AMYLOID FIBERS BASED ELECTRODES

Abstract

Amyloid fibers-based electrodes and apparatuses comprising the same. Additionally, methods for manufacturing amyloid fibers-based electrodes.

Claims

1. An electrode comprising at least one electrically conducting layer of amyloid fibers.

2. The electrode according to claim 1, wherein the layer of amyloid fibers covers a surface or an extremity of the electrode that is positioned in contact with a biological tissue.

3. The electrode according to claim 1, wherein the electrode provides a bidirectional communication between a biological tissue and a medical apparatus.

4. The electrode according to claim 1, wherein the layer of amyloid fibers is in the form of a film.

5. The electrode according to claim 1, wherein the layer of amyloid fibers has a thickness not exceeding 1000 μm, when the layer of amyloid fibers is in an aqueous environment.

6. The electrode according to claim 1, wherein the amyloid fibers are aggregates of monomers of a protein or a peptide selected from the group comprising: HET-s prion domain, a-lactalbumin, Lysozyme, Aβ1-42, htau, α-synuclein, TTR (V30M), IAPP, β-Lacloglobulin, HAS, Insulin, CSNNFGA, NNLAIVTA, CsgA, PMEL17, Ure2p, Orb2 (CPEB), SH3 domain PI3 kinase, Acylphosphatase, Prion Protein, Islet Amyloid PolyPeptide, Prolactin, Galectin 7, Corneodesmosin, Lactadherin, Kerato-epithelin, Lactoferrin, Semenogelin, Enfuvirtide, APP, Apolipoproteins, Gelsolin, β2-microglobulin, Transthyretin, TasA, FapC, SP-C, LECT-2, Proteins S100A8/A9, Huntingtin exon-1, Fragment of Immunoglobulin Heavy Chain, Fragment of Immunoglobulin Light Chain, ABri Peptide, ADan Peptide, N-Terminal fragments of Serum amyloid A protein, ChpD-H (Streptomyces coelicolor), MSP2, Spidroin (spider silk), Sup35p, Rnq1p, Swi1p, Cyc8p and MspA.

7. The electrode according to claim 1, the electrode being implantable, preferably in a nervous tissue.

8. The electrode according to claim 7, the nervous tissue being selected among the group comprising: brain, spinal cord or nerves.

9. An apparatus configured to detect, record, stimulate, simulate, or a combination thereof, electrophysiological signals within living organisms comprising at least one electrode as defined in claim 1.

10. The apparatus according to claim 9, the apparatus comprising two or more electrodes as defined in claim 1.

11. The apparatus according to claim 9, the apparatus being a medical device.

12. A method of detecting, recording, stimulating, simulating or a combination thereof, electrophysiological signals with the apparatus of claim 9, the method comprising: applying the electrode to a biological tissue; and forming an electrically conducting interface between the biological tissue and the apparatus with the at least one layer of amyloid fibers.

13. (canceled)

14. A method for manufacturing an electrode comprising: applying at least one electrically conducting layer of amyloid fibers on a surface or an extremity of an electrode.

15. The electrode according to claim 1, wherein the layer of amyloid fibers has a thickness not exceeding 100 μm when the layer of amyloid fibers is in an aqueous environment.

16. The electrode according claim 1, wherein the layer of amyloid fibers has a thickness not exceeding 10 μm when the layer of amyloid fibers is in an aqueous environment.

17. The electrode according to claim 1, wherein the amyloid fibers are aggregates of monomers of a protein or a peptide selected from the group comprising HET-s prion domain, α-lactalbumin and Lysozyme.

Description

FIGURE LEGENDS

[0089] FIG. 1. Molecular model of 10 subunits of HET-s amyloid fibers. Attached water molecules are indicated by black beads.

[0090] FIG. 2. Conductivity dependence with ambient relative humidity. Example taken from alpha-lactalbumin fibers dry films. RH: relative humidity.

[0091] FIG. 3. Electrical setup scheme used in the experiments. F=amyloid fiber film. R=100 kΩ resistor. G=Generator. O=Oscilloscope. 1 & 2 illustrate measurements points for two different oscilloscope channels.

