EP4314836A1 - Nanostructured biomimetic neuromorphic system - Google Patents

Abstract

The present invention relates to a nanostructured biomimetic neuromorphic system including a solution comprising inverted micelles (14) in contact with each other so as to form lipid bilayers (13) at contact points, at least part of said contact points comprising at least one ion transporting membrane protein (15) allowing at least one ion transport from an inverted micelle (14) to another in return for a reverse exchange of at least one ion, wherein said solution comprising inverted micelles (14) is a compacted emulsion (10). Such system permits the creation of an ionic gradient that permits to generate a voltage.

Description

Description

Title : IManostructured biomimetic neuromorphic system

Field of the invention

[001 ] The present invention relates to a nanostructured biomimetic neuromorphic system based on biological components such as inverted micelles.

Background of the invention

[002] The state-of-the-art electronic neuromorphic systems aim to reproduce the complete functionality of a synapse using electronic components. Those devices have some limitations in achieving the typical synaptic process that underlies learning in biological systems (e.g. activity-dependent synaptic plasticity). In order to mimic synaptic plasticity, the state-of-the-art electronic device is assumed to be able to change its resistance (synaptic strength, or weight) upon proper electrical stimuli (synaptic activity) and show several stable resistive states throughout its dynamic range (analog behavior). Moreover, those electronic systems aim to perform spike timing dependent plasticity (STDP), an associative homosynaptic plasticity learning rule that attempts to mimic biological neuronal function by utilizing the delay time between multiple firing neurons connected to a synapse in order to influence the learning responses of the output signal from the connected synapse. For example, Covi et al. 2016 demonstrated that an analog, rather than a binary, memristive synaptic element in a small-scale spiking neuromorphic network was capable of unsupervised learning for character recognition.

[003] Memristors provide the complementary metal oxide semiconductor-based electronic building blocks for such state-of-the-art electronic devices capable of pattern-learning and recognition (Ziegler et al. 2015, Li et al. 2014, Sa^'ighi et al. 2015). In 2018, the use of multiple memristive devices made a significant advance in creating the basis for building the next- generation of intelligent computing systems. In Boybat et al. 2018, a neuromorphic system utilized more than 1 million phase-change memristive devices to demonstrate an efficient spiking neural network that was capable of unsupervised learning, particularly for learning temporal correlations. Moreover, they demonstrated a significant step towards building large-scale and energy-efficient neuromorphic computing systems.

[004] Since memristors are primarily targeted toward future high-density nanoscaled arrays, complementary metal oxide semiconductor (CMOS) driver circuits need to be scaled to these dimensions as well. However, developing appropriate and highly scaled driver circuits for memristive synapses, which do not bring large overheads, is a significant challenge for state-of-the-art research in designing CMOS-based systems. This is especially true for systems that exploit passive crossbar integration. Such circuit topology is particularly appealing for neuromorphic engineers as it offers a direct equivalent for the neuron/synapse circuit with high parallelism and high integration density in which a single device is associated to a single synapse between two neurons (input line and output column). However, it brings circuit challenges (e.g. crosstalk, sneak path, or impedance mismatch) that need to be overcome (Sa^'ighi et al. 2015).

[005] Despite the considerable advances in electronic design to develop the memristor, those electronic devices still consume significantly greater energy to perform neuromorphic computing functions and remain much larger in size than biological neurons and synapses. Furthermore, emulating brain function with neuromorphic systems requires electronic artificial synapses to be constructed in 3-dimensions. However, the fabrication complexity of complementary metal-oxide-semiconductor architectures impedes the achievement of three-dimensional interconnectivity. The use of Field Programmable Gate Arrays (FPGAs) provides specific hardware technology, which can also be reprogrammable and thus provide a reconfigurable sensor system. Thus, the corresponding circuit can be modified to adapt its functionality to perform different tasks (Garcia et al. 2014). Nonetheless, systems designed using FPGAs continue to be large and require additional complex Digital Signal Processors (DSPs), VLSI chips or microcontrollers (Shimonouura et al. 2008).

[006] A solution to this problem has been attempted with flexible three- dimensional artificial chemical synapse networks in which two-terminal memristive devices are connected by vertically stacking crossbar electrodes (Wu et al. 2017). Flexibility in artificial electronic synapses has also been attempted using biopolymers (Wu et al. 2017, Yu et al. 2018, Hu et al. 2018). However, the operation of these organic memristors relies either on the slow kinetics of ion diffusion through a polymer to retain their states or on charge storage in metal nanoparticles, which inherently limits performance and stability.

