivity” mentioned here 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 of power to achieve power parameters that achieve stable hydrogel construction.

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

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

[0109] d. Find the parameters of excess thermal effect leading to carbonization of PEDOT.

[0110] 2. The results show that 250 is the lowest parameter that can induce hydrogel construction, and 400 or more will lead to PEDOT carbonization. Therefore, after screening between 250-400, it was found that 350 is the most suitable parameter, which can ensure hydrogel construction, high degree of phase separation, PSS thermal decomposition, and PEDOT retention.

[0111] Scan speed filter:

[0112] 1. Purpose:

[0113] a. Neurons are tiny, and electrode recording sites smaller than 100 microns are sufficient for recording action potentials. Different scanning speeds will affect the thickness of the electrode line and the size of the electrode points.

[0114] b. Combination with the substrate: Too thin or too small a size will result in a small bonding area between the electrode and the PSFD substrate, leading to unstable bonding.

[0115] 2. The results show that the speed of 20 mm / s is the most suitable, the binding with the substrate is stable, and the size is suitable for neurons. Figure 2 .

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

[0117] Since laser can provide extremely high energy injection and fine processing accuracy, it can simultaneously meet the design of micropatterns in flexible conductive polymer PEDOT:PSS and the regulation of conductive / insulating phase content in the system. By programming and controlling the scanning path (the pattern is a cat), laser-etched PEDOT:PSS (LPP) film ( Figure 3 , Figure 7 A. Figure 7 B).

[0118] During the laser etching process, the thermal effect caused by it will reduce the electrostatic force between PEDOT and PSS, causing part of the PEDOT phase to separate from the PSS phase ( Figure 7 C). At the same time, its thermal effect will cause the insulating PSS phase with a lower decomposition temperature to thermally decompose. High-energy lasers will also stimulate the ionization of the surrounding air, forming an electric field composed of plasma ( Figure 7 D). The partially separated two phases are in this electric field, and because the two phases have opposite charges, they will further separate. After the induction of laser photothermal / photogenerated electric field, the π-π stacking of PEDOT domains and the interchain entanglement in PSS chains in the system are adjusted and balanced. After contact with water, the adjusted PEDOT stable domains form a gel skeleton, and because the residual hydrophilic PSS phase is rich in water, the laser scanning part is transformed into a cat-shaped mode hydrogel.

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

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

[0121] 4. Dual-strategy treatment to induce phase separation in PEDOT: PSS

[0122] Laser can induce phase separation of PEDOT:PSS, but this single treatment mode is limited and it is difficult to further improve the conductivity. The combination of chemical doping and laser treatment can synergistically enhance phase separation and achieve extensive regulation of PEDOT:PSS conductivity. Considering the biocompatibility of flexible bioelectronic devices, non-toxic DA is used as a chemical doping agent to further induce phase separation in PEDOT:PSS.

[0123] First, the effect of DA alone on the phase separation of PEDOT:PSS was evaluated. PEDOT:PSS was doped with DA and named PPD. After several screening and characterizations, the DA doping concentration was set at 10 mg / ml (the doped product was named PPD10) for further laser treatment ( Figure 8-Figure 13 ).like Fig.18 As shown in Figure A, in principle, the electrostatic contact between the primary amine groups of DA and the sulfonate ions 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 the action of π-π conjugation to form PEDOT-enriched domains, which is accompanied by the conformational transition of PEDOT from benzene (random spiral shape) to quinone (regular linear shape) ( Fig.18 B). It was confirmed again that the laser intensity of 350 was also the optimal parameter for inducing phase separation of PEDOT and PSS in the PPD10 group, showing the largest PEDOT peak in the XPS S2p spectrum and the most obvious red shift of the PEDOT peak in the Raman spectrum. Therefore, for the DA / laser dual strategy treatment of PEDOT:PSS, the DA concentration was 10 mg / mL and the laser intensity was 350, and the product after the dual strategy treatment was called LPPD10.