[0092] FIG. 4. Examples of α-lactalbumin 500 μM dry film responses (output current-upper graphs) to neuron-like signal stimulations (input current-lower graphs). A. Spike duration: 1 ms, Min=−60mV/Max=20 mV, interspike duration: 4 ms to 40 ms. B. Zoom on A.

[0093] FIG. 5. Examples of lysozyme 500 μM dry film responses (output current-upper graphs) to neuron-like signal stimulations (input current-lower graphs). A. Spike duration: 1 ms, Min=−60mV/Max=20 mV, interspike duration: 4 ms to 40 ms. B. Zoom on A.

[0094] FIG. 6. Examples of HET-s 280 μM dry film responses (output current-upper graphs) to neuron-like signal stimulations (input current-lower graphs). A. Spike duration: 1 ms, Min=−60mV/Max=20 mV, interspike duration: 4 ms to 40 ms. B. Zoom on A.

[0095] FIG. 7. Graph representing the current transferred from pulses (100 mV) with variable duration (abscissa) for dry films made from 3 types of amyloid fibers (α-lactalbumin, lysozyme or HET-s).

[0096] FIG. 8. Graph representing the ratio (in %) between the measured current and the current without deformation (maximum) from pulses (100 mV) with variable duration (abscissa) for dry films made from 3 types of amyloid fibers (α-lactalbumin, lysozyme or HET-s).

[0097] FIG. 9. Macrophotography of rat brains with mandrels (M) implanted in the right cerebral hemisphere: brain having received a 316L stainless steel mandrel (a), brain with stainless steel mandrel+amyloid fibers (e). Microphotography of rat brain sections stained with cresyl violet (Niss1 staining) after implantation of a 316L stainless steel mandrel (b) or a stainless steel mandrel coated with amyloid fibers (f). Microphotography of sections of rat brains labeled with the antibodies directed against the GFAP and Iba-1 proteins: with 316L stainless steel mandrels (respectively c and d for GFAP and Iba-1) and 316L stainless steel mandrels+amyloid fibers (respectively g and h for GFAP and Iba-1).

[0098] FIG. 10. Setup for action potential recording. A. Microelectrode arrays (MEA). B. Photography of the electrode-recording site below the neuronal culture.

[0099] FIG. 11. A. Example of action potential recording with the setup shown in FIG. 10. Diamond marks represents the locations were pulses have been detected. B. Pulse-like signals observed in the same range of voltage than regular modern electrode recordings, and with a pulse morphology as expected for typical action potential with polarization and depolarization phases.

EXAMPLES

Example 1. Artificial Neuron-Like Spike Detection by Amyloid Fiber Film

Materials

[0100] Oscilloscope: Keysight InfiniiVision DSOX1102A/G 2-Channel Oscilloscope, 70/100 MHz, 2 GS/s

[0101] Generator: Keysight ARB GENERATOR 33512B, 20MHz, 2 Output, 16 bits, 160 M Samples/s

[0102] Electrode: Novocontrol Interdigitated Gold Electrode, BDS 1410-15-150

[0103] Temperature/Relative Humidity Sensor: Sensirion SHT75

[0104] Diluted mother solutions of amyloid fibers: [0105] Lysozyme: 20 g/L protein [1399 μM]. 3 mM HCl (pH=2.5)/90 mM NaCl

[0106] HET-s: 289 μM protein. 0.1 mM HCl (pH=4)/No salts

[0107] α-lactalbumin: 35 g/L protein [2500 μM]. 10 mM HCl (pH=1.7)/No salts

Method

[0108] The goal of the experiment was to inject voltage patterns and record output current passing through amyloid fiber films.

[0109] Because of the nature of amyloid fibers, charges carriers responsible for charge transport are protons and ions: ions enter the fibers core through their terminal ends or their lateral faces and are guided along its axis. Protons are carried along the fiber by another mechanism. Indeed, amyloid fibers possess water channels resulting from their sophisticated hierarchical structures (FIG. 1). When the percolation threshold is reached, protons are able to “jump” from one building block to the other (Grothuss mechanism) until the end of the fiber. By the combination of those two phenomena fiber to fiber, a current is created to form a closed-loop circuit.