[007] Recently, van de Burgt et al (2017) described an ENODe device, which is a new organic electronic device that functions as an artificial synapse and is constructed from inexpensive and commercially available polymers. The ENODe artificial synapse exhibits a large number of non-volatile and reproducible states (>500) and operates at very low voltages. The ENODe device utilizes two poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS)/Poly(ethyleneiminie) (PEI) electrodes (“presynaptic” and “postsynaptic”) separated by an electrolyte solution, from which cations penetrate the postsynaptic electrode and protons penetrate the presynaptic electrode. Applying a charge to the presynaptic electrode stimulates diffusion of the ions in the PEDOT:PSS/PEI electrodes, which provides the switching capability for this artificial synapse and the ability to charge to be retained in each electrode. That lack of volatility depends on the separating electrolyte solution, which is not an electron conductor. The ENODe mechanism differs fundamentally from that of existing organic-electronic neuromorphic devices.

Summary of the invention

[008] As such the purpose of the present invention is to overcome the above- mentioned disadvantages by proposing a nanostructured biomimetic neuromorphic system including a solution comprising inverted micelles in contact with each other so as to form lipid bilayers at contact points, at least part of said contact points comprising at least one ion transporting membrane protein allowing at least one ion to transport from an inverted micelle to another in return for a reverse exchange of at least one ion, wherein said solution comprising inverted micelles is a compacted emulsion. [009] The present invention advantageously allows to create a voltage or to create an ionic signaling pathway that can be recognized by living cells or body tissues.

[0010] Preferentially, said ion transporting membrane protein is an electrogenic antiporter allowing protons H⁺ to transport from an inverted micelle to another in return for a reverse exchange of at least one ion. The use of such an electrogenic antiporter would preferentially be used to create a voltage and electricity.

[0011] In a preferred embodiment, said ion transporting membrane protein is an electrogenic antiporter allowing calcium ions Ca²⁺ to transport from an inverted micelle to another in return for a reverse exchange of at least one ion. The use of such an electrogenic antiporter would preferentially be used to create an ionic signaling pathway that can be recognized by cells or tissues of the body.

[0012] An ion transporting membrane protein is a protein configured to be embedded in the membrane of a cell or a lipid bilayer. This membrane or lipid bilayer is practically impermeable to the transport of ions. An ion transporting membrane protein usually permits the transport of ions across the membrane or lipid bilayer in order to control cellular uptake and efflux of ions.

[0013] An electrogenic antiporter is an ion transporting membrane protein that allows the exchange of ions to occur across the membrane or lipid bilayer. This usually proceeds with the exchange of one or several inwardly transporting ions with one or several outwardly transporting ions. This exchange is electrogenic due to an imbalance between the number of inwardly transporting ions compared to the outwardly transporting ions. The results of such an electrogenic exchange is the creation of a gradient of those ions across the membrane or lipid bilayer into which the electrogenic antiporter is embedded.

[0014] An inverted micelle is a spheroidal aggregate of amphiphilic molecules, that is to say molecules having a hydrophilic polar head directed towards the interior of the sphere and a hydrophobic chain directed towards the exterior of the sphere, that is to say towards a fatty solvent. The term oil-phase is also used to describe this fatty solvent. Thus, the solution comprising inverted micelles is considered as an emulsion. Said emulsion comprises an aqueous solution (also called aqueous phase of the emulsion) inside the inverted micelles and a lipidic solution (also called hydrophobic phase or oil- phase of the emulsion) outside the inverted micelles.

[0015] The inverted micelles in contact with each other with contact points comprising at least one ion transporting membrane protein are considered as interconnected. When ions that can be specifically exchanged against protons H⁺ by the ion transporting membrane protein are brought to an edge of the solution comprising inverted micelles, an ionic gradient of said ions is then created across said solution from inverted micelles to inverted micelles. Advantageously, when such ionic gradient is created a transfer of protons H⁺ (a gradient of H⁺ ions) is created in said solution from inverted micelles to inverted micelles in the opposite way of the ionic gradient, thus generating a voltage.

[0016] A compacted emulsion is an emulsion wherein the inverted micelles are compacted against each other so as to increase the surface of lipid bilayers in the emulsion. The surface of lipid bilayers in the emulsion corresponds to the contact area between inverted micelles. The compacted state of the emulsion implies that the inside of the emulsion and also the edge of the emulsion are compacted, thus the inside and the edge of the compacted emulsion share the same shape/aspect. The contact area between inverted micelles inside and at the edge of the compacted emulsion is larger than the one of a non-compacted emulsion, that we could call free emulsion. In a non-compacted state of an emulsion comprising inverted micelles (free emulsion), the inverted micelles are in contact with each other but the contact area between them is smaller than in the compacted state and the edge of the free emulsion has a different shape/aspect from the inside of the free emulsion with a contact area between inverted micelles that is smaller than the one inside the free emulsion and thus, less surface of lipid bilayers.