[0124] Phase separation regulation and electrochemical properties of PPD hydrogels with adjustable dopamine concentration, e.g. Figure 8As shown in Figure 1, the introduction of dopamine (DA) leads to phase separation of PEDOT and PSS, and in the 1HNMR spectrum, peaks of catechol rings appear in the DA-doped PEDOT:PSS groups (PPDs). The degree of phase separation in PPDs increases with the gradual increase of DA concentration, while excessive DA concentration (15 mg / mL) may lead to obvious aggregation of PEDOT:PSS ( Fig. 9 ). Subsequently, XPS analysis was performed to study the phase separation between PEDOT and PSS in the system at different DA doping concentrations. The results showed that the degree of phase separation gradually increased with the increase of DA concentration ( Fig.10 ). As the phase separation increases, the increased concentration of free PEDOT chains forms denser crystalline domains through π-π conjugated interactions. This transition is manifested as a conformational change of PEDOT from a benzene structure to a quinone structure, which is analyzed by Raman analysis ( Fig.11 ) is strongly confirmed. In addition, the more crystalline domains produced due to the enhanced phase separation lead to an increase in the decomposition temperature of PEDOT ( Fig.12 ).

[0125] Subsequently, the electrochemical characterization of PPDs with different DA concentrations was carried out. The cyclic voltammetry (CV) curves showed that DA doping into PEDOT:PSS triggered a quasi-reversible redox reaction, manifested as a new oxidation peak at about 0.3V. In addition, the area enclosed by the CV curve was positively correlated with the increase in DA content, indicating a better charge storage capacity ( Fig.13 A). Fig.13 As shown in Figures B and C, the PPDs exhibit reduced impedance and a reduced Nyquist plot radius in the frequency range of 1–10 MHz compared to untreated PEDOT:PSS. In addition, the observed reduction in phase angle indicates that DA doping effectively enhances the synchronization of current-voltage changes ( Fig.13 D). These changes indicate that 10 mg / ml DA concentration is the optimal parameter for DA doping PEDOT:PSS without causing aggregation.

[0126] AFM phase images of untreated PEDOT:PSS show large PSS-rich domains (yellow or green) encapsulated in small PEDOT-rich domains (purple), while PPD-doped or laser-treated PEDOT:PSS (LPP) both show an increase in purple PEDOT-rich domains ( Fig.18 C and Fig.14). Moreover, the area of ​​the conductive PEDOT-enriched region in LPPD10 treated with the dual strategy increased most significantly, indicating that the dual strategy significantly improved the PEDOT:PSS phase separation. The increase in PEDOT aggregation domains also promoted the improvement of modulus, and the appropriate modulus is the key to maintaining the structural integrity of the material under cyclic compressive stress (see Fig.18 D). Thermogravimetric analysis (TGA) results show that LPPD10 exhibits the highest decomposition temperature (see Fig.18 E). During the pyrolysis of PSS, unbound PEDOT chains with high decomposition temperatures will recrystallize under π-π interactions, thereby forming a wider range of PEDOT-enriched domains. This change leads to a gradual increase in the decomposition temperature of PEDOT:PSS materials from untreated to single treatment and then to dual strategy treatment. With the increase of DA doping concentration, the increasing trend of decomposition temperature becomes more and more significant ( Fig.15 XPS detection further confirmed that the dual-strategy processed LPPD10 has a higher PEDOT / PSS ratio than PPD10 and LPP (see Fig.18 F and G). Raman spectroscopy analysis also confirmed a similar trend through the transformation of PEDOT domain conformations. Notably, LPPD10 showed the highest quinone / benzyl ratio, which clearly pointed out the effective transformation of benzyl structure to quinone structure under the dual strategy treatment (see Fig.18 H and I). In addition, by adjusting the concentration of DA in the dual-treated LPPD group, the PEDOT / PSS ratio and the quinone / benzoyl ratio can be regulated to a certain extent, thereby achieving fine regulation of the phase separation of PEDOT and PSS (see Fig.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 the possibility of constructing a more differentiated gradient conductive structure. This method indicates great potential in the manufacture of neural electrodes with higher precision recording capabilities.