[0110] 200 μL of buffer solution was applied onto the interdigitated electrode surface to wet it. The protein mother solution was diluted to achieve a concentration of 500 μM (except with HET-s).

[0111] After one hour, the buffer solution was removed and 200 μL of the diluted protein solution was dropped onto the electrode surface. It was left to dry for one or two days until water has evaporated.

[0112] Once the thin film of protein has dried (thickness of ca. 10 μm), the electrode was placed into a closed box. Because amyloid fiber conductivity depends highly on ambient humidity (FIG. 2), holes were made into the box to apply a continuous humid airflow. Temperature and ambient moisture were monitored by a sensor.

[0113] To achieve it, a 100 kΩ resistor was directly connected to the film. By measuring voltage drop at its terminal, the input current of the resistor can be determined (Ohm's Law), which is also the transferred current of the amyloid fiber film.

[0114] Keysight generator+terminal was connected to one end of the electrode, −terminal (which is linked to ground) to one end of the resistor. Keysight oscilloscope possess two channels: the first channel was branched over the resistor (output signal), the second channel over the generator (to verify and record the input signal). The whole setup scheme is represented FIG. 3.

[0115] Neuron-like pulse patterns were created using a handmade Matlab program coupled with BenchLink Waveform Generator software for signal integration (within Keysight generator).

[0116] Spike parameters were chosen carefully in order to have to the same structure as in vivo neuron action potentials. The goal was more to have the same generic properties (frequency, amplitude, patterns) than to recreate perfect in vivo signals.

[0117] Spike parameters are as follows: [0118] Amplitude: 80 mV+Offset −60mV [0119] Pulse Width: 1 ms [0120] Inter-Pulse Width: Ranging from 2 to 6 ms [0121] Noise: White noise added [0122] Recurring Patterns: Active/Passive Behavior

[0123] Once the signal loaded in the generator, ambient humidity was set at ca. 70% in order to have a good protonic conduction. No temperature change needs to be done; the experiment was conducted at ambient temperature.

Results

[0124] The results obtained with the films of amyloid fibers of α-lactalbumin (FIGS. 4, 7 and 8), of lysozyme (FIGS. 5, 7 and 8) and of HET-s (FIGS. 6, 7 and 8) demonstrate that amyloid fibers are able to conduct and detect neuron-like signals. Indeed, these results show that the amyloid fibers are able to receive and transmit such signals with minimal alteration of the imposed signal patterns.

[0125] The protein-based active layers did not reach their limits during the above-described first experiments. Indeed, further tests were performed to determine their temporal detection limit. It was observed that amyloid fibers dry films were able to detect spikes down to the microsecond timescale (for a Vpp=80 mV; Vpp total amplitude of the spike).

[0126] Moreover, the charge transport properties of amyloid fibers films can be fine-tuned by adjusting selected parameters, relative humidity (RH) being the most important one. Because their conductivity as function of relative air humidity has an exponential dependence, a small change in ambient moisture will translate into a substantial variation of its conductivity level. These features lead to an active layer with an easily tunable conductivity. All the previous experiments shown above were made with about 70% relative humidity (RH). For example, if one would like to increase the sensibility of the film, tuning the RH to 75% would double the output amplitude (and so signal-to-noise ratio). Controlling moisture around a device is not an easy task, but neuronal extracellular medium consists in 99% of water. This should allow the film to conduct enough to have an ultra-high resolution.

[0127] The resistance of the amyloid fiber film to degradation is also an important parameter. It was tested by forcing a 300 Hz, 1 ms continuous spike signal (⅓ duty cycle) over long period of time [˜20k spikes/min]. After 4 hours, no distinct changes in amplitude output were noticed [˜6 million spikes]. The day after, the film was again submitted to the same test and showed no change.