[0017] The compacted state of the emulsion thus advantageously allows to obtain a larger surface of lipid bilayer and thus to accommodate in the compacted emulsion more ion transporting membrane proteins than in a free emulsion, thus allowing a greater capacity of ion exchange between the inverted micelles.

[0018] The compacted state of the emulsion allows to reach a total bilayer area greater than or equal to 1.5 m² per ml_ of compacted emulsion, preferably greater than or equal to 1.75 m²/ml_, preferably greater than or equal to 1.9 m²/ml_, more preferably greater than or equal to 2 m²/mL. Such an important amount of bilayer area advantageously permits to integrate more ion transporting membrane proteins than in a free emulsion.

[0019] The compacted state of the emulsion allows to reach a number of molecules of ion transporting membrane proteins per pm² of emulsion that is greater than or equal to 100 molecules/pm², preferably greater than or equal to 120 molecules/ pm², more preferably greater than or equal to 140 molecules/pm². Such a number of molecules of ion transporting membrane proteins per pm² advantageously allows a capacity of ions transport between inverted micelles that can’t be reached with a free emulsion.

[0020] Preferably, the compacted emulsion comprises more than 90% of aqueous solution and less than 10% of lipidic solution. In a special embodiment the compacted emulsion comprises at least 92% of aqueous solution and 8% or less of lipidic solution. In a special embodiment the compacted emulsion comprises at least 94% of aqueous solution and 6% or less of lipidic solution.

[0021] The compacted state of the emulsion may be obtained by centrifugation of the emulsion and release of the supernatant (most part of the oil-phase). Further, a compacted state may be obtained by including a protein or peptide anchoring molecule in the solution comprising inverted micelles. Such protein or peptide anchoring molecule will be posted in the lipid bilayers maintaining the inverted micelles against each other in a compacted state.

[0022] The design of the nanostructured biomimetic neuromorphic system in this invention utilizes biological components to construct neuromorphic systems of a smaller size than can be achieved using the state-of-the-art electronic neuromorphic engineering design. Moreover, the ion transporting membrane proteins (ion channels) of this invention have memristive functions and the use of arrays of inverted micelles enables the assembly of very large 3-dimensional arrays that are on the order of nanoscale to microscale dimensions to infinite dimensions. Indeed, a very large number of inverted micelles can be assembled into 3-dimensions to provide the nanostructured biomimetic neuromorphic system of the present invention.

[0023] The ion transporting membrane proteins control the diffusion of ions throughout the nanostructured biomimetic neuromorphic system of interconnected inverted micelles, which provides the system with capabilities to generate electrical voltages. Due to the utilization of biological components, this biological nanostructured biomimetic neuromorphic system operates with extremely low input power requirements as compared to state-of-the-art electronic neuromorphic engineering systems.

[0024] In particular embodiments, the invention furthermore meets the following characteristics, implemented separately or in each of their technically operative combinations.

[0025] Advantageously, the efficiency of the invention to generate large gradients of for example H⁺ ions (essentially generating differences in pH) or Ca²⁺ ions and to sustain such large gradients at a steady-state is enhanced by increasing the number of inverted micelles that contact each other in the nanostructured biomimetic neuromorphic system. Increasing the number of contacting inverted micelles increases the number of compartments available to conduct the transport processes in the nanostructured biomimetic neuromorphic system (inverted micelles being seen as compartments). This increased compartmentalization both increases the amount of ions (for example H⁺ or Ca²⁺ ions) available to be transported and also increases the number of membranes that contains and increased number of ion transporting membrane proteins in order to achieve an increased movement of said ions. Such a compartmentalization creates a nanostructured biomimetic neuromorphic system of the invention that is capable of generating a larger gradient of ions, for example H⁺ ions (that is larger gradient of pH) or Ca²⁺ ions, than is the situation for a system that contains only a single inverted micelle with the same number of ion transporting membrane proteins as in a multi-inverted-micelle compartmentalized system. [0026] In a preferred embodiment, the nanostructured biomimetic neuromorphic system includes at least 0.7 nanoliter, preferably 1 nanoliter of the solution comprising inverted micelles. Such a small volume would allow a device or electrode to include a small cluster of inverted micelles to be included on the surface, or inside said device or electrode, so as to generate a local gradient of pH.