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

[0128] 1. Characterization

[0129] The phase separation in PEDOT:PSS has been rigorously characterized using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Raman spectroscopy, and electron spin resonance (ESR). In addition, the material has been analyzed using scanning electron microscopy (SEM). In addition, dynamic mechanical analysis (DMA) has been 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 analysis of variance (ANOVA). **p<0.05, ***p<0.01, ***p<0.001, ****p<0.0001 were statistically significant.

[0132] 3. Results and Discussion

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

[0134] To prepare the highly conductive hydrogel layer, preliminary phase separation was first achieved via PPD. Fig.19 A shows the interaction between the positively charged primary amine groups on DA and the negatively charged sulfonic acid groups on PSS. This interaction weakens the Coulomb force between PEDOT and PSS, freeing the PEDOT chain from the entanglement of PSS and causing a transition from curled to 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 ( Fig.19 B). During this process, the laser-induced photon vibration and thermal effect destroyed the interaction between PEDOT and PSS, while promoting the thermal decomposition of PSS. Subsequently, the released PEDOT chains formed 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, whose pattern design was consistent with the laser scanning path.

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

[0136] Atomic force microscopy (AFM) test results show that PEDOT:PSS significantly changes from a helical structure to a linear structure of PPD ( Fig.19 C), confirming the effect of DA doping. Electron spin resonance (ESR) analysis showed that the ESR signal in LPPD decreased significantly, which was attributed to the increase in the content of bipolarons after laser treatment, which was closely related to the improvement of PEDOT crystallinity and the pyrolysis process of PSS ( Fig.19 D).

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

[0138] Raman spectroscopy further confirmed the molecular conformational transition and crystallization process of PEDOT chains after phase separation ( Fig.19 F). PEDOT:PSS is dominated by benzene ring structures, while the quinone structures increase significantly after phase separation, indicating an increase in crystallinity. The PEDOT / PSS ratio and quinone / benzoyl ratio calculated from 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-treatment achieves efficient phase separation ( Fig.19 G, Fig.19 H).

[0139] As a hybrid ion-electron conducting polymer, the free delocalized electrons of PEDOT-based materials migrate on the o-quinone structure of DA to form an electron transfer pathway. Cyclic voltammetry (CV) analysis confirmed the electron transfer process between PEDOT and DA, and LPPD showed higher charge storage capacity and conductivity ( Fig.19 I, Fig.19 J, Fig.19 K). In particular, the electrical conductivity of LPPD is as high as 4176 s / cm, which is about 14 times that of pure PEDOT:PSS, showing excellent conductive properties.

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

[0141] SF, as a naturally derived polymer, is known for its excellent biocompatibility and is widely used as a neural electrode encapsulation material. To ensure the tight integration of the layers in the neural electrode, this study used DA to modify SF and selected PEGDE as a crosslinker to successfully prepare the PSFD hydrogel encapsulation layer ( Fig.26 A). Fig.26 The 1H NMR spectrum in B shows the characteristic peaks of the catechol ring and the -CH group protons, confirming the successful synthesis of SFD. At the same time, the peak at 1055 cm-1 in the FTIR spectrum indicates the successful cross-linking of PSF (PEGDE cross-linked SF) and PSFD hydrogel ( Fig. 20 ).

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

[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. Fig.26 F shows the process of preparing MHD2 hydrogel by multiple cross-linking of Mxene and HAD using HRP / H2O2. Fig.26 G elaborated on the shielding mechanism of MHD, whose open pore structure allows electromagnetic waves to penetrate, and then disperse and reflect through the 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 flake-like Mxene ( Fig.26 I), while the XRD pattern confirmed the successful introduction of Mxene into MHD hydrogel ( Fig. 22 ). The SEM images reveal the porous structure of the MHD hydrogel, supporting the hypothesis that electromagnetic waves penetrate the shielding layer.