Example 2. Biocompatibility of Amyloid Fibers

Material

[0128] The biocompatibility of amyloid fibers was assessed using an in vitro mammalian cell culture. The study was based on the requirements of the International Organization for standardization 10993, Biological Evaluation of Medical Devices, Part 5 (2009): Tests for in vitro Cytotoxicity and Part 12 (2007) : Test article preparation and reference materials.

[0129] Mammalian cell culture monolayer consisting of L-929 mouse fibroblast cells was used. 200 μL of Amyloid protein solution (500 μM) were mixed with 2 mL of PBS 1× for hydrogel formation. Amyloid fibers-based hydrogels were then washed three times with a PBS solution. A single preparation of the amyloid fibers-based hydrogel was extracted in single strength Eagle Minimum Essential Medium (EMEM10) at 37±1° C. for 24±2 hours. A negative control, reagent control and a positive control were similarly prepared. Following extraction, triplicate monolayers of L-929 mouse fibroblast cells were dosed with the extracts (100% for the controls and 100%, 50%, 10% and 1% for the amyloid fibers-based hydrogel) and incubated at 37±1° C. for 24±2 hours in presence of 5±1% CO.sub.2. Following incubation, 20 μL of the MTS/PMS solution, prepared just before use, were dispensed in each well and incubated during 120-135 minutes at 37±1° C. in 5±1% CO.sub.2.

[0130] The percent viability for the amyloid fiber-based hydrogel was determined as compared with the reagent control. A decrease/increase in the number of living cells results in a decrease/increase in the metabolic activity in the sample. This decrease/increase directly correlates to the amount of brown formazan formed, as monitored by the optical density at 492 nm.

[0131] The hydrogel of amyloid fibers was prepared based on a ratio of 3 cm.sup.2/mL. The hydrogel was used at 100%, 50%, 10% and 1%.

[0132] The reagent control article was Eagle Minimum Essential Medium (EMEM1X) supplemented with 10% fetal bovine serum, 1% glutamine.

[0133] The negative control (High density polyethylene sheet/Hatano Research Institute) was prepared based on a ratio of 6 cm.sup.2/mL. A single preparation of the material was made and extracted at 37±1° C. for 24±2 hours. The negative control was tested at 100%.

[0134] The positive control was segmented polyurethane film containing 0.1% zinc diethyldithicarbamate (ZDEC/Hatano Research Institute) and was prepared based on a ratio of 6 cm.sup.2/mL. A single preparation of the material was made and extracted using the same conditions as described for the amyloid fibers-based hydrogel. The positive control was used at 100%.

[0135] The percent viability is compared to the reagent control by using the following formula:

Percent Viability=100×OD.sub.c492e/OD.sub.c492rc

[0136] ODc (corrected OD)=OD.sub.Amyloid fibers/negative control/positive control−OD.sub.CM

[0137] OD.sub.c492rc is the mean value of the measured optical density of the Amyloid fibers or negative control or positive control extract.

[0138] OD.sub.c492rc is the mean value of the measured optical density of the reagent control.

[0139] OD.sub.CM is the mean value of the measured optical density of culture medium without cells (CM)

Results

[0140] All system suitability criteria were met indicating a valid test assay.

[0141] All Amyloid fibers extracts showed no cytotoxic potential to L-929 mouse fibroblast cells as shown in Table 1.

TABLE-US-00001 TABLE 1 Percent viability of amyloid fibers extracts, negative control and positive control and cytotoxic potential of Amyloid fibers extracts. Percent Viability of Material Control Articles System Suitability Positive control (100%)  3% Met criteria Negative control (100%) 97% Met criteria Percent Viability of Material Amyloid fibers Cytotoxic Potential Amyloid fibers (100%) 103% No cytotoxic potential Amyloid fibers (50%) 100% No cytotoxic potential Amyloid fibers (10%) 100% No cytotoxic potential Amyloid fibers (1%)  97% No cytotoxic potential

Example 3. In Vivo Testing of Amyloid Fiber

Material

[0142] The experimental protocol was as follows:

[0143] This in vivo study was carried out according to the ISO 10993-6: 2016 standard which specifies the test methods for evaluating the local effects after implantation of biomaterials intended for use in medical devices.