[0027] In a preferred embodiment, the concentration of ion transporting membrane proteins in the solution comprising inverted micelles is at least 10 nM.

[0028] In an embodiment of the above-defined system, said system comprises at least 15 inverted micelles in contact so as to form a line of inverted micelles, each contact points between inverted micelles comprising at least one electrogenic antiporter. The line of 15 inverted micelles will produce more than 90% of the maximally attainable response for either the gradient of pH or the gradient of voltage. The system will function with less than 15 inverted micelles in contact to form a line of inverted micelles, but with a reduced proportion of the maximally attainable output. Conversely, the system will function with more than 15 inverted micelles in contact to form a line of inverted micelles, but with an output so as to reach closer to the maximally attainable output.

[0029] In another embodiment of the above-defined system, said at least one ion that can be transported by the ion transporting membrane proteins in exchange of protons is chosen from sodium (Na⁺), lithium (Li⁺).

[0030] In another embodiment of the above-defined system, the ion transporting membrane protein is the transmembrane protein NhaA. The protein NhaA is an electrogenic antiporter. In a specific embodiment the ion transporting membrane protein is the protein NhaA from Escherichia coli. Escherichia coli NhaA orthologs may be used in the system of the present invention, for example NhaA protein from Helicobacter pylori, Catenulispora acidiphila, Salinispora arenicola, Deferribacter desulfuricans, AcidithiobacHlus ferrivorans ou Halorubrum vacuolatum. The organisms producing NhaA protein live in different conditions of salinity, pH and temperature. The use of their NhaA protein should allow an adaptation to less stringent conditions for systems of the present invention, for example to be able to work with a wider range of pH. [0031] In another embodiment of the above-defined system, the ion transporting membrane protein is the A167P mutant of Escherichia colis NhaA protein. This mutation corresponds to the substitution of alanine (A) at position 167 of the NhaA protein sequence by a proline (P). This mutant allows advantageously an exchange of 1 Li⁺ against 7.5H⁺, thus generating a stronger charge gradient. The sequences of NhaA protein from Escherichia coli is known and available in NCBI GenBank database under the accession number NC_000913, version NC_000913.3 (nucleotides sequence) and the accession number NP_414560, version NP_414560.1 (peptides sequence). The sequence of A167P mutant of Escherichia coil’s NhaA protein is SEQ ID NO: 1 given in the sequence listing filed with the present patent application.

[0032] In another embodiment the above-defined system comprises a first tank configured to supply ions that can be transported by the ion transporting membrane protein in exchange of at least one ion, for example H⁺ ion or Ca²⁺ ion and a second tank configured to sequester said ions that can be transported by the ion transporting membrane protein, said first tank and said second tank being positioned on either side of the solution comprising the inverted micelles. The presence of these first and second tanks is one of the possibilities to create a gradient of ions in the solution containing the inverted micelles and thus, for example, to create a voltage thanks to the ion transporting membrane proteins that exchange protons H⁺ and said ions in the presence of such gradient.

[0033] In a specific embodiment the ions supplied by said first tank and sequestered by said second tank are sodium ions (Na⁺) or lithium ions (Li⁺). These first and second tanks supplying and sequestering Na⁺ or Li⁺ permit to create a gradient of sodium ions or lithium ions in the solution containing the inverted micelles and thus to create a voltage thanks for example to the ion transporting membrane proteins that exchange protons H⁺ and sodium or lithium ions in the presence of such gradient, like for example NhaA antiporters.

[0034] In another embodiment of the above-defined system, said system comprises a cathode and an anode. Said cathode and anode are capacitive or electrochemical oxidation/reduction electrodes that permit to translate the ionic gradient produced into electronic current.

[0035] The system of the present invention is capable of forming stable and biocompatible connections to living cells (such as nerves and muscles) for an implanted usage of the system, since it is constructed from biological components that use biological signaling mechanisms that allow the self- assembly of synaptic connections between the system and living cells. Thus, under another aspect, the present invention relates to the nanostructured biomimetic neuromorphic system of the present invention for its use as voltage source for an implanted medical device, a non- implanted medical device, or a nomadic consumer electronic device.

[0036] In a specific embodiment, the present invention relates to the nanostructured biomimetic neuromorphic system for its use to contact living cells and tissues of a mammal body so as to sense the ionic and chemical responses of cells and tissues, or to provide an ionic signal so as to influence the biological responses of cells and tissues.