[0145] The EMI shielding performance of MHD2 hydrogel was evaluated by fixing the Mxene concentration of MHD2 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). Fig.26 L shows the contribution of total SE (SET), SE caused by absorption (SEA), and SE caused by reflection (SER), among which SET is dominated by SEA, reflecting the high conductivity of MHD2 hydrogel (1.62S / m, Fig.23 ) promotes the mobile carriers, interface polarization and dipole polarization.

[0146] By comparing the SE of wet MHD2 hydrogels and dehydrated MHD2 foams, it was found that the water area within the hydrogel contributes to the enhancement of polarization loss and dielectric loss ( Fig.26 M). The EMI SE of wet MHD2 is significantly higher than that of dehydrated foam material (43.2dBvs 22.5dB), indicating that a humid environment is more conducive to EMI shielding performance ( Fig.26 N). In addition, the MHD2 hydrogel can still maintain stable shielding properties after 500 stretching cycles ( Fig.24 ) and exhibits excellent EMI shielding capabilities in the GHz frequency range (including X, Ku, K and Ka bands) Fig.25 ), which has potential application value in reducing environmental noise and improving the signal-to-noise ratio of signal acquisition. In the following chapters, MHD will be referred to as MHD2 group.

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

[0148] The preparation process of all-hydrogel neural electrodes is as follows: Fig.29 A. First, a PPD film is deposited on the PSFD hydrogel layer and a 16-channel pattern is formed by laser etching ( Fig. 27 ). Subsequently, the untreated part was removed, and the remaining LPPD membrane absorbed water to form a conductive MEAs layer. Next, the insulating PSFD hydrogel layer was covered on top of the LPPD MEAs layer and laser etched again to expose 16 electrode points. Finally, the MHD shielding layer was fixed to form a four-layer all-hydrogel neural electrode ( Fig.29 B). This electrode outputs electrical signals through the FPC connector ( Fig.29 C).

[0149] In the entire electrode structure, the introduction of DA ensures the tight integration and flexible interface compliance between layers through multiple interactions, which is crucial for the reliability and stability of neural signal acquisition ( Fig.29 D). In addition, the electrode exhibits tight adhesion to the target tissue, improving the signal-to-noise ratio and reducing motion artifacts ( Fig.29 EF). Lap shear tests showed that PSFD hydrogel had higher adhesion strength than PSF hydrogel without DA doping ( Fig.29 E). This property enables the all-hydrogel neural electrode to adhere firmly to brain tissue ( Fig.29 G), and independent LPPD MEAs also show excellent conformality to complex surfaces such as fingers ( Fig.29 H).

[0150] The multiple dynamic bonds of DA significantly enhance the flexibility and binding stability of the electrode, and it can maintain elasticity and structural integrity even after deformation such as stretching and twisting ( Fig.29 This flexibility is partly due to the porous structure of the LPPD hydrogel, which not only facilitates stress dissipation ( Fig.29 L), and also meets the cell growth requirements, ensuring the high biocompatibility of the overall device ( Fig.29 M, Fig.28 ).

[0151] Given that the electrochemical properties and interface integration of flexible electronic devices are easily affected by mechanical stimulation, the mechanical stability of all-hydrogel neural electrodes was evaluated. At 1kHz, by applying deformations such as stretching, twisting, and bending, it was found that the electrodes introduced with DA showed smaller impedance changes and maintained good electrochemical properties and interface integration under different external pressures, verifying its superiority and reliability in neural recording ( Fig.29 NO).

[0152] 3.4. In vivo multi-channel monitoring of local field potential (LFP)

[0153] In vivo electrocorticogram (ECoG) recording is one of the key techniques for studying neural activity (Buzsaki et al. 2012). Fig.36 A explained 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). The conductive channels constructed by microelectrode arrays (MEAs) provide an efficient electronic transmission interface for ECoG recording (Rivnay et al. 2017). The local field potential (LFP) is generated by the coordinated oscillation of neuronal action potentials, reflecting a large range of ECoG activities (Buzsáki et al. 2012).