[0144] The implantation surgery was performed according to the following protocol:

[0145] The implanted samples were of 2 types:

[0146] 1. cylindrical-shaped mandrels in 316L stainless steel (length of 6 mm and diameter of 800 pin), n=3,

[0147] 2. cylindrical-shaped mandrel in 316L stainless steel coated with amyloid fibers, n=6.

[0148] The first step consisted of performing a craniotomy according to the following steps: [0149] induction of the animal by gas anesthesia, [0150] installation of the animal in a Kopf type stereotaxis frame, [0151] midline incision of the scalp and resection of the subcutaneous tissue, [0152] drilling of the cranial box (diameter approximately 2 mm), [0153] incision of the dura, [0154] implantation of mandrels within the right cerebral hemisphere in the caudate nucleus/putamen (mandrel inserted 5 mm high in the cerebral parenchyma), [0155] sealing performed with aseptic surgical wax, [0156] suture of the scalp and disinfection.

[0157] Postoperative pain was taken care of by intramuscular administration of 0.1 mg/kg of Buprenorphine (Vetergesic®).

[0158] No major problems were encountered during the surgeries which were performed by the same person.

[0159] Monitoring of rats during the month of implantation of the mandrels: weight, behavior, physical appearance.

[0160] Following the surgeries, the rats were followed during the month of accommodation, with daily monitoring of the animals according to the score and cut-off point application defined below.

[0161] The animals were examined daily and a clinical score was assigned to them according to the grid below (see Table 2). Any score greater than or equal to 3 result in euthanasia of the animal to the detriment of the experimental protocol.

TABLE-US-00002 TABLE 2 Grid for clinical score. Change in initial Physical body weight appearance Behavior 0 Normal 0 Normal 0 Normal 1 Weight loss < 1 Lack of 1 Minor changes: limping 10% grooming gait, wound protection 2 Weight loss be- 2 Ruffled coat, 2 Abnormal: reduced tween 10 and 15% runny nose/eye mobility, inactive 3 Weight loss > 3 Very disheveled 3 Vocalizations, self- 20% coat, abnormal mutilation, very agitated posture or immobile

[0162] This follow-up includes weighing of each rat, at least once a week. The rats were all weighed according to the same protocol and on the same scale throughout the month of accommodation. All of the rats had constant weight curves. The behavior of the rats (activity, movements) as well as their physical appearance (state of the coat, posture) remained normal throughout this in vivo study, not reflecting any discomfort or suffering.

[0163] At the end of the study, the rats were euthanized and then exsanguinated in order to better preserve the brains for the histological study:

[0164] 1) Fixed anesthesia by Exagon (intraperitoneal injection)

[0165] 2) Intracardiac infusion of 0.9% NaCl (200 ml)

[0166] 3) Intracardiac formalin infusion (400 ml)

[0167] 4) Collection of the anatomical part (brain) containing the mandrel

[0168] The brain preparation protocol is as follows: [0169] Fixation of the brain in formalin [0170] 15% sucrose impregnation [0171] Sucrose impregnation 30% [0172] Removal of chucks [0173] Freezing of the anatomical part in liquid nitrogen vapor [0174] Brain sections made perpendicular to the axis of the mandrel, 35 μm floating sections made with a cryostat and placed in a cryopreservation solution at −20° C.

Staining of Rat Brain Sections with Cresyl Violet

[0175] Cresyl violet staining is a histological staining method used to reveal the cellular architecture of nervous tissues of which it stains the cell bodies and more particularly the rough endoplasmic reticulum and makes it possible to identify phenomena of cell proliferation or loss (necrosis) around the implanted area. The protocol used is as follows: [0176] Degreasing: successive baths of different concentrations of ethanol [0177] Washing with water [0178] Coloring of sections in a solution of cresyl violet [0179] Fixation with 70% and 95% ethanol [0180] Washing with water [0181] Immersion of the slides in a differentiation solution [0182] Washing with water [0183] Succession of ethanol bath of different concentrations [0184] Xylene bath

[0185] The acquisitions were made on a bright field microscope.