[0037] Under another aspect, the present invention relates to a method for manufacturing the nanostructured biomimetic neuromorphic system of the present invention comprising the following steps of:

- preparing a lipidic solution;

- adding drop by drop an aqueous solution comprising ion transporting membrane proteins into the lipidic solution wherein the volume ratio lipidic solution/aqueous solution is 2, so as to create a solution comprising inverted micelles that is an emulsion;

- fragmenting the inverted micelles so as to reduce the size of inverted micelles, to increase their number and the emulsion homogeneity;

- compacting the inverted micelles against each other so as to increase the surface of lipid bilayers created.

[0038] When adding drop by drop the aqueous solution into the lipidic solution, the drops of aqueous solution are immediately surrounded by bipolar lipids and therefore remain individualized to create inverted micelles. Thus, the inverted micelles are in the lipidic solution (known as hydrophobic phase of the emulsion) and can be defined as vesicles containing the aqueous solution (known as aqueous phase of the emulsion) delimited by a lipid layer.

[0039] The preparation of the lipidic solution may be realized by adding lipids to oil such as mineral oil. As an example, the lipids are supplied by a solution of asolectin dissolved in hexadecane. An example of aqueous solution is Tris HCI buffer 25 mM and Tris KCI buffer 250 mM with 10 to 30 pg/mL of ion transporting membrane proteins. Preferably, the aqueous solution comprises a surfactant. The surfactant advantageously stabilizes the emulsion. Advantageously, a surfactant with a Hydrophilic-Lipophilic Balance (HLB) between 1 and 9 is used. In a particular embodiment the surfactant used is the n-Dodecyl b-D-maltoside (DDM) (20-30 mM). Stability of the emulsion may also be obtained by using a mixture of highly concentrated salts (for example 250 mM KCI) and with the presence of MgCL or glycerol. The Pickering effect may also be used to enhance the stability of the emulsion.

[0040] Fragmenting the inverted micelles may be realized by agitating the solution. For example, a mechanical agitation may be applied, such as the flushing technique made with a pipette. The fragmenting step permits a reduction in the size of inverted micelles, an increase in their number and the emulsion homogeneity. The inverted micelles obtained are very heterogeneous in size and of the order of a few hundred micrometers.

[0041] A second fragmentation step can be applied in order to obtain an emulsion that contains inverted micelles whose polydispersity of surface area is between a fraction of 0.015 pm and 150 pm, preferably of 0.020 pm and 150 pm, more preferably of 1 and 150 pm. This second step of fragmentation can for example be a mechanical fragmentation such as a rapid transfer of the solution including the inverted micelles between two syringes.

[0042] Compacting the inverted micelles against each other permits to increase the number of inter-micellar lipid bilayers and thus to increase the general surface of lipid bilayers in the solution comprising inverted micelles. It also makes it possible to increase the number of ion transporting membrane proteins placed in the lipid bilayers and thus a better circulation of protons between the micelles. In a preferred embodiment, the step of compacting the inverted micelles against each other is realized in order to reach a total bilayer area greater than or equal to 1.5 m² per ml_ of compacted emulsion, preferably greater than or equal to 1.75 m²/ml_, preferably greater than or equal to 1.9 m²/ml_, more preferably equal to 2 m²/mL

[0043] According to a method of implementation, the compacting step may be realized by centrifugation of the emulsion. For example, a centrifugation at 13,400 RPM during 3 minutes may be applied. Then, the supernatant (mostly lipidic solution) is discarded.

[0044] Under another aspect, the present invention relates to a method for generating a voltage using the nanostructured biomimetic neuromorphic system of the present invention, including the step of supplying to the solution comprising inverted micelles, ions that can specifically be exchanged against a proton H⁺ by the ion transporting membrane proteins present in said solution so as to apply an ionic gradient of said ions in the solution comprising inverted micelles.

[0045] In a special embodiment of said method for generating a voltage, the ion transporting membrane proteins comprised in the nanostructured biomimetic neuromorphic system is the transmembrane protein NhaA from Escherichia coli or A167P mutant of Escherichia col is NhaA protein, and the ions supplied in the solution comprising inverted micelles are sodium ions or lithium ions so as to apply respectively a sodium gradient or a lithium gradient in said solution.

[0046] Under another aspect, the present invention relates to a fuel-cell comprising the nanostructured biomimetic neuromorphic system of the present invention. Such a fuel-cell can also be termed a battery in common usage.