[0154] To verify the LFP capture capability of the present neural electrode, electrodes were implanted on the surface of rat cortex ( Fig.30 ), recording signal changes in the 4-aminopyridine (4-AP)-induced epileptic seizure model, covering the resting state, epileptic state, and recovery stage after electrical stimulation treatment ( Fig.37 B). Fig.37 C shows the representative LFP signals of channel 1 in different states: the amplitude is low at rest; during an epileptic seizure, the amplitude fluctuates rapidly from the early to late stages, showing typical sharp epileptic waves; after electrical stimulation, the amplitude is stable and basically returns to normal levels.

[0155] Fig.37 DG and Figure 31-Figure 34 The time-frequency spectrogram of multi-channel (channels 1 to 8) LFP signals was displayed, revealing the spectral power enhancement caused by abnormal neuronal discharge during epileptic seizures and the energy reduction corresponding to voltage-gated ion channel activity after electrical neuromodulation (Zhang et al. 2023). At the same time, LFP power spectral density (PSD) analysis showed that the energy released by abnormal neuronal discharges in the epileptic state was weakened ( Fig.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 ( Fig.37 I). The results showed that the two were similar in signal amplitude and waveform, but the background noise of the LPPD hydrogel electrode was significantly lower, and the signal-to-noise ratio was almost 10 times that of the Ag electrode ( Fig.37 J). In addition, the electromagnetic shielding effect of MHD hydrogel further improves the signal-to-noise ratio ( Fig.35 ). It is worth noting that 2 weeks after implantation, the signal-to-noise ratio and amplitude of the LFP signal remained at a high level ( Fig.36 ).

[0157] 3.5. In vivo multi-channel monitoring of action potentials in single neurons

[0158] Monitoring action potentials is essential for accurately capturing neural activity as small as a single neuron (Bean, 2007). With the high precision of laser processing, the recording electrodes (diameter ~15μm) made in this study match the size of neurons (≤100μm) (Hong & Lieber, 2019), laying the foundation for monitoring action potentials of single neurons. Fig.40 A shows the recording electrode layout. Fig.40 B is the time series of action potentials recorded within 100 seconds. The waveforms of each potential are similar, but the amplitude fluctuates between 35 and 78 μV due to the relative position or direction of the electrode and the neuron. Fig.40 C and 40D show that when the electrode points contact multiple neurons, action potentials with different peak values ​​can be recorded simultaneously. PCA analysis ( Fig.40 E) and autocorrelation plot ( Fig.40 F, G) further confirmed that these signals originated from different neurons, indicating that the 16-channel neural electrode has the ability to record single-neuron or multi-neuron signals.

[0159] In the 4-AP-induced rat epilepsy model, the threshold method was used to analyze neuronal spiking activity. The results showed that nearly synchronous specific action potentials were recorded in multiple channels, reflecting the synchronous discharge of abnormal neurons ( Fig.40 H,I). By extracting the peak time ( Fig.40 J) and compared the action potentials recorded from different channels, and found that there was a time lag in the epileptic state ( Fig.40 K). Calculate the time delay of 50 spikes ( Fig.40 L) revealed its propagation characteristics in cortical areas, consistent with the neuronal firing pattern in status epilepticus. Electrical neuromodulation effectively inhibited epileptic-like discharge activity ( Fig.40 M, 40N).

[0160] Long-term biocompatibility studies showed that the all-hydrogel neural electrodes did not cause significant tissue damage or inflammatory response after being implanted under the rat skull for two weeks, while the rigid Ag electrode control group showed obvious damage ( Fig.40 O, Fig.38 ). In addition, the electrode maintained stable action potential capture capabilities two weeks after implantation ( Fig.39 ), indicating 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 prepared, which combines the PEDOT:PSS phase separation technology with the enhanced adhesion properties of polyphenol (DA). Through the synergistic effect of laser and DA, the conductive layer achieved an ultra-high conductivity of 4176S / cm and had a fine resolution of 15μm, laying a solid foundation for the recording of weak neural signals. At the same time, a DA-optimized insulating packaging layer was integrated to ensure the independence and effective isolation of each recording channel. In addition, the introduction of an EMI shielding layer composed of HAD and MXene significantly improved the electrode's resistance to electromagnetic interference (EMI).