[0186] The “316L stainless steel mandrel” control allowed to evaluate the effects of implantation of the mandrel within the cerebral parenchyma and constitutes a negative control given that the characteristics of the mandrel (dimensions, shape and material) give it an implantable character not inducing an inflammatory reaction.

[0187] Nissl staining showed that for all the cerebral hemispheres implanted with 316L stainless steel mandrels, no proliferation or cell loss was observed around the implanted area (marked with the letter M and delimited by a dotted line on the FIG. 9b). For the cerebral hemispheres having received the 316L stainless steel mandrels coated with amyloid fibers, the cerebral parenchyma located near the implanted area did not show any tissue damage (proliferation or necrosis) and remain similar to those of the control condition (FIG. 9f).

[0188] These results suggest that amyloid fibers appear to be well tolerated by the cerebral parenchyma for a contact duration of 1 month.

Anti-GFAP and Anti-Iba1 Immunohistochemistry

[0189] Glial fibrillar acidic protein (GFAP) is an intermediate filament present in certain glial cells of the central nervous system, in particular astrocytes.

[0190] The Iba1 labeling is specific for the protein Ionized calcium-binding adapter molecule 1 (Iba1). Among brain cells, the Iba1 protein is specifically expressed in microglia. Microglial activation is another type of inflammatory reaction that involves the activation of microglia, the immune cells in the brain. Microglial cells go through different stages of activation, the extensions of the cells widen more and more until they form a sphere (macrophage stage).

[0191] The protocol followed for the GFAP and Iba-1 immunolabeling is as follows:

[0192] DAY 1: -Washing of sections in 1×PBS [0193] -PBS-Triton-NGS membrane permeabilization [0194] -Saturation with PBS-BSA 3% [0195] -Incubation with the primary antibody overnight

[0196] DAY 2: -PBS-BSA-Tween wash [0197] -Incubation with the secondary antibody [0198] -PBS-BSA-Tween wash [0199] -Assembly of the cuts between blade and coverslip

[0200] The acquisitions were carried out under a confocal microscope after a selection of the slides under an epi-fluorescence microscope.

[0201] The antibodies used are:

[0202] GFAP: specific for glial fibrillar acid protein (GFAP) which is an intermediate filament present in certain glial cells of the central nervous system, in particular astrocytes.

[0203] Iba1: specific for the ionized calcium-binding adapter molecule 1 protein (Iba1). Among brain cells, the Iba1 protein is specifically expressed in microglia (immune cells of the brain).

Anti-GFAP Immunohistochemistry for the Detection of Astrocytic Gliosis

[0204] GFAP labeling is specific for glial fibrillar acidic protein (GFAP) which is an intermediate filament present in certain glial cells of the central nervous system, in particular astrocytes.

[0205] Astrocytic gliosis is an inflammatory reaction that involves the activation of astrocytes within brain tissue. When these astrocytes are activated, their cell bodies become rounded and their cytoplasmic extensions thicken.

[0206] The immunostaining directed against the GFAP protein showed that for all the cerebral hemispheres implanted with the 316L stainless steel mandrels, no proliferation or astrocyte morphological modification was observed around the implanted area (marked with the letter M and delimited by a dotted line in FIG. 9c). For the cerebral hemispheres having received the 316L stainless steel mandrels coated with amyloid fibers, the results obtained were comparable to those of the control condition (FIG. 9g). The absence of astrocytic gliosis around the area implanted with the mandrel coated with amyloid fibers suggests that the latter do not induce an inflammatory reaction after a period of contact with the brain parenchyma for 1 month.

Anti-Iba1 Immunohistochemistry for the Detection of Microglia Activation

[0207] The immunostaining directed against the Iba-1 protein showed that for all the cerebral hemispheres implanted with the 316L stainless steel mandrels no microglial activation was observed around the implanted area (marked with the letter M and delimited by a line dotted in

[0208] FIG. 9d). Histologically, the microglial cells around the implanted area exhibit a small cell body (low volume cytoplasm) with some fine cytoplasmic extensions, characterizing resting microglia. For the cerebral hemispheres having received the 316L stainless steel mandrels coated with amyloid fibers, the results obtained were comparable to those of the control condition (FIG. 9h) and it was not observed any reactive microglial cells in the zone of the trajectory of the mandrel.

[0209] The absence of microglial activation around the area implanted with the mandrel coated with amyloid fibers suggests that the latter do not induce an inflammatory reaction after a contact period of 1 month with the brain parenchyma.

[0210] In conclusion, the implantation of the mandrel coated with amyloid fibers does not induce any tissue damage or any inflammatory reaction after 1 month of contact with the brain parenchyma. These explorations have demonstrated an adapted response of the host tissue and demonstrate the implantable nature of amyloid fibers at a cerebral site and allow their use in implantable devices to be considered.

Example 4. Testing with Real Physiological Pulses

[0211] After the experiment on artificial signals, the material made of HET-s proteins in amyloid fiber form was tested with real physiological pulses.

[0212] To do so, microelectrode arrays (MEA) bought from Multichannel Systems, which are widely used for simultaneous recordings on neuronal cells, were used (FIG. 10A). A drop of 100 μL HET-s proteins in amyloid fiber form was casted on the MEA and left to dry overnight. Then, the film was washed several times with the cell culture medium before adding a primary culture of mouse hippocampal neurons. Because the culture medium is around pH7, HET-s dry film transforms into a consistent hydrogel.

[0213] FIG. 10B shows an electrode-recording site below the neuronal culture. The hydrogel is invisible because the dimension of the fiber network is well beyond the resolution of the image, but it is located between the electrode array and the neurons. On regular MEA's measurements, monolayer of Poly-D-Lysine (PDL) and/or Poly-L-Lysine (PLL) are often used as cell adhesion enhancers but are not fit to be used for middle/long term in in vivo application due to a fast degradation of the layer. It is noticed here that, while the PLL layer thickness is few nanometers, the Het-s hydrogel is several micrometers.

[0214] FIG. 11A present an example of action potential recording with the setup described in FIG. 10. Diamond marks represents the locations were pulses have been detected. As criteria, we selected pulses that were 1 ms wide (typical neuron action potential) and at least 20 μV deep. Despite the presence of an HET-s hydrogel between the neurons and the electrode, we were able to detect plenty of pulse-like signals in the same range of voltage than regular modern electrode recordings, and with a pulse morphology as expected for typical action potential with polarization and depolarization phases (FIG. 11B). Therefore, it is possible to affirm that HET-s coating onto an abiotic electrode allows the charges to be passed through, resulting in the detection of physiological neuronal pulses. At the end, it allows the use of a biocompatible, biodegradable material that possesses an ionic/protonic conductivity, which makes it a great pretender for electrode or electrode coatings technologies.

Concluding Remarks

[0215] The purpose of HET-s coating on inorganic electrodes lies in multiple aspects:

[0216] 1) Without adding any external growth factors, primary neurons, which are extremely sensitive to their environment, are able to develop very well on HET-s hydrogels, until DIV10 from the last results. Indeed, similarly to carbon nanotubes, HET-s amyloid fibers possess a very high aspect ratio (˜1000), thus making it very well fitted to promote growth and development on cells that attach to these kind of structure.

[0217] 2) Action potentials made of ion and proton gradients are transmitted through the amyloid network from the cells to the electrode. HET-s hydrogel acts as an intermediary that is able to relay ionic information. That protonic conductivity is uncommon and lowers the overall contact resistances of the measure.

[0218] 3) HET-s amyloid fibers are biodegradable and biocompatible as shown above with the cytotoxicity results. Moreover, the hydrogel do not dilute itself in aqueous medium, which is essential to be used as electrode or electrode coating.