[0047] Such battery can be used as a voltage source for an implanted medical device, a non-implanted medical device, or a nomadic consumer electronic device.

Description of the figures

[0048] The invention will be better understood by reading the following description, given as a non-limitative example, and made with reference to the figures that represent: [0049] [Fig. 1] represents a drawing of the emulsion obtained after fragmenting step of the method for manufacturing the nanostructured biomimetic neuromorphic system of the present invention (A), a drawing of the compacted emulsion obtained after the compacting step of the method for manufacturing the nanostructured biomimetic neuromorphic system of the present invention without revealing the ion transporting membrane proteins (B), a drawing of the compacted emulsion obtained after the compacting step of the method for manufacturing the nanostructured biomimetic neuromorphic system of the present invention revealing the non-activated (black squares) ion transporting membrane proteins (C), and a drawing of the compacted emulsion obtained after the compacting step of the method for manufacturing the nanostructured biomimetic neuromorphic system of the present invention revealing the non-activated (black squares) and activated (white squares) ion transporting membrane proteins in the presence of an ionic gradient of the ion that can specifically be exchanged against a proton H⁺ by said ion transporting membrane proteins (D) ;

[0050] [Fig. 2] represents a drawing of an embodiment of the present invention in which the compacted emulsion of the nanostructured biomimetic neuromorphic system of the present invention is positioned between a first tank to supply ions (Na⁺) that can be transported by the ion transporting membrane protein (NhaA) in exchange of protons and a second tank configured to sequester said ions (A), and a drawing of the same embodiment as in (A) wherein an ionic gradient (Na⁺ gradient) is in place in the compacted emulsion thanks to ion transporting membrane proteins (NhaA proteins) present in the compacted emulsion and performing Na⁺ transfer from first tank to second tank (B) ;

[0051] [Fig. 3] represents an image of the compacted emulsion taken with a confocal microscope with an objective of 63x (the diameter of the micelles in the microscope field are between 1 and 5 pm) on which is drawn an electrogenic antiport (NhaA protein) in a polarized lipid bilayer between two inverted micelles as well as the movement of protons 2H⁺ and Na⁺ ions at the electrogenic antiport when Na⁺ ions are supplied to the compacted emulsion by a cathode (playing role of first tank) and sequestered by an anode (playing role of second tank) (A), an image of said compacted emulsion taken with a confocal microscope with an objective of 63x (the diameter of the micelles in the emulsion are between 1 and 5 pm) on which is drawn the movement of protons 2H⁺ and Na⁺ ions (B), an image of said compacted emulsion taken with a confocal microscope with an objective of 63x on which is drawn the movement of protons 2H⁺ and Na⁺ ions (C), a schematic view of said compacted emulsion with a smaller zoom than in (C) on which is drawn the movement of protons 2H⁺ and Na⁺ ions and where Na⁺ ions are supplied to the compacted emulsion by a cathode and sequestered by an anode (D) ;

[0052] [Fig. 4] represents a drawing according to a longitudinal section of a fuelcell comprising a hermetically sealed envelope enclosing the nanostructured biomimetic neuromorphic system of the present invention in the form of a compacted emulsion, a cathode and an anode (A) and a drawing according to a cross-section of the same battery (B).

[0053] [Fig. 5] represents a drawing illustrating a mathematical model of ions transport by NhaA protein as ion transporting membrane protein installed in a lipid bilayer between two inverted micelles.

[0054] [Fig. 6] illustrates a graphic representation of the gradient of Fl⁺ ions (ApFI) that is achieved at a steady-state regarding the number of contacting inverted micelles in a line from a model of the nanostructured biomimetic neuromorphic system.

[0055] [Fig. 7] illustrates a graphic representation of the gradient of voltage (AVm) that is achieved at a steady-state regarding the number of contacting micelles in a line from a model of the nanostructured biomimetic neuromorphic system.

[0056] [Fig. 8] illustrates a graphic representation of the distribution of the cross- sectional surface area of the micelles in the nanostructured biomimetic neuromorphic system of the present invention.

[0057] In these figures, identical numerical references from one figure to the other designate identical or similar elements. Furthermore, for reasons of clarity, the drawings are not to scale, unless otherwise stated. Detailed description of the invention

[0058] The following description describes the details of producing the nanostructured biomimetic neuromorphic system wherein the solution comprising inverted micelles is an emulsion. The protocols described in the following description are the preferred protocol for the purposes of this invention disclosure. In the following detailed description, the embodiment that is described is an embodiment of the invention wherein the ion transporting membrane protein is an electrogenic antiporter allowing protons H⁺ to transport from an inverted micelle to another in return for a reverse exchange of at least one ion.