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

[0164] In in vivo experiments, the 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 has the ability to distinguish and record spike signals from two different neurons, highlighting its great potential in capturing neural signals from small neuronal clusters.

[0165] More importantly, the all-hydrogel neural electrode can also apply artificial electrical stimulation to the rat cerebral cortex during epileptic seizures, showing its application prospects in therapeutic intervention. In summary, the all-hydrogel neural electrode prepared by the present invention is expected to become a powerful tool for exploring the development mechanism, functional characteristics and treatment strategies of brain diseases and neurodegenerative diseases.

Claims

1. A fully hydrogel neural electrode, characterized in that: It is composed of four layers of hydrogel, namely: PSFD hydrogel layer-LPPDMEAs layer-PSFD hydrogel layer-MHD layer; The PSFD hydrogel layer is an insulating encapsulation layer; The LPPDMEAs layer is a conductive layer; The MHD layer is a shielding layer; in, The PSFD hydrogel is a hydrogel prepared by mixing degummed silk fibroin SF modified with dopamine DA and polyethylene glycol diglycidyl ether PEGDE with an average Mn of 500; The LPPDMEAs layer is a multi-electrode array formed by depositing a PPD aqueous solution on a PSFD hydrogel layer and then laser-etching; 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 preparation method of the PPD aqueous solution is: DA·HCl was added to a PEDOT:PSS aqueous solution (10 mL, 10 mg / mL) to synthesize, and after stirring at room temperature for 30 minutes, a PPD aqueous solution was obtained.

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

4. The all-hydrogel neural electrode according to claim 1, characterized in that: The preparation method of the MHD hydrogel is: EDC and NHS were added to a 10wt% hyaluronic acid HA solution to activate the carboxyl group, and then DA·HCl was added; the reaction solution was stirred at room temperature overnight under nitrogen protection; then dialyzed in deionized water for 3 days using a 10000Da dialysis membrane, and the pure product of HAD was obtained by freeze drying; Then, 2.5 wt% HAD solution was mixed with MXene with concentrations of 0, 1, 1.5 and 2 mg / mL, respectively, and 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 an environment of 40°C 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: 8 wt% degummed silk fibroin SF solution was placed in an ice bath, followed by the addition of EDC and NHS, and after stirring for 30 minutes, DA·HCl was added to the solution; after one hour of reaction, the mixture was dialyzed with deionized water using a 14000Da dialysis bag, and pure SFD was obtained by freeze drying; Polyethylene glycol diglycidyl ether PEGDE with an average Mn of 500 was added to a 5wt% SF-DA solution, which was then dynamically stirred under heating at 60°C; the solution was poured into a substrate, and a film was formed after the water evaporated in the ambient air; after absorbing water, a PSFD hydrogel was obtained.

6. The method for preparing the all-hydrogel neural electrode according to any one of claims 1 to 6, characterized in that: It includes the following steps: a. Deposit the PPD film on the PSFD hydrogel layer and then perform laser etching to form a 16-channel pattern; b. Wash with water to remove the untreated part, and the remaining LPPD membrane absorbs water to form an LPPDMEAs layer; c. Place another insulating PSFD hydrogel layer on top of the LPPD MEAs hydrogel layer, followed by secondary laser etching to expose 16 electrode sites; d. Add an MHD shielding layer on top and link them through the 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 conditions of the laser etching described in step a are: laser power 0.185 W; scanning speed 20 mm / s. The laser etching described in step c is to perform laser etching on the electrode points separately after the second layer of PSFD is encapsulated, selectively etching away the second layer of PSFD to expose the electrode points for contact with the tissue.

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

Images