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WO2024215255A1 - Électrode neuronale, dispositif neuronal et leurs procédés de fabrication - Google Patents

Électrode neuronale, dispositif neuronal et leurs procédés de fabrication Download PDF

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WO2024215255A1
WO2024215255A1 PCT/SG2024/050237 SG2024050237W WO2024215255A1 WO 2024215255 A1 WO2024215255 A1 WO 2024215255A1 SG 2024050237 W SG2024050237 W SG 2024050237W WO 2024215255 A1 WO2024215255 A1 WO 2024215255A1
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rgo
fiber core
neural
neural electrode
probe
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John S. Y. Ho
Yunxia JIN
Yuxin Liu
Chwee Teck Lim
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National University of Singapore
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/25—Bioelectric electrodes therefor
    • A61B5/263—Bioelectric electrodes therefor characterised by the electrode materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/25—Bioelectric electrodes therefor
    • A61B5/279—Bioelectric electrodes therefor specially adapted for particular uses
    • A61B5/291—Bioelectric electrodes therefor specially adapted for particular uses for electroencephalography [EEG]
    • A61B5/293—Invasive
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/25—Bioelectric electrodes therefor
    • A61B5/279—Bioelectric electrodes therefor specially adapted for particular uses
    • A61B5/294—Bioelectric electrodes therefor specially adapted for particular uses for nerve conduction study [NCS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/316—Modalities, i.e. specific diagnostic methods
    • A61B5/369—Electroencephalography [EEG]
    • A61B5/37—Intracranial electroencephalography [IC-EEG], e.g. electrocorticography [ECoG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
    • A61B5/316—Modalities, i.e. specific diagnostic methods
    • A61B5/388—Nerve conduction study, e.g. detecting action potential of peripheral nerves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/05—Electrodes for implantation or insertion into the body, e.g. heart electrode
    • A61N1/0551—Spinal or peripheral nerve electrodes
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/182—Graphene
    • C01B32/194—After-treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
    • A61B2562/12—Manufacturing methods specially adapted for producing sensors for in-vivo measurements
    • A61B2562/125—Manufacturing methods specially adapted for producing sensors for in-vivo measurements characterised by the manufacture of electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01G—CAPACITORS; CAPACITORS, RECTIFIERS, DETECTORS, SWITCHING DEVICES, LIGHT-SENSITIVE OR TEMPERATURE-SENSITIVE DEVICES OF THE ELECTROLYTIC TYPE
    • H01G11/00—Hybrid capacitors, i.e. capacitors having different positive and negative electrodes; Electric double-layer [EDL] capacitors; Processes for the manufacture thereof or of parts thereof
    • H01G11/22—Electrodes
    • H01G11/30—Electrodes characterised by their material
    • H01G11/32—Carbon-based
    • H01G11/36—Nanostructures, e.g. nanofibres, nanotubes or fullerenes
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01G—CAPACITORS; CAPACITORS, RECTIFIERS, DETECTORS, SWITCHING DEVICES, LIGHT-SENSITIVE OR TEMPERATURE-SENSITIVE DEVICES OF THE ELECTROLYTIC TYPE
    • H01G11/00—Hybrid capacitors, i.e. capacitors having different positive and negative electrodes; Electric double-layer [EDL] capacitors; Processes for the manufacture thereof or of parts thereof
    • H01G11/84—Processes for the manufacture of hybrid or EDL capacitors, or components thereof
    • H01G11/86—Processes for the manufacture of hybrid or EDL capacitors, or components thereof specially adapted for electrodes

Definitions

  • the present invention relates, in general terms, to a neural electrode, a neural device and their methods of fabrication thereof.
  • Neural electrodes provide an interface for the effective information transmission between the nervous system and external devices, which not only can be used in fundamental neuroscience research, such as exploring the mechanisms of cognitive processes and the neural basis of sensory information processing, but also help find cures for many neurological diseases. For example, recording brain activity by neural electrodes may identify the neural firing patterns related to epileptic activity, which can be used to locate epileptic lesions. In terms of neuromodulation, cochlear implants help deaf patients restore their hearing. Deep brain stimulations are used to relieve Parkinson's symptoms, and spinal cord stimulators help relieve neuropathic pain.
  • a functional microelectrode is required to communicate with an individual neuron to record bio-signals, while delivering sufficient amount of electrical charge to depolarize the neural tissue and initiate a response.
  • the ideal neural electrodes should have good biocompatibility, low impedance, and high charge injection capacity for the high-quality, low-damage, and long-term stable neural recording and regulation.
  • Electrode impedance is strongly correlated with the effective surface area of the electrode. Increasing its value can reduce the impedance and lower the thermal noise amplitude. A small metal electrode may have a large electrochemical impedance. resulting in a low signal-to-noise ratio.
  • the electrophysiological recording should identify the densely-packed nearby neurons over chronic timescales with minimum invasion. It requires invasive probe to directly interface with individual neuron near 1 :1 size ratio in each channel and assemble all the channels at neuronal density.
  • broad adoption of such ideal design in neural electrophysiological recording probe is limited by the poor electrochemical performance of current metallic electrodes. This is due to the high interface impedance between neurons and metal induced low coupling efficiency causing low signal-to-noise ratio of recording signals.
  • metal wire tetrode of 17 urn 90%platinum/10% iridium wires, a gold standard of the smallest unit of triangulation analysis of single-unit sorting, requires electroplating of platinum nanoparticles or PEDOT:PSS to decrease the impedance. It is challenging to perform stable chronic recording from such probes for months with minimum immunohistochemical response as it has high chance of modification layer delamination due to the weak interfacial adhesion and low environmental stability of coating layer or metal nanoparticle dissolution due to high relative surface area exposed.
  • the latest silicon depth probe has achieved dense coverage along a plane perpendicular to the brain surface. While other geometries including Utah array and microelectrode array have been targeting to sample across a plane parallel to the brain surface, which offer important advantages in precise electrophysiology towards cellular medicine and brain functional organization. The areal spacing of recording sites has been narrowed down to 100 ⁇ m in large array.
  • An alternative to commonly used metallic electrode is the graphene-based fiber probes, which has been validated for in vivo electrophysiology, and particularly attractive because of electrochemical inertness and competence, biocompatibility, as well as versatile fabrication and integration to flexible electronics or biomarker sensors.
  • the present disclosure relates to a neural electrode comprising at least one probe, the probe having a reduced graphene oxide (rGO) fiber core and a sheath encapsulating the rGO fiber core; wherein the rGO fiber core is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; wherein an end of the rGO fiber core is substantially exposed from the sheath and is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; and wherein the end of the rGO fiber core comprises a nano-textured morphology in order to increase its surface area thereof.
  • rGO reduced graphene oxide
  • the rGO fiber core comprises intercalated and/or stacked rGO sheets, wherein the rGO sheets are aligned parallel to a longitudinal axis of the rGO fiber and wherein the rGO sheets are folded onto each other in a perpendicular direction relative to the longitudinal axis of the rGO fiber.
  • the rGO fiber core is characterised by a cross sectional diameter of about 10 ⁇ m to about 20 ⁇ m. In some embodiments, the cross sectional diameter of the rGO fiber core is characterised by a relative standard deviation of less than 0.15.
  • the rGO fiber core comprises nanotunnels between the rGO sheets, wherein the nanotunnels are characterised by a width of about 0.5 nm to about 500 nm.
  • the end of the rGO fiber core has a cross sectional diameter which is substantially similar to the core of the rGO fiber.
  • the end of the rGO fiber core comprises rGO sheets folded onto each other in a perpendicular direction relative to the longitudinal axis of the rGO fiber.
  • the end of the rGO fiber core is characterised by an exposed cross sectional area of about 50 ⁇ m 2 to about 1000 ⁇ m 2 .
  • the nano-textured morphology comprises a tip, wherein the tip is characterised by a morphology selected from a cone, or a pyramid.
  • the rGO fiber core is characterised by a Young's modulus of about 10 GPa to about 50 GPa.
  • the rGO fiber core is characterised by a cross sectional area of about 50 ⁇ m 2 to about 3500 ⁇ m 2 .
  • the sheath comprises an insulator.
  • the insulator is selected from parylene C, SU-8, polyimide, polyurethane, or a combination thereof.
  • the sheath is characterised by a thickness of about 0.5 ⁇ m to about 10 ⁇ m.
  • the probe is electrically connected to a wire or metal trace.
  • the neural electrode comprises an array of probes, the probes spaced apart from each other and are adhered together by a hard polymer or a soft polymer.
  • the neural electrode comprises at least 4 probes.
  • the neural electrode is characterised by a cross sectional diameter is about 80 ⁇ m to about 100 ⁇ m.
  • the neural electrode is characterised by an areal density of about 500 electrodes/mm 2 to about 4000 electrodes/mm 2 .
  • the neural electrode is characterised by an impedance of about 0.0005 M ⁇ to about 1 M ⁇ .
  • the neural electrode is characterised by a charge injection capacity of about 1.5 mC/cm 2 to about 10 mC/cm 2 .
  • the neural electrode comprises an array of probes, wherein the array of probes is characterised by a probe density of about 500 probe/mm 2 to about 4000 probe/mm 2 .
  • the present disclosure also relates to a neural device, comprising : a) a neural electrode as disclosed herein; and b) a microdrive electrically connected to the neural electrode.
  • the neural device further comprises computing means having a data processing algorithm.
  • the present disclosure also relates to a method of fabricating a neural electrode, the neural electrode comprising at least one probe, the probe having a reduced graphene oxide (rGO) fiber core and a sheath encapsulating the rGO fiber core, the method comprising : a) loading a solution of graphene oxide (GO) sheets and a reducing agent into a tubing in order to form a rGO gel; b) evaporating the rGO gel in order to form a rGO fiber; c) coating the rGO fiber with an insulator in order to form a rGO fiber core encapsulated with a sheath; and d) breaking the rGO fiber core and sheath in order to substantially expose an end of the rGO fiber core from the sheath; wherein the rGO fiber core is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; wherein an end of the rGO fiber core is characterised by a cross sectional diameter of about 5
  • the reducing agent is ascorbic acid.
  • method further comprises a step after step a) of washing the rGO gel.
  • the method further comprises a step after step c) of elastocapillary assembling at least one probe in order to form the neural electrode.
  • the breaking step comprises: a) partially embedding the rGO fiber core and sheath in a polymer; b) cryo-freezing the partially embedded rGO fiber core and sheath; c) breaking the partially embedded rGO fiber core and sheath at an interface of the embedment; and d) dissolving the polymer of step a) in a solvent.
  • the polymer is selected from OCTTM, polyvinyl alcohol, and/or polyethylene glycol.
  • the method further comprises a step of adhering the probes together with a hard polymer or a soft polymer.
  • the present disclosure also provides a method of isolating a neuron using the neural device as disclosed herein.
  • Figure 1 shows stress-concentrated breaking-down technique to expose the electrode tip.
  • a Schematics of stress-concentrated breaking-down method (Natural cut (Ncut)). It involves partially coating the frozen optimal cutting temperature (OCT) compound on probe, increasing the diameter at one end, and hence causing significant bending stiffness difference between coating and uncoating part. It leads to naturally breakdown of the probe from the coating-uncoating point upon applying force, leaving a sharp exposure of graphene edge with well-preserved nanotexture and nanotunnels
  • b-c SEM images of cross-section of the bare microfiber (b) and microfiber with parylene C (c) exposed by Ncut.
  • d Enlarged SEM images in c by viewing from top and tilted angle.
  • Figure 2 is an overview of the monolithic high-density NeuroEdge
  • a The schematic of NeuroEdge array interface with individual neurons at near 1:1 number ratio within an overall cross-section dimension of less than 100 ⁇ m.
  • b Scanning electron microscope (SEM) image of cross-section of a 20-channel NeuroEdge array.
  • the NeuroEdge is coated with 2- ⁇ m parylene C for insulation and the NeuroEdge array is bundled via elastocapillary force-induced self-assembly from photoresist SU-8 bath followed by UV curing
  • c Confocal image of neuron in the cortical area of a mouse brain labelled with neuronal nuclei (NeuN).
  • Neuronal nuclei Neuronal nuclei
  • the NeuroEdge and neurons show a similar density, d, Schematic of NeuroEdge tip. It featurs nanotexture, nanoedges and nanotunnels at the tip allowing for large relative electrochemical surface area and ion/electron coupling along nanotunnels, e, SEM images of the cross-section of NeuroEdge.
  • the tip of NeuroEdge is sharply exposed using stress-concentrated breaking technique
  • I, II, III mark the nanotexture, nanotunnel and nanoedge, respectively
  • f Comparison of the probe density of NeuroEdge, multielectrode array (MEA), and silicon probe regarding in the lateral direction within 100 ⁇ m x 100 ⁇ m region
  • g Comparison of impedance (Z) of NeuroEdge, metal with a coating (ME-coat) and bare metal (ME-bare) with electrode dimensions comparable to the neuron cell body ( ⁇ 10 ⁇ m).
  • the ideal impedance for electrophysiological recording is below 106 ⁇ .
  • the NeuroEdge is within the ideal working range.
  • the ME-coat initially falls in the working range, but moves out of the range over time due to the delamination and degradation of the modification layer.
  • the ME-bare is far from the ideal working range.
  • Scale bar is 25 ⁇ m in b and c, and 0.5 ⁇ m in e.
  • FIG. 3 shows the fabrication process of NeuroEdge. Fabrication includes four key steps, incorporating solution-processable self-assemblies and stress-concentrated break-down. Specifically, in step I, to maximize the in vivo biocompatibility and realize the small form factor while streamline fabrication, solution-processable graphene oxide (GO) sheets were employed to form reduced graphene oxide (rGO) gel fiber through in situ 3D self-assembly and reduction with only water and vitamin C, a natural antioxidant that is widely employed as a food additive and can compete with the highly toxic hydrazine in terms of its reducing capability.
  • GO graphene oxide
  • Step II involves restricted self-alignment, driven by water evaporation induced self-assembly, to form a straight microelectrode fiber (II) from the gel fiber.
  • the photograph reveals the prepared microfiber is significantly slimmer than the hair and fingerprint.
  • step III to maximize array density, elastocapillary self-assembly was employed, merging the parylene C insulated microfibers into a bundle during the withdrawal from a bath of diluted SU-8 solution.
  • the small form factor of microfiber enabled the elastocapillary self-assembly by providing a balanced adhesion energy arising from van der Waals force and probe stiffness.
  • step IV we developed a stress-concentrated break down method to expose the bundle tip end.
  • the rGO sheet stack are well exposed preserving the nanoedge, nanotexture and nanotunnels, which accounts for the low impedance.
  • the absence of toxic or organic solvent aligned with small size of probe prioritizes the biocompatibility essentials for implantable probes.
  • the solution processable technology allows for precise tuning of the probe to any desirable size.
  • FIG. 4 shows step II of fabrication process of NeuroEdge.
  • the gel fiber was exposed to air and underwent natural evaporation of water within the gel fiber.
  • the evaporation process allows the rGO sheets to contact each other and shrink.
  • the uneven evaporation rate influenced by distinct radii of curvature at different locations along the gel fiber, led to uneven movement of rGO sheets, resulting in varying diameters at different locations.
  • the SEM shows deep folding or crumpling of rGO sheets
  • b Schematic of wet-mounting approach and the SEM images of the prepared fiber.
  • the gel fiber was placed on a glass substrate. As water evaporated, the high surface tension of water caused the gel fiber to wet the hydrophilic glass, expanding its axial footprint.
  • the bottom layer of rGO sheets were adhered to the glass due to interface tension between water and glass, minimizing crumpling and allowing for the stacking of rGO sheets layer-by-layer.
  • the SEM images show the outer rGO sheets appear much flatter compared to those in a. with a footprint of 70 ⁇ m.
  • c Schematic of restricted solvent evaporation induced self-assembly (SEIA) and the SEM images of the resulting fiber.
  • SEIA restricted solvent evaporation induced self-assembly
  • the gel fiber was anchored at both ends on a plastic substrate, and underwent solvent evaporation induced self-assembly of the rGO sheets along the anchoring direction.
  • the absence of interface tension allowed the rGO sheets to freely assembly while being confined to the mounting direction, resulting in a small diameter and a straight longitudinal alignment.
  • the SEM images showed the diameter is only 12 ⁇ m which is 5- fold smaller compared to b.
  • d-e Schematics of viewing direction and the SEM images from cross-section for fiber that was prepared by methods in b (d) and c (e).
  • Figure 5 shows a-b, Raman characterization (a) and X-ray photoelectron spectroscopy (XPS) (b) for NeuroEdge.
  • the inset in a is the Raman scan of the exposed tip end of NeuroEdge.
  • the D/G ratio is predominantly around 1.3.
  • the XPS result showed a significantly increased ratio of carbon-to-oxygen compared to graphene oxide (GO). It indicates that the GO has been successfully converted to reduced graphene oxide (rGO).
  • Figure 6 shows electrochemical characterization of NeuroEdge, a, Impedance at 1 kHz of NeuroEdge compared with reported metal electrodes (Metal), surface modified metal electrodes (Modified), and carbon-based electrodes (Carbon), b Impedance of NeuroEdge compared with that of conventional metal wires Pt/Ir (iridium 10%) in different diameters, c, Specific impedance at 1 kHz of NeuroEdge compared with Pt/Ir and PEDOT:PSS modified Pt/Ir. Values are mean ⁇ s.d. d, Specific impedance at 1 kHz of NeuroEdge as a function of charging time compared with PEDOT:PSS modified Pt/Ir under a biphasic pulse.
  • the minimum current was relatively stable with a change of ⁇ 10% at 6-month.
  • the scale bar is 1 ⁇ m in d and e.
  • Figure 7 shows in vivo high-density and high-quality recording and spike-sorting with NeuroEdge
  • a Schematics of a mouse brain with implantation showing the location of 16-channel NeuroEdge, ground screw (GND), and supporting screw, as well as a 16- channel NeuroEdge neural interface (right side)
  • b Representative 10-min neural recording from channel 1 (Chi) in the cortex shows both local field potential (LFP) (0.1- 9000 Hz) and spontaneous spiking signal (300-9000 Hz)
  • LFP local field potential
  • HP high-pass
  • the waveforms before and after filtering are almost identical in shape with a high amplitude of 600 ⁇ V. d, e. Power spectrum density (PSD) (d), and spectrogram (e) of the recorded signal (0.1-9000 Hz).
  • PSD Power spectrum density
  • e spectrogram
  • the smooth average value of PSD shown in dark line in d and continuous horizontal PSD at different frequency demonstrate no significant external environment noise in the recording, f, g, Amplitude (f) and SNR (g) in maximum and average of the recorded signal after 300-9000 Hz band-pass filtering, h-j.
  • Triangulation spike sorting using Offline Sorter It mainly includes three steps. First, filter the raw data with a second order Butterworth HP filtering (h).
  • spike clustering based on principle components analysis (PCA) (j). It groups spikes with similar feature vectors that represent the activity of a single neuron. Each point on the plot represents a spike exceeding the defined threshold.
  • PCA principle components analysis
  • N1 to N7 Seven clusters are projected onto the first two principal components and marked as N1 to N7.
  • k Average spike waveforms on 4 channels for each putative neuron.
  • I Representative dense neural recording from 16-channel in cortex revealed clear and strong LFP and spiking signal (0.1-9000 Hz, left) and filtered spontaneous spiking signal (300-9000 Hz, right), m, n, Raster plot (m) and average waveforms (n) of the sorted units from the recording in I.
  • Figure 8 shows implantation of NeuroEdge, a. Schematics of fabrication and implantation of the NeuroEdge drive into mouse brain, b, Photograph of one representative probe, c, Photograph of the implantation of the probe into brain. NeuroEdge was attached onto a movable drive. With a clockwise turn of the screw on drive, the electrode was loaded down around 180 ⁇ m.
  • Figure 9 shows NeuroEdge resolves heterogeneity in the acoustic frequency response of neighboring neurons in mice auditory cortex, a, Schematic set-up for the auditory stimuli and recordings.
  • the sound card supplies two channels. One is the click channel to align the timeline of the electrophysiological recording and the stimuli, and another is the tone channel to deliver auditory stimuli. There are 78 frequencies spanning 0.5- 42 kHz in 1 /2 octave with a duration of 100 ms and interstimulus interval of 1.3 s.
  • b Representative recording and its spectrogram during the stimulus duration in a marked by red background
  • c Representative electrophysiological recording from NeuroEdge during pre-stimulation and stimulation spanning 0.5-42 kHz. d.
  • the red lines are the fitted curves, f, Average SD ratio for each putative neuron responding to full frequency from 0.5 to 42 kHz (All), lower frequency marked in f1 and higher frequency marked in f2 in d.
  • the center frequency of f1 and f2 is 2.6 kHz and 9.5 kHz, respectively.
  • the black solid line represents the SD ratio from the whole channel including all the neurons, g, Representative cross-correlograms of neurons under no stimulation and stimulation with full frequency of 0.5-42 kHz, f1and f2 h, Cross-correlation matrices of pairwise neurons under no stimulation and stimulation with full frequency of 0.5-42 kHz, f1 and f2.
  • Figure 10 shows auditory stimulation setup and data processing
  • a Schematics of the auditory stimulation setup. It contains two channels, with the trigger channel marking the timestamp of the stimulation tone aligned with the recorded signal. Each frequency is stimulated for 100 ms, and interstimulus interval is 1.3 s to leave enough rest time for the neuron to recover from the last stimulus, b. Schematics of data processing. The filter signal was realigned by interval of 1.3 s, and each corresponds to a sound frequency, c. Peristimulus raster plot.
  • Figure 11 shows chronic assessment of recording stability, functionality and biocompatibility of NeuroEdge, a, A photograph of a mouse implanted with NeuroEdge and a schematic of the chronic recording timeline, b, Representative electrophysiological recording from NeuroEdge at Ml, M3 and M6. BP filtering of 300-9000 Hz was applied, c-d, Performance of the electrophysiological recording stability. The average values of SNR and noise level (c), NeuroEdge impedance and spike amplitude (d) all remained stable for 6 months, e-f, Peristimulus spike raster plot (e) and spike density (f) in response to 78 frequencies spanning 0.5-42 kHz at month 1, 3 and 6.
  • Neurons, microglia and astrocytes are immunohistochemically stained with NeuN (red), Ibal(red) and GFAP (green), respectively, i, Analysis of the neuron cells (NeuN), activated microglia (Ibal) and astrocyte cells (GFAP) around insertion position across the entire immunohistochemical images. Values are shown mean ⁇ s.d. using one-way t-test (*P ⁇ 0.05, **P ⁇ 0.01).
  • graphene-based neural electrodes Owing to its excellent bio and neuroelectronic properties, graphene is an ideal material for future neural probes.
  • Graphene is a two-dimensional nanomaterial with a single atomic layer composed of sp 2 -hybridized carbon atoms.
  • graphene As an active material for neural electrodes, graphene has many advantages over other materials that include: (1) high mechanical flexibility that allows close contacts with soft brain tissues and formation of a stable electrode-neuron interface; (2) good electrical conductivity and excellent carrier mobility (up to 100,000 cm 2.
  • V -1 'S -1 which promote the highly sensitive detection of neuroelectric signals by a graphene field-effect transistor; (3) a single atomic layer thickness and ultra-high specific surface area combined with unique electrical properties, which ensure low electrochemical impedance and high charge injection capabilities of graphene neural electrodes that lay the foundation for the effective electrical stimulation of neural tissues; and (4) high transparency and transmittance of a single-layer graphene (up to 97.3%).
  • a porous electrode materials may be used to obtain a neural electrode with a larger effective surface area but smaller geometric size.
  • traditional porous electrode materials has bad mechanical property, which is not easy to handle or insert into tissue.
  • graphene sheets may be obtained by exfoliation, and graphene fiber electrodes with highly active graphene edge and large relative surface area are produced after tip exposure. Therefore, graphene electrodes can record electrophysiological signals while observing changes in cells and blood vessels under the electrodes with electrophysiological activity. Owing to their high transparency, graphene neural electrodes can also be combined with other technologies (such as optogenetics) for the optical regulation of neuroelectric activities.
  • the present disclosure concerns a graphene fiber based pristine neural electrode and neural probe array, which may enable ultralow-impedance and ultrahigh-density neuronal recording and modulation achieving a neuron-to-neuron functionality discrimination and accurate electronic medicine therapy for months. It may be used for chronically stable neural interface for lots of promising application that could alter our living status. For instance, the brain-machine interface, neuronal modulation for deepbrain disease, prosthesis helping disables.
  • the probe array may record the signal from nervous system with high signal-to-noise ratio and long-term stability.
  • the individual probe can be down to single neuronal size, and more than 15 vertical aligned probes can be bonded together in the horizontal direction at an area of cross-section of individual human hair, which enables the probe density close to the neuron density in the cortex area of brain.
  • the unique texture structure on the exposed cross-section end has high electrochemical activity and large surface area, resulting in an ultra-low impedance, which is even better than modified probe with metal nanoparticles or conducting polymer.
  • Such high electrochemical activity at extremely low exposed area allows for high-quality recording and modulation at high-density, which turns out a high- quality spike-sorting to identify single-units at higher yield and higher density. It helps further allocate the functionality of brain at higher resolution to individual near-by neurons.
  • the present disclosure relates to a neural electrode comprising at least one probe, the probe having a reduced graphene oxide (rGO) fiber core and a sheath encapsulating the rGO fiber core; wherein the rGO fiber core is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; wherein an end of the rGO fiber core is substantially exposed from the sheath and is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; and wherein the end of the rGO fiber core comprises a nano-textured morphology in order to increase its surface area thereof.
  • rGO reduced graphene oxide
  • the cross sectional diameter (and hence cross sectional area) of the end of the rGO fiber core is maintained relative to the body of the rGO fiber core.
  • the end of the rGO fiber core is not deformed.
  • nano-textured morphology may be created at the end of the rGO fiber core which may increase its surface area, higher electrochemical activity and thus low impedance is enabled for large array or individual fiber.
  • a normal cut using scissors will cause the tib end to collapse and the sheath on the surface of fiber to deform and cover the end of the rGO fiber, causing large impedance.
  • ultralow-impedance recording and ultrahigh charge injection capacity may be obtained.
  • the rGO fiber has a diameter of less than 100 ⁇ m, or preferably less than 20 ⁇ m, which is similar to neuronal size, this further adds to the improved quality for recording and modulation.
  • pure metal probe cannot achieve neuronal size while meeting the impedance requirement, and modified probe can reach this size but with extra risk of modification layer detach causing worse long-term stability.
  • the electrode (and probe) as disclosed herein does not need an additional modification layer to lower down impedance or improve charge injection capacity.
  • the modified layer has a high chance to delaminate or fatigue with time. This will cause failure in recording, and at the same time damage the tissue to induce immune response.
  • the probe is pristine. This means that the probe is not further functionalised or modified. In some embodiments, the probe is void of surface modifications.
  • the rGO fiber core comprises intercalated and/or stacked rGO sheets.
  • the sheets are physically connected together but leave nanovoids between the rGO sheets.
  • the rGO sheets are characterised by an intersheet distance of about 0.5 nm to about 500 nm.
  • the rGO sheets are axially aligned. The alignment may be along a longitudinal direction of the rGO fiber core.
  • the probe comprises nanotunnels between the rGO.
  • the nanotunnels may be formed due of the spacing between the intercalated and/or stacked rGO sheets.
  • the nanotunnels may be about 0.5 nm to about 500 nm in width.
  • the intersheet distance and/or nanotunnels is about 0.5 nm to about 450 nm, about 0.5 nm to about 400 nm, about 0.5 nm to about 350 nm, about 0.5 nm to about 300 nm, about 0.5 nm to about 250 nm, about 0.5 nm to about 200 nm, about 0.5 nm to about 150 nm, about 0.5 nm to about 100 nm, or about 0.5 nm to about 50 nm.
  • rGO fiber core comprises a single linear rGO fiber.
  • the rGO fiber core may be formed as a straight fiber.
  • the rGO fiber core is characterised by a uniform and straight morphology. This is due to the restricted selfassembly of rGO sheets, and optionally the restricted drying of the rGO gel in order to form the rGO fiber. As shown in Figure 9, in the restricted self-assembly of rGO sheets, the rGO sheets in the rGO gel are induced to self-assemble along an anchoring direction.
  • the resulting rGO fiber has a small diameter (at least 5 times smaller than a fiber formed from a wet mounting approach) as a result of the rGO sheets stacking more closely to each other.
  • the rGO sheets are also aligned within the rGO fiber in a straight longitudinal alignment relative to the rGO fiber.
  • a cross sectional view of the rGO fiber shows that the rGO sheets are folded on each other resulting in a wavy pattern as observed under SEM, which indicates that the rGO sheets curled onto each other.
  • rGO fiber comprises rGO sheets, wherein the rGO sheets are aligned parallel to a longitudinal axis of the rGO fiber. In some embodiments, the rGO sheets are folded onto each other in a perpendicular direction relative to the longitudinal axis of the rGO fiber. In some embodiments, a cross section of the rGO fiber comprises folded rGO sheets.
  • the rGO fiber core is characterised by a Young's modulus of about 10 GPa to about 50 GPa.
  • the Young's modulus is about 10 GPa to about 45 GPa, about 10 GPa to about 40 GPa, about 10 GPa to about 35 GPa, about 10 GPa to about 30 GPa, about 10 GPa to about 25 GPa, about 10 GPa to about 20 GPa, or about 10 GPa to about 15 GPa.
  • the rGO fiber core is characterised by a bending stiffness of about 2 x 10- 2 nN m 2 to about 8 x I0 -2 nN m 2 . In some embodiments, the rGO fiber core is characterised by a bending stiffness of about 2 x 10' 2 nN m 2 to about 7.5 x 10" 2 nN m 2 , about 2 x 10' 2 nN m 2 to about 7 x 10' 2 nN m 2 , about 2 x 10' 2 nN m 2 to about 6.5 x 10' 2 nN m 2 , about 2 x 10' 2 nN m 2 to about 6 x 10' 2 nN m 2 , about 2 x 10' 2 nN m 2 to about 5.5 x 10 2 nN m 2 , about 2 x 10 2 nN m 2 to about 5 x 10 2 nN m 2 , about 2 x I0 -2
  • the rGO fiber core is characterised by a cross sectional area of about 50 ⁇ m 2 to about 3500 ⁇ m 2 .
  • the cross sectional area is about 100 ⁇ m 2 to about 3500 ⁇ m 2 , about 200 ⁇ m 2 to about 3500 ⁇ m 2 , about 300 ⁇ m 2 to about 3500 ⁇ m 2 , about 400 ⁇ m 2 to about 3500 ⁇ m 2 , about 500 ⁇ m 2 to about 3500 ⁇ m 2 , about 600 ⁇ m 2 to about 3500 ⁇ m 2 , about 700 ⁇ m 2 to about 3500 ⁇ m 2 , about 800 ⁇ m 2 to about 3500 ⁇ m 2 , about 900 ⁇ m 2 to about 3500 ⁇ m 2 , about 1000 ⁇ m 2 to about 3500 ⁇ m 2 , about 1500 ⁇ m 2 to about 3500 ⁇ m 2 , about 2000 ⁇ m 2 to about 3500 ⁇ m 2 , about 2500 ⁇ m 2 to about 3500 ⁇ m 2 ⁇
  • the cross sectional diameter of the rGO fiber core is about 10 ⁇ m to about 100 ⁇ m, about 10 ⁇ m to about 90 ⁇ m, about 10 ⁇ m to about 80 ⁇ m, about 10 ⁇ m to about 70 ⁇ m, about 10 ⁇ m to about 60 ⁇ m, about 10 ⁇ m to about 50 ⁇ m, about 10 ⁇ m to about 40 ⁇ m, about 10 ⁇ m to about 30 ⁇ m, or about 10 ⁇ m to about 20 ⁇ m.
  • the cross sectional diameter of the rGO fiber core is about 12 ⁇ m to about 15 ⁇ m.
  • the cross sectional diameter of the rGO fiber core is characterised by a relative standard deviation of less than 0.15. A consistent diameter was found to be advantageous for the low impedance.
  • the end of the rGO fiber core is undeformed. This is shown in Figure 6.
  • the end of the rGO fiber retains a cross sectional diameter similar to the core of the rGO fiber.
  • the end of the rGO fiber has a cross sectional diameter which is substantially the same as the core of the rGO fiber.
  • the present method of forming the probe does not deform the sheath nor the rGO fiber core.
  • the end of the rGO fiber is undeformed in that the intersheet spacing of rGO sheets and/or nanotunnels is substantially similar to that in the rGO fiber core.
  • the rGO sheets also maintain its folded configuration onto each other.
  • the edge or end is also not covered by the sheath if present. As this edge is maintained similar to the core of the fiber, electrical signals from a brain may be transferred at low impedance.
  • the end of the rGO fiber core is characterised by a surface unevenness of about 5 nm to about 900 nm.
  • the end of the rGO fiber core comprises a tip. The tip may protrude from the sheath, or may be levelled with an interface of the sheath.
  • the tip is characterised by a morphology selected from a cone, or a pyramid. Other shapes may also be possible. It was found that a protrusion is sufficient to provide a low impedance, as it improves contact with the neural tissue.
  • the end of the rGO fiber core is characterised by an exposed cross sectional area of about 50 ⁇ m 2 to about 3500 ⁇ m 2 .
  • the exposed cross sectional area is about 100 ⁇ m 2 to about 3500 ⁇ m 2 , about 200 ⁇ m 2 to about 3500 ⁇ m 2 , about 300 ⁇ m 2 to about 3500 ⁇ m 2 , about 400 ⁇ m 2 to about 3500 ⁇ m 2 , about 500 ⁇ m 2 to about 3500 ⁇ m 2 , about 600 ⁇ m 2 to about 3500 ⁇ m 2 , about 700 ⁇ m 2 to about 3500 ⁇ m 2 , about 800 ⁇ m 2 to about 3500 ⁇ m 2 , about 900 ⁇ m 2 to about 3500 ⁇ m 2 , about 1000 ⁇ m 2 to about 3500 ⁇ m 2 , about 1500 ⁇ m 2 to about 3500 ⁇ m 2 , about 2000 ⁇ m 2 to about 3500 ⁇ m 2 , about 2500 ⁇ m 2 to about
  • the end of the rGO fiber core is characterised by a cross sectional diameter of about 10 ⁇ m to about 100 ⁇ m, about 10 ⁇ m to about 90 ⁇ m, about 10 ⁇ m to about 80 ⁇ m, about 10 ⁇ m to about 70 ⁇ m, about 10 ⁇ m to about 60 ⁇ m, about 10 ⁇ m to about 50 ⁇ m, about 10 ⁇ m to about 40 ⁇ m, about 10 ⁇ m to about 30 ⁇ m, or about 10 ⁇ m to about 20 ⁇ m.
  • the end of the rGO fiber core is characterised by a cross sectional diameter of about 12 ⁇ m to about 15 ⁇ m.
  • the sheath comprises an insulator.
  • An electrical insulator is a material in which electric current does not flow freely. The atoms of the insulator have tightly bound electrons which cannot readily move. The property that distinguishes an insulator is its resistivity; insulators have higher resistivity than semiconductors or conductors.
  • the insulator is selected from parylene C, SU-8, polyimide, polyurethane, or a combination thereof. In some embodiments, the insulator is parylene C.
  • the sheath is characterised by a thickness of about 0.5 ⁇ m to about 10 ⁇ m. In other embodiments, the thickness is about 0.5 ⁇ m to about 9 ⁇ m, about 0.5 ⁇ m to about 8 ⁇ m, about 0.5 ⁇ m to about 7 ⁇ m, about 0.5 ⁇ m to about 6 ⁇ m, about 0.5 ⁇ m to about 5 ⁇ m, about 0.5 ⁇ m to about 4 ⁇ m, about 0.5 ⁇ m to about 3 ⁇ m, about 0.5 ⁇ m to about 2 ⁇ m, or about 0.5 ⁇ m to about 1 ⁇ m.
  • the probe comprises nanotunnels between the rGO, wherein the nanotunnels are filled with an electrolyte.
  • the electrolyte may be a salt solution.
  • the electrolyte may be phosphate buffered saline or body fluid.
  • the nanotunnels may be filled with an electrolyte when used, or the probe may be dry without electrolyte inside nanotunnels if not in use.
  • the probe is electrically connected to a wire or metal trace. For example, a stainless steel wire may be used. The probe may be connected to the wire via the other end of the rGO fiber.
  • the neural electrode comprises an array of probes.
  • the probes may be adhered together by a hard polymer or a soft polymer.
  • a single sheath may be used to encapsulate the probes while still maintaining electrical separation relative to each other.
  • epoxy, polyimide, polyurethane, PEG, PVA and/or PDMS may be used.
  • the epoxy may be SU-8.
  • the neural electrode comprises at least 4 probes. In some embodiments, the neural electrode comprises at least 16 probes.
  • the neural electrode may comprise an array of probes. The array may be a square array or a circular array. The probe may be arrayed such that they are spaced apart from each other.
  • ultrahigh density array may be achieved. This improves the recording or modulation resolution to be individual neuron cell.
  • the neural electrode is characterised by a cross sectional diameter of about 10 ⁇ m to about 100 ⁇ m, about 20 ⁇ m to about 100 ⁇ m, about 30 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 50 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, about 70 ⁇ m to about 100 ⁇ m, or about 80 ⁇ m to about 100 ⁇ m.
  • the cross sectional diameter is about 100 ⁇ m.
  • the neural electrode is characterised by an areal density of about 500 electrodes/mm 2 to about 4000 electrodes/mm 2 .
  • the areal density is about 800 electrodes/mm 2 to about 4000 electrodes/mm 2 , about 1000 electrodes/mm 2 to about 4000 electrodes/mm 2 , about 1500 electrodes/mm 2 to about 4000 electrodes/mm 2 , about 2000 electrodes/mm 2 to about 4000 electrodes/mm 2 , about 2500 electrodes/mm 2 to about 4000 electrodes/mm 2 , about 3000 electrodes/mm 2 to about 4000 electrodes/mm 2 , or about 1000 electrodes/mm 2 to about 3500 electrodes/mm 2 .
  • the neural electrode is characterised by an impedance of about 0.0005 M ⁇ to about 1 M ⁇ .
  • the impedance is about 0.0005 M ⁇ to about 0.9 M ⁇ , about 0.0005 M ⁇ to about 0.8 M ⁇ , about 0.0005 M ⁇ to about 0.7 MQ, about 0.0005 M ⁇ to about 0.6 M ⁇ , about 0.0005 MQ to about 0.5 MQ, about 0.0005 MQ to about 0.4 MQ, about 0.0005 MQ to about 0.3 MQ, about 0.0005 MQ to about 0.2 MQ, about 0.0005 MQ to about 0.1 MQ, about 0.0005 MQ to about 0.09 MQ, about 0.0005 MQ to about 0.08 MQ, about 0.0005 MQ to about 0.07 MQ, about 0.0005 MQ to about 0.06 MQ, or about 0.0005 MQ to about 0.05 MQ.
  • the impedance is about 0.04 MQ.
  • the impedance is substantially the same over a period of at least 50 days.
  • the impedance is less than about 0.5 MQ.
  • the neural electrode is characterised by a charge injection capacity of about 1.5 mC/cm 2 to about 10 mC/cm 2 .
  • the charge injection capacity is about 2 mC/cm 2 to about 10 mC/cm 2 , about 3 mC/cm 2 to about 10 mC/cm 2 , about 4 mC/cm 2 to about 10 mC/cm 2 , about 5 mC/cm 2 to about 10 mC/cm 2 , or about 6 mC/cm 2 to about 10 mC/cm 2 .
  • the neural electrode is characterised by a specific impedance around 20 MQ ⁇ m 2 over 3000 min.
  • the neural electrode is characterised by a phase angle at 1 kHz of about -20°.
  • the neural electrode is characterised by a depolarization, repolarization and/or refractory period with amplitude of about 100 ⁇ V to about 800 ⁇ V.
  • the depolarization, repolarization and/or refractory amplitude is about 100 ⁇ V to about 750 ⁇ V, about 100 ⁇ V to about 700 ⁇ V, about 100 ⁇ V to about 650 ⁇ V, about 100 ⁇ V to about 600 ⁇ V, about 100 ⁇ V to about 550 ⁇ V, about 100 ⁇ V to about 500 ⁇ V, about 100 ⁇ V to about 450 ⁇ V, about 100 ⁇ V to about 400 ⁇ V, about 100 ⁇ V to about 350 ⁇ V, or about 100 ⁇ V to about 200 ⁇ V.
  • the neural electrode is characterised by a signal to noise ratio (SNR) of about 20 dB to about 40 dB.
  • SNR signal to noise ratio
  • the signal to noise ratio (SNR) is about 20 dB to about 38 dB, about 20 dB to about 36 dB, about 20 dB to about 34 dB, about 20 dB to about 32 dB, about 20 dB to about 30 dB, about 20 dB to about 28 dB, or about 20 dB to about 26 dB.
  • the neural electrode comprises an array of probes, wherein the array of probes is characterised by a probe density of about 500 probe/mm 2 to about 4000 probe/mm 2 .
  • the present disclosure also relates to a neural device, comprising : a) a neural electrode as disclosed herein; and b) a microdrive electrically connected to the neural electrode.
  • the neural device further comprises a control system.
  • the control system may be a computer implemented system. This may involve a computer programme which regulates the generation of electric signals to the neuron, and/or receive a feedback signal from the neuron.
  • the control system may be configured with data processing means, such as data processing algorithm.
  • the data processing algorithm may involve clustering a signal obtained from the neural electrode into at least 3 clusters, at least 4 clusters, or at least 7 clusters. Principle component analysis may be used.
  • the electrode and/or device may be used as a tool or therapy media to explore the function of our brain or do precision medicine. This may unlock more secrets of our brain and improve the quality of electronic medicine.
  • the lowest impedance achieved enable the high SNR recording at the neuronal size without any additional modification.
  • the recording site should be small enough to be close to the neuron size, but the impedance of probe will be inversely spiked, which will fail the recording as there is big noise caused by the large impedance.
  • Traditional metal wire all need to be surface-modified (PEDOT or metal nanoparticle) before using as singunit recording probe.
  • the modification because the weak interface adhesion between the original probe and the modification layer will cause the detachment and the modification layer itself is not stable in the in vivo environment. Additionally, the raw material may be obtained cheaply, is biocompatible and is environmental friendly to fabricate while maintain high performance. In contrast, current commercial probes cost thousands of dollars.
  • the present disclosure also relates to a method of fabricating a neural electrode, the neural electrode comprising at least one probe, the probe having a reduced graphene oxide (rGO) fiber core and a sheath encapsulating the rGO fiber core, the method comprising : a) loading a solution of graphene oxide (GO) sheets and a reducing agent into a tubing in order to form a rGO gel; b) evaporating the rGO gel in order to form a rGO fiber; c) coating the rGO fiber with an insulator in order to form a rGO fiber core encapsulated with a sheath; and d) breaking the rGO fiber core and sheath in order to substantially exposed an end of the rGO fiber core from the sheath; wherein the rGO fiber core is characterised by a cross sectional diameter of about 5 ⁇ m to about 100 ⁇ m; wherein an end of the rGO fiber core is characterised by a cross sectional diameter of about 5
  • the rGO gel is formed in the tubing. After the rGO gel is formed, it is removed from the tubing and the aqueous medium evaporated in order to allow for restricted selfassembly of the rGO sheets to form a straight thin rGO fiber.
  • the rGO gel may be tethered or fixed to a substrate at only its ends thereof with its body remain unsupported. This removes external forces which may influence the self-assembly of the rGO sheets.
  • the reducing agent is a non-toxic reducing agent. In some embodiments, the reducing agent is ascorbic acid. In some embodiments, the graphene oxide is reacted with the reducing agent at about 95 °C for at least 1 h.
  • the rGO gel is cylindrical.
  • the tubing is a glass capillary tubing with an inner diameter of about 200 ⁇ m. This will provide a rGO fiber with a core of about 10-15 ⁇ m in diameter.
  • the rGO gel is washed in a water bath.
  • the rGO gel may be washed through a repeat exchange of an aqueous medium.
  • the rGO gel may be washed for at least 2 to 3 times.
  • the method further comprises a step after step c) of elastocapillary assembling the at least one probe in order to form the neural electrode.
  • elastocapillary assembly the fibers are immersed in an adhesive solution (SU-8 solution) and upon withdrawing the fibers from the adhesive solution, the fibers are bonded together as a bundle due to the elastocapillary force.
  • the small form factor of microfiber enabled the elastocapillary self-assembly by providing a balanced adhesion energy arising from van der Waals force and probe stiffness. Such assembly cannot be extended to larger metal probes as their stiffness will exceed the capillary adhesion force, preventing the formation of bundles.
  • the method further comprises a step after step b) of electrically connecting the rGO fiber to a wire or metal trace.
  • the breaking step comprises: a) at least partially embedding the rGO fiber core and sheath in a polymer; b) cryo-freezing the at least partially embedded rGO fiber core and sheath; c) breaking the partially embedded rGO fiber core and sheath at an interface of the embedment; and d) dissolving the polymer of step a) in a solvent.
  • the polymer is a water soluble polymer. In some embodiments, the polymer is OCTTM. OCT may be dissolved in an aqueous medium or an organic solvent. In some embodiments, the polymer is polyvinyl alcohol and/or polyethylene glycol.
  • step c) comprises fracturing the rGO fiber core to form an end.
  • a rapid force may be applied. Due to the bending stiffness mismatch at the coating boundary, stress is created which results in a natural fracture. This exposes the graphene edge nanotexture with well-preserved nanoedge-nanotunnel architecture.
  • the method further comprises a step of adhering the probes together with a hard polymer or soft polymer.
  • the hard polymer may be epoxy.
  • the plurality of probes is bonded using SU-8.
  • the present disclosure also concerns a method of fabricating a neural device, comprising electrically connecting a neural electrode as disclosed herein to a microdrive.
  • the method further comprises electrically connecting the neural device to a control system.
  • the control system may be configured with data processing means, such as data processing algorithm.
  • the data processing algorithm may involve clustering a signal obtained from the neural electrode into at least 3 clusters, at least 4 clusters, or at least 7 clusters. Principle component analysis may be used.
  • the neural electrode and device may be used to track neuron activity chronically with almost maximum SNR for spontaneous electrophysiological signal, and high resolution to record and identify the evoked signal, as month long stable recording with high quality may be obtained.
  • the present disclosure also provides a method of isolating a neuron using the neural device as disclosed herein.
  • the method may be an in vivo method, or an in vitro method.
  • the method of isolating a neuron may be in a subject in need thereof.
  • the neuron may be located in the auditory cortex.
  • the neuron may be located at cortical laminae in the auditory cortex.
  • the method further isolates neighbouring neurons.
  • the present disclosure also provides a method of diagnosing a neural condition or disease in a subject in need thereof using the neural device as disclosed herein.
  • the method may comprise a step of connecting the neural device to a brain of the subject.
  • the neural device may be connected to the brain of the subject via an end of the probe, which has the rGO fiber exposed.
  • the probe may be implanted into the cortex, hippocampus, and/or thalamus.
  • the cortex may be an auditory and/or visual cortex.
  • the neural device may be connected to a cortical laminae in the auditory cortex.
  • the method may comprise a step of collecting neural signals obtained from the subject.
  • the neural signals may be obtained by stimulating the subject visually or by sound.
  • the signals may then be analysed using principal component analysis. In this regard, the signals may be categorised into single unit events based on the unequal response to different stimuli.
  • the neural device is connected to the subject for about 5 days to about 6 months. In other embodiments, the duration is for at least 1 month.
  • High-density single-unit recording among closely-spaced neurons over long durations is crucial for understanding the cellular-level functional architecture of the brain.
  • Existing brain probes laterally sample neurons in the same cortical layer far more sparsely than neuronal density, preventing precise interrogation of neuron-to-neuron functional connectivity, and suffer from long-term instability due to electrode surface modification.
  • a monolithic graphene-edge probe that achieves monthlong single-unit recording at 2000 electrodes/mm 2 , approaching cortical neuronal density. The probe directly interfaces with neurons through exposed nanoedges and electrolyte-filling nanotunnels constructed by self-aligned reduced graphene oxide nanoflakes.
  • the monolithic electrode structure ensures a low specific impedance of 20 M ⁇ ⁇ m 2 without any surface modification.
  • the improved density and impedance enable stable electrophysiological recording with high signal-to-noise ratio (> 20 dB) as well as discrimination of individual neuron spikes over 6 months.
  • the probe provides a tool for the precise interrogation of local microcircuitry at the neural density of the brain.
  • Optical readout techniques such as calcium imaging are limited by poor temporal resolution or low signal-to-noise ratio (SNR).
  • SNR signal-to-noise ratio
  • microelectrode arrays such as the Utah array, possessing an 100 ⁇ m interelectrode spacing, which is still much larger than the average neuron-to-neuron distance ( ⁇ 25 ⁇ m).
  • SNR signal-to-noise ratio
  • PEDOT:PSS poly(3,4- ethylenedioxythiophene) polystyrene sulfonate
  • nanoparticle-coated platinum/iridium-based tetrodes which are widely used for neural recording and triangulation of spike sorting, have limited effective lifetimes because of corrosion of the nanoparticle-based modification layer and associated cytotoxicity to surrounding tissue. These factors limit the long-term use of microelectronic probes in brain-machine interfaces and neuroscience studies, where high-quality and precise chronic interrogation of electrophysiological activities is essential.
  • a monolithic graphene nanoedge probe (NeuroEdge) that achieves a channel density of >2000 electrodes/mm 2 at ⁇ 25 ⁇ m interelectrode spacing, approaching cortical neuronal density, and long-term recording stability in the brain.
  • the probe integrates more than 16 channels within a diameter of 100 ⁇ m, comparable to human hair, using a self-assembly fabrication strategy.
  • the probe array was designed to be neuronal size in each channel with equivalent circular diameter of 12-15 ⁇ m and inter-channel spacing below cortical neuron-to- neuron distance (Fig. 2a-c, Fig. 3). This configuration allows each channel to access an overlapping volume of neurons, enabling the triangulation sorting of single neuron across multiple channels, which allows a high-quality single-unit discriminability compared to single-channel based spike-sorting. Besides, the array was designed to be less than 100 ⁇ m in cross-section for each bundle to minimize tissue displacement and implant-induced inflammatory response.
  • NeuroEdge array has a high density of more than 2000 electrodes/mm 2 in the intralaminar cortical plane, which is 4-fold denser than the tetrodes, 8 times higher than Neuropixels and 16 times higher than the Utah arrays across the lateral region of 100 ⁇ m x 100 ⁇ m (Fig. 2b and f).
  • the NeuroEdge has a significantly lower electrochemical impedance compared with bare metallic electrodes and modified electrodes (Fig. 2g).
  • the electrolyte-filled nanotunnel serves as an additional ionic conduction pathway in parallel with the electronic conduction pathway formed via stitched graphene nanoflakes. The ionic and electronic interactions between these two pathways are likely to further reduce the impedance of the monolithic electrode, which can be interpreted with a modified transmission line model.
  • the NeuroEdge electrode despite having a small size comparable to an individual neuron, can achieve a low impedance of ⁇ 0.2 M ⁇ at 1 kHz, which is 20-fold lower than platinum/iridium (Pt/Ir) electrodes, 5 times lower than PEDOT:PSS modified electrodes and beyond other modified electrodes with CNT, porous TiN and IrO? etc. (Fig. 2g, 6a-c).
  • Pt/Ir platinum/iridium
  • NeuroEdge offers high electrochemical stability by eliminating surface modifications such as PEDOT:PSS coating, porous TiN deposition or platinum nanoparticle plating, which are prone to delamination or degradation under chronic electrochemical and physiological stress.
  • NeuroEdge maintains a specific impedance around 20 M ⁇ ⁇ m 2 over 3000 min of current pulses at a charge density of 1 mC cm 2 , which is among the typical activation thresholds for cortical microstimulation.
  • PEDOT:PSS coated Pt/Ir electrode exhibits a transient increase of 2.3 times in impedance within 5 min under the same current pulsing condition.
  • NeuroEdge shows stable CV and EIS curves over 6 months (Fig. 6g and h).
  • the capacitive behaviour of NeuroEdge evidenced by the lack of redox peak in the CV curve, ensures minimal toxic side product from redox reaction at the electrode interface in a physiological environment. It is in agreement with reported results that carbon usually has much greater resistance to electrochemical corrosion than metals used in neural probes.
  • a single bare NeuroEdge fiber which is only 1.5% of human hair in axial area, can mechanically support a load 1000 times its own weight.
  • the tensile measurement shows a modulus of 19.5 ⁇ 3.4 GPa, and a bending stiffness of (4.0 ⁇ 0.4) x 10' 2 nN m 2 for NeuroEdge, which is 10-fold softer than the metallic electrode while still providing adequate mechanical strength for surgical procedures.
  • Fig. 7a, 8 We assessed the high-density and high-fidelity recording capability of NeuroEdge by implanting a 4-channel and a 16-channel ( ⁇ 100 ⁇ m in axial diameter) bundle into a mouse cortex, respectively (Fig. 7a, 8). The recordings were acquired at 4-day postimplantation. Each channel of NeuroEdge was registered with strong electrophysiological signals.
  • Fig. 7b shows a typical 10-min raw electrophysiological data from a 4-channel NeuroEdge, including both high-fidelity local field potential (LFP) and action potential (AP). The spiking signal after band-pass filtering at 300-9000 Hz is consistently stable (Fig. 7b). The average AP amplitude is 150 ⁇ V.
  • the combination of superior electrode density and high SNR enable NeuroEdge to simultaneously oversamples the same neurons with higher fidelity, particularly lower- amplitude distant neurons, and more channels compared with existing neural probe, such as tetrode and Neuropixel.
  • NeuroEdge has higher discriminability of neighbouring neuron population for investigating microcircuit functional organization.
  • the independently extracted individual spikes from each channel are shown in Fig. 7h and i, which are represented in raster plots. It shows simultaneous recording of individual neurons on all four channels, evidenced by similar cross-channel firing patterns, laying the foundation for highly accurate triangulation.
  • the spike clustering projections based on the 4-channel recording are illustrated in Fig. 7j, showing 2.25 ⁇ 0.75 single units per channel on average.
  • PCA Principal components analysis
  • a 16-channel NeuroEdge was used for hours-long electrophysiological recording.
  • Representative LFP (Fig. 71) shows similar wave patterns across all channels and the AP exhibits close spike timing, indicating localized high-density electrophysiological recording.
  • the ⁇ 100- ⁇ m array identified more than 40 well-isolated single units across multiple channels. Each unit has no overlap in spike timings (Fig. 7m) and shows distinct average waveforms (Fig. 7n), suggesting an absence of duplicated units.
  • the 16-channel NeuroEdge allows for a laminar recording at density of more than 4000 units per electrode mm 2 , approaching the neuronal density.
  • Frequency is the main acoustic parameter that is thought to be topographically mapped in most auditory cortex.
  • the existence of local heterogeneity in frequency preference across neighboring neurons within a cortical layer remains inconclusive or incomplete. This is largely because there is a lack of tools or methods that combine both high temporal resolution and high lateral spatial resolution (matching cellular density).
  • We implanted NeuroEdge (with sub-25 ⁇ m spatial resolution) into mouse auditory cortex to record closely spaced neurons in response to tone frequencies spanning 0.5-42 kHz in Vi octaves (Fig. 9a and 10).
  • the recorded local field potential shows an increased negative deflection near tone onset, which is sustained for the entire 100-ms stimulus window (Fig. 9b).
  • the filtered spiking signal reveals that the neurons have unequal responsivity to different sound frequencies, with distinct variation in firing rate from the highest of 16 Hz to almost zero (Fig. 9c).
  • Fig. 9d illustrates the characteristic frequency response in peristimulus raster plot from 12 representative sorted single units. It shows an average 2.7-fold increase in spike density, computed as the ratio of peak over baseline, during the stimulus window. However, all units exhibit different spike density increases ranging from 1 to 5 (Fig. 9d- f). For instance, unit 7 (N7) shows a 4.4-fold spike density increase, while unit 1 (Nl) exhibits almost no change. The diversity in spike density changes of near-by neurons within the stimulus window indicates that the near-by neurons are not equally activated by the external acoustic stimulus.
  • each unit has its own frequency preference. For instance, units 7 and 12 (N7, N12) show strong response to low frequencies ( ⁇ 4 kHz), while units 6, 10 and 11 (N6, N10, Nil) show elevated response to higher frequencies (>4 kHz), and unit 9 (N9) presents response both below and above 4 kHz (Fig. 9g).
  • Such frequency preference suggests heterogeneous responsivity among near-by neurons, agreeing with previously reported results using optical methods.
  • NeuroEdge probes demonstrate the capability of NeuroEdge probes to discriminate substantial heterogeneity of neuronal tuning at cellular level within a localized cortical region which has been previously understudied, and reveal the dynamic functional connectivity with frequency specificity among neighboring neurons within a functional microcircuit in the auditory cortex.
  • NeuroEdge provides a versatile tool for investigating the neural basis of behavior, extending beyond organization across the depth of the cortex to within the intralaminar cortical layer.
  • FIG. 11a Representative electrophysiological recordings at month 1 (Ml), month 3 (M3), and month 6 (M6) all exhibit prominent and high-amplitude action potential.
  • Fig. 11b The SNR was consistently above 20 dB with a noise level below 7 ⁇ V across the entire experimental duration of 6 months (Fig. 11c). The average spike amplitude remained stable.
  • NeuroEdge with size comparable to neuron also maintained a low impedance in vivo of around 0.5 M ⁇ over 6 months (Fig. lid).
  • the sustained high SNR and low impedance of NeuroEdge enable neural activities monitoring with high-fidelity over an extended period without deterioration in yield and quality.
  • NeuroEdge that can measure the longitudinal neuronal activity of closely spaced neurons.
  • NeuroEdge can overcome two key limitations of state-of-the-art silicon probes such as NeuroPixels.
  • NeuroPixels can directly interface with neurons through exposed nanoedges and electrolyte-filling nanotunnels constructed by self-aligned reduced graphene oxide nanoflakes, enabling a channel density of >2000 electrodes/mm 2 that approaches cortical neuronal density.
  • NeuroEdge exploits graphene edges to achieve a low specific impedance of 20 M ⁇ ⁇ m 2 without relying on unstable surface modification, thereby achieving long-term stability over six months.
  • NeuroEdge enables electrophysiological recording with a high signal-to-noise ratio (>20 dB), with the ability to distinguish individual neuron spikes over six months.
  • NeuroEdge resolves heterogeneities in the acoustic frequency response and reveals functional connectivity among neighboring neurons in the mouse auditory cortex, providing a fundamentally new brain probe that can precisely interrogate local microcircuitry at the neural density of the brain.
  • NeuroEdge which has the capability of millisecond temporal resolution, cellular-scale spatial resolution, and monolithic-design- enabled chronic stability, will significantly contribute to the study of neural microcircuits and their functional architecture in many brain regions, especially those with dense neuron nuclei or fine-grained functional division, such as the hippocampus, visual cortex and thalamus. It can be broadly applied to investigate functional connectivity and plasticity in microcircuits and how they are affected by neurological disorders. The capabilities of NeuroEdge also position it as a highly promising solution for clinical bioelectronics applications. Particularly, it could facilitate the develo ⁇ ment of braincomputer interfaces, where precise control of closely spaced neurons is required for the restoration of fine motor and sensory functions. Further improvements will be dedicated to exploring new configurations with more channels to cover larger brain areas. Additionally, we envision that the NeuroEdge can be used for detection of both action potentials and neurochemical concentrations by leveraging the inherently exceptional electrochemical activity of graphene edges.
  • Aqueous dispersion of graphene oxide (GO) (Suzhou Tanfeng Graphene Technology CO., LTD, 10 mg ml 1 ) was diluted to 6 mg mF 1 with water. Then, 85 mg of ascorbic acid (Sigma-Aldrich) was added to 1 ml of the GO dispersion and ultrasonicated for 5 min. The resulting GO/ascorbic acid mixture dispersion was loaded into glass capillary tubing, sealed with Teflon tape, and reacted at 95 °C for 1 h, resulting in a reduced graphene oxide (rGO) gel fiber in the capillary tubing.
  • GO graphene oxide
  • rGO reduced graphene oxide
  • the gel fiber was extracted, and soaked in a water bath for 10 min, followed by changing the water and repeating for another 10 min.
  • the gel fiber was then placed on a flat plastic polystyrene substrate to constrict the self-assembly alignment of rGO fiber during water-evaporation driven drying.
  • the diameter of the rGO fiber was determined by the inner diameter of the glass capillary tubing, with a diameter of 10-15 ⁇ m synthesized from 200 ⁇ m of capillary tubing.
  • a 50 ⁇ m stainless steel wire (793400, A-M system) was connected to the fiber using silver nanowire ink.
  • the bare rGO fiber was coated with a 1-2 ⁇ m thick of parylene C to insulate the side wall of the fiber.
  • the tip of the fiber was exposed via a strain-concentrated breaking.
  • optimal cutting temperature (OCT, PVA solution) compound was coated on the surface of parylene C-coated rGO fiber, and frozen in liquid nitrogen.
  • the OCT liquid solidified at the low temperature, encapsulating the entire rGO fiber, leaving a short fiber exposed at the tip end.
  • the sample was referred to as a graphene edge probe (NeuroEdge).
  • the rGO fibers coated with parylene C were bundled with elastoca pi llary self-assembly by withdrawing the free-moving end of the fibers from the bath of diluted SU-8 solution (SU-8 2002, Kayaku Advanced Materials, Inc.). The assembly was baked in oven at 100 oC for 10 min to evaporate the solvent in SU-8 and then exposed to 365 nm UV lamp for 5 min to bond the rGO fibers permanently to a thin array. The tip end of the array was exposed using strain-concentrated breaking method as described in the previous section.
  • the stainless steel wire that is connected to NeuroEdge was tightened inside receptacle (ED85100-ND, Digi-key) by putting back the original pin. Two holes in receptacle were left to connect with the reference and ground wires for in vivo electrophysiological recording.
  • the moving stage inside trimmer potentiometer was exposed to place the PI tubing part of the NeuroEdge array which was secured with adhesive (Loctite 404, Ted Pella). To create a strong bond of NeuroEdge on the moving stage, the adhesive was left overnight to allow full curing.
  • the NeuroEdge tip end was adjusted to pass the bottom of the potentiometer by 1-2 mm to facilitate implantation into the brain.
  • the receptacle was secured on the top of the trimmer potentiometer. Moreover, the stainless steel wires were left bend to facilitate the moving down of the NeuroEdge.
  • the size and morphology of the NeuroEdge tip end were analyzed with a Field-emission Scanning Electron Microscopy (FESEM) (FEI Verios460).
  • FESEM Field-emission Scanning Electron Microscopy
  • the NeuroEdge array was attached to a 70° SEM stub with its tip slightly higher than the top side and grounded using silver ink on the bottom end of NeuroEdge to ensure electrical connection to double-sided carbon conductive tape adhered on the stub.
  • the sample was not coated with additional Pt or Au to allow detection of the nanoscale graphene edges, the nanotextures and the nanotunnels.
  • the sample holder stage was tilted to allow for the tip end to be viewed vertically or at a 45-degree angle relative to the FESEM detector.
  • Electrochemical impedance spectroscopy was conducted over a frequency range of 100 to 100k Hz with a perturbation of 10 mV RMS sinusoidal excitation voltage (Biologic SP200). Cyclic voltammetry (CV) curves were acquired at a sweep rate of 100 mV/s between negative and positive potential limits of -1.6 and 1.3 V versus Ag/AgCI.
  • the current pulse was delivered by applying a rectangular charge- balanced biphasic current pulse with the cathodic phase applied first which is a common practice for in vivo stimulation(Gamry reference 600+).
  • the interpulse delay between cathodal and anodal phase was set to 50 us to visualize the interpulse potential.
  • the pulse width was 1 ms, and the charge density was controlled to be 1 mC cm-2.
  • the long-term stability study of NeuroEdge was performed by soaking it in 150 mM PBS for half a year.
  • a metal electrode was electroplated with poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (PEDOT:PSS) from a 0.1 M PSSNa (Sigma-Aldrich) aqueous solution with 0.01 M EDOT (Sigma-Aldrich) under galvanostatic conditions using an Ag/AgCI electrode as a reference and a platinum wire as a counter.
  • the current density was controlled to be around 0.4 mA/cm2 to allow slow electrodeposition for enhanced adhesion and total electrodeposition charge density was around 600 mC/cm2 to minimize the risk of PEDOT:PSS layer cracking.
  • the electrode was immersed in DI water for 15 min to wash away any weakly attached PEDOT:PSS on the surface.
  • the electrochemical performance was characterized using EIS, CV and CIC measurements, with potential limits of -0.6 to 0.8 V versus Ag
  • mice All animal procedures are approved by the Institutional Animal Care and Use Committee (IACUC) at National University of Singapore and were conducted in accordance with NACLAR guidelines.
  • IACUC Institutional Animal Care and Use Committee
  • mice were anesthetized via intraperitoneal injection of ketamine/xylazine mixture in saline (100 and 10 mg per kg mouse, respectively), and subcutaneous injection of analgesic buprenorphine (0.1 mg per kg mouse). The depth of anesthesia was monitored with the toe pinch reflex. The top of the head of the mice were shaved, and then placed in a stereotaxic frame (David Kopf Instruments), with ear bars securing the head and gel applied on the eyes to keep them moist. The exposed skin on the top of the head was cleaned with alcohol and iodine, and cut along the midline to expose the skull.
  • the drive was then lowered down to the brain with the NeuroEdge tip facing towards the coordinates relative to bregma of -1.9 mm AP, 1.4 mm ML, 0.5-1.0 mm DV.
  • the dura was partially removed surrounding the NeuroEdge penetration point, and sterile saline was applied to keep the craniotomy moist.
  • the drive was fixed onto the skull using cement (C&.B Metabond, Parkell Inc.) with Kwik-Sil (Word Precision Instruments Inc.) applied to the craniotomy area. After the cement and Kwik-Sil had complete cured, the insertion tool was removed, and a plastic tubing was placed outside the drive to protect it.
  • mice with implantation were returned to their cage after waking up from anesthesia. The mice were closely monitored for 3 days after surgery, and the NeuroEdge was moved down around 100 ⁇ m on the third day.
  • An Intan recording system which includes an RHD USB interface board and RHD headstage with an RHD2132 chip (Intan Technologies), was connected to the receptacle connector of the drive using an in-house adaptor.
  • the mice were maintained at an anaesthetized state using 1-2 % isoflurane (Baxter Healthcare Corporation). This helped to minimize the trial-to-trial variation arising from the effects of attention, arousal, and body movements to facilitate the performance investigation of electrode itself.
  • the mice were placed inside a Faraday cage, and the cage was grounded via the ground connector in the USB interface board.
  • the RHX Data Acquisition Software was used to record the local field potential (LFP) and action potential (AP). The data acquisition was performed in the frequency width of 0.1- 9000 Hz with a sampling rate of 30 kHz, and the in vivo impedance was measured at 1 kHz before recording.
  • the NeuroEdge was implanted into the left-side auditory cortex (AC) at coordinates relative to bregma of -2.2 mm AP, -4.0 mm ML, 0.6 mm DV, using the same recording and implantation setup as previously described.
  • Pure tones were generated with a sound card (Sound Blaster Audigy Rx) and Matlab software, and amplified via an audio amplifier (Samson Servo 120a) before being presented to the mice's ear via loudspeaker (XT25TG30-04, Peerless by Tymphany) at a distance of 15 cm.
  • the output of the speaker was measured with a free-field microphone (4939-A- 011, Briiel & Kjaer) placed at the same location as the mouse's head facing the speaker.
  • the output was read in dB sound pressure level (SPL).
  • SPL sound pressure level
  • the pure tones were arranged from 0.5 to 42 kHz in 1/12 octave steps.
  • the duration was 100 ms including 6 ms cosine2 onset/offset ramp and inter-tone interval of 1300 ms.
  • the click channel from the same connection ports on the sound card with the same onset and offset timeline settings as the tone channel was directly connected to an analog-to-digital converter (ADC) port in the Intan USB interface board and the voltage of the acoustic click channel was recorded. Recording sessions lasted up to 2 h. To allow investigation of electrode performance only, the mice were kept under shallow anesthesia, responding with a light withdrawal reflex to tail or toe pinch but remaining quiet and motionless without indication of pain or distress. Deep anesthesia was maintained with a higher concentration of isoflurane, and the mice showed no response to tail or toe pinch.
  • ADC analog-to-digital converter
  • the signal-to-noise ratio (SNR) was expressed in decibels from 20 x In (RMS(S)/RMS(N)), where S is the waveform peak and N is the noise of the recording after removing the detected spikes.
  • the SNR was evaluated based on the average spike amplitude over a 10-min recording.
  • For sorting and clustering into OFS the raw recording data was converted to a binary data format and imported to OFS. The data was then filtered with 2-pole Butterworth high- pass filter at a cutoff frequency of 500 Hz. Spikes at different channels were detected and extracted in a 1.13 ms time window with a prethreshold period of 0.26 ms and dead time of 1.03 ms.
  • mice were implanted with NeuroEdge and NeuroEdge with thin coating of 100 nm Au/1-2 ⁇ m PEDOT:PSS for fair comparison. After 3 months of implantation, the mice were perfused with saline and 4% paraformaldehyde (PFA) (Sigma-Aldrich) in 0.1 M PB. The fixed brain was extracted and post-fixed in 4% PFA overnight at 4 oC, then immersed in 30% sucrose in 0.1 M PB until the brain sunk to the bottom. The brain was frozen with OCT and sectioned to 45 ⁇ m lateral slices at -20 oC using a Cryostat (Leica CM3050s).
  • PFA paraformaldehyde
  • the individual serial sections were collected into 96-well plates filled with cryoprotection solution and stored at -20 oC until use.
  • the slices were washed with tris-buffered saline (TBS) twice and incubated in blocking solution containing 0.2% Triton X-100 (Sigma- Aldrich) and 3% donkey serum (GeneTex GTX73205) in TBS for 1 h at room temperature.
  • TBS tris-buffered saline
  • the slices were then incubated with primary antibodies, including mouse anti-NeuN (MAB377, 1 :100, Sigma), chicken anti-GFAP (ab4674, 1 : 1000, Abeam) and rabbit anti-Ibal (019-19471, 1: 1000, Wako) in blocking solution for 1 day at 4 oC.
  • the slices were washed with TBS twice for 10 min each, and incubated with secondary antibodies at room temperature for 4 h, including donkey anti-mouse Alexa Fluro 647 (715-605-151, 1:250, Jackson ImmunoResearch), donkey anti-chicken Alexa Fluro 488 (703-545-155, 1 :250, Jackson ImmunoResearch) and donkey anti-goat cy3 (705-165- 147, 1 :250, Jackson ImmunoResearch).
  • the slices were mounted on polylysine coated glass slide (Epredia Polysine) with Antifade Mounting Medium (Vector Laboratories, H-1200-10) and coverslips. Images were acquired using a confocal laser scanning microscope with a 20x air objective lens (Olympus FV3000). Quantification of expressed area was analyzed with ImageJ.

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Abstract

La présente divulgation concerne une électrode neuronale comprenant au moins une sonde, la sonde comportant un cœur de fibre d'oxyde de graphène réduit (rGO) et une gaine encapsulant le cœur de fibre de rGO ; le cœur de fibre de rGO étant caractérisé par un diamètre de section transversale d'environ 5 µm à environ 100 µm ; une extrémité du cœur de fibre de rGO étant sensiblement exposée à partir de la gaine et étant caractérisée par un diamètre de section transversale d'environ 5 µm à environ 100 µm ; et l'extrémité du cœur de fibre de rGO comprenant une morphologie nano-texturée afin d'en augmenter la superficie. La présente divulgation concerne également un dispositif neuronal, des procédés de fabrication et des méthodes d'utilisation du dispositif neuronal associé.
PCT/SG2024/050237 2023-04-11 2024-04-08 Électrode neuronale, dispositif neuronal et leurs procédés de fabrication Pending WO2024215255A1 (fr)

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US20220305253A1 (en) * 2018-11-21 2022-09-29 Board Of Regents, The University Of Texas System Methods of making and bioelectronic applications of metalized graphene fibers

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JI QINGHUA, ZHAO XU, LIU HUIJUAN, GUO LIN, QU JIUHUI: "Facile Synthesis of Graphite-Reduced Graphite Oxide Core–Sheath Fiber via Direct Exfoliation of Carbon Fiber for Supercapacitor Application", APPLIED MATERIALS & INTERFACES, vol. 6, no. 12, 25 June 2014 (2014-06-25), US , pages 9496 - 9502, XP093225374, ISSN: 1944-8244, DOI: 10.1021/am5018682 *
JUNGGEON PARK; JIN JEON; BYONGYEON KIM; MIN SUK LEE; SIHYEON PARK; JUHAN LIM; JONGDARM YI; HWANGJAE LEE; HEE SEOK YANG; JAE YOUNG : "Electrically Conductive Hydrogel Nerve Guidance Conduits for Peripheral Nerve Regeneration", ADVANCED FUNCTIONAL MATERIALS, vol. 30, no. 39, 12 August 2020 (2020-08-12), DE , pages 1 - 14, XP072414396, ISSN: 1616-301X, DOI: 10.1002/adfm.202003759 *
NICHOLAS V. APOLLO; MATIAS I. MATURANA; WEI TONG; DAVID A. X. NAYAGAM; MOHIT N. SHIVDASANI; JAVAD FOROUGHI; GORDON G. WALLACE; STE: "Soft, Flexible Freestanding Neural Stimulation and Recording Electrodes Fabricated from Reduced Graphene Oxide", ADVANCED FUNCTIONAL MATERIALS, vol. 25, no. 23, 4 May 2015 (2015-05-04), DE , pages 3551 - 3559, XP072291277, ISSN: 1616-301X, DOI: 10.1002/adfm.201500110 *

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