[0059] Protocol to obtain isolated NhaA proteins from E. Coli

[0060] The protein production followed a protocol based on Kubicek et al. (“ Expression and purification of membrane proteins”, Methods in Enzymology 2014, 541 , 117-140). The NhaA protein gene was introduced into the E. coli strain C43(DE3) using the plasmid vector pET15b (Novagen^®) for overexpression. The cells were cultivated in ampicillin containing LB medium at 37 °C until the optical density at 600 nm (OD600) reached 0.4. Next 200 mM IPTG (lsopropyl-p-D-1-thiogalactopyranoside) was added to the culture medium to induce overexpression of the NhaA gene, followed by further cultivation for five hours. The resulting cells were harvested by centrifugation at 8000 rpm for 5 minutes to form pellets.

[0061] For purification, the pellets were first incubated with binding buffer containing 20 mM TRIS, 500 mM KCI, 10 mM imidazole, and 12.6 % (v/v) glycerol as well as lysozyme and benzonase nuclease for 30 minutes. The incubated cells were disrupted in a French pressure cell, followed by centrifugation at 14000 rpm for 20 minutes and subsequent ultracentrifugation at 36000 rpm for two hours to separate the membrane fraction from soluble components. The membrane fraction was then resuspended in binding buffer with additional 20 mM DDM and incubated overnight. Purification was carried out using an immobilised metal affinity chromatography column (Ni-NTA agarose, Qiagen). The column was first equilibrated with binding buffer followed by incubation of the membrane fraction for two hours. After rinsing with a washing buffer, the purified NhaA was recovered in elution buffer of 20 imM TRIS, 500 imM KCI, 300 imM imidazole, 12.6 %(w/v) glycerol and 225 mM DDM at concentrations between 0.5 to 2 mg/mL, preferably 0.75 mg/mL.

[0062] Protocol to obtain the lipidic solution

[0063] The lipidic solution consist in lipids, first dissolved in hexadecane at a concentration between 350 mg/mL and 1 g/L, for example 500 mg/mL. The lipids used can be a standard mix of lipids like asolectin (Phosphatidylethanolamine (PE), Phosphatidylinositol (PI), Phosphatidylcholine (PC)) or pure lipids, or a mix of lipids at different range concentration (i.e PC/Cardiolipin (CL)) or plant lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG, sulfoquinovosyldiacylglycerol (SQDG) or lipids from Archaea (diphytanyl lipids, ether lipids) or synthetic lipids. Lipids from Archaea advantageously bring more stability to the lipidic solution and the emulsion. Asolectin is an emulsifier.

[0064] This first solution is then dissolved 1/100 in mineral oil to obtain a lipid concentration of minimum 3.5 mg/mL, typically 5 mg/mL. The mineral oil comprises a mixture of saturated alkanes (C7 to C40).

[0065] Protocol to obtain the aqueous solution

[0066] To obtain the aqueous solution, 10 to 60pL of NhaA protein (ideally 40pL) from a 0.75mg/mL obtained in the purification step in elution buffer are added to 500pL of a previously prepared solution comprising:

- Tris 25 mM pH7 (other kind of buffer can also be used, like potassium phosphate buffer or Hepes, but should not contain Na⁺, so avoiding NaOH to equilibrate the pH)

- KCI 250 mM (from 150 to 300 mM, other salts can be used except Na⁺ or Li⁺). With more than 300 mM, there is a risk of destabilizing the lipids, with less than 150 mM the risk is to modify the global osmolarity when adding Na⁺.

- Glycerol 3% (from 0 to 3%)

- MgS04 1% (from 0 to 1%) [0067] Preparation of the nanostructured biomimetic neuromomhic system wherein the solution comprising inverted micelles is a compacted emulsion

[0068] First, 1 ml_ of lipidic solution prepared as described above, is added in a tube. This lipidic solution represents the hydrophobic phase of the emulsion in progress.

[0069] Then 500mI_ of the aqueous solution comprising NhaA proteins (ion transporting membrane proteins) prepared as described above, is added in the tube drop by drop with a pipette. The aqueous solution represents the aqueous phase of the emulsion in progress. The aqueous phase is the discontinuous phase of the emulsion. A volume ratio lipidic solution/aqueous solution equal to 2 is respected.

[0070] The drops of aqueous solution are immediately surrounded by bipolar lipids and therefore remain individualized at the bottom of the tube. This step guides the creation of the inverted micelles in a solution that can be called emulsion. The NhaA electrogenic proteins present in the aqueous solution, in the presence of the DDM surfactant, localize spontaneously in the lipid bilayers formed at contact points between the inverted micelles. They are randomly oriented.

[0071] The next step corresponds to a first fragmentation step to fragment the inverted micelles so as to reduce their size and increase their number and the emulsion homogeneity. To realize this step the solution comprising the inverted micelles is agitated mechanically using the flushing technique made with a pipette. A number of about 20 flushes is made with the pipette. The emulsion takes on a lactescent/milky aspect. The inverted micelles obtained are very heterogeneous in size and of the order of a few hundred micrometers. The emulsion is now homogeneous.

[0072] A second fragmentation step is applied in order to obtain an emulsion whose polydispersity is between a fraction of 1 and 150 pm which has the shape of a bell curve with a maximum close to 1 pm. This second fragmentation is realized mechanically by a rapid transfer of the emulsion between two syringes. [0073] The emulsion 10 obtained (Figure 1A) comprises an amount in lipidic solution 11 that is twice the amount of aqueous solution 12 (33% of aqueous solution and 66 % of lipidic solution).

[0074] To increase the number of lipid bilayers 13 and thus to increase the general surface of lipid bilayers 13 in the emulsion 10 and the number of ion transporting membrane proteins 15 placed in said lipid bilayers (enhancing the circulation of protons between the inverted micelles 14), a step of compacting the inverted micelles against each other is then applied. This step is realized by centrifuging the emulsion in the tube at 13,400 RPM during 3 minutes. Then, the supernatant (mostly lipidic solution) is discarded. What remains in the tube is a compacted emulsion 10 (Figures 1 B, 1C) with ion transporting membrane proteins 15 placed in the lipid bilayers 13. This compacted emulsion 10 comprises 92% of aqueous solution 12 and 8 % of lipidic solution 11. In the compacted emulsion 10 the compacted inverted micelles 14 have a polyhedral shape. A logarithmic- distribution of inverted micelles surface area is obtained from analyzing images of the emulsion (Figure 8).

[0075] Generating a voltage with the compacted emulsion of the present invention

[0076] As written above, the lipidic solution 11 was almost completely discarded. Only 8% of it remain in the compacted emulsion 10 and represent the hydrophobic phase of it. The hydrophobic phase is represented by the interfaces between the inverted micelles. These interfaces are lipid bilayers 13 connected in a network. The hydrophobic phase is dielectric and since the conductive aqueous phase, represented by the aqueous solution 12, is isolated in the inverted micelles 14, the nanostructured biomimetic neuromorphic system under the form of the compacted emulsion 10 is also dielectric.

[0077] The ion transporting membrane proteins 15 provided by the aqueous solution are localized in the lipid bilayers 13 formed at contact points between the inverted micelles 14. In the absence of ionic difference Li⁺ or Na⁺ and Fl⁺ on either side of the lipid bilayers 13, no ionic transfer is performed. The ion transporting membrane proteins 15 are not activated (the black squares in figure 1C). [0078] When ions that can specifically be exchanged against a proton H⁺ by the ion transporting membrane protein 15, for example a Na⁺ or Li⁺ for NhaA proteins (Figure 1 D), are supplied to the compacted emulsion 10, the ion transporting membrane proteins 15 are activated (the white squares in figure 1 D) only in the case of an ionic difference on both sides of the lipid bilayers 13. The ion transporting membrane proteins 15 located at the level of lipid bilayers 13 separating two areas of equal ionic concentrations remain inactive (the black squares in figure 1 D). When present, the difference in ionic concentration generates oriented ionic transfers and thus generate an ionic gradient and a voltage.

[0079] In a special embodiment, to apply the ionic gradient to the solution comprising the inverted micelles (here the compacted emulsion 10) and generate a voltage, the nanostructured biomimetic neuromorphic system of the present invention comprises a first tank 16 configured to supply ions that can specifically be exchanged against a proton H⁺ by said ion transporting membrane proteins 15 present in the compacted emulsion 10 and a second tank 17 configured to sequester said ions (Figure 2A), said first tank 16 and said second tank 17 being positioned on either side of the compacted emulsion 10. Of course, said first tank 16 is connected to the compacted emulsion 10 so that the ions it supplies may go from the first tank 16 to the compacted emulsion 10. Said second tank 17 is also connected to the compacted emulsion 10 so that ions that have passed through the compacted emulsion 10 by the ion transporting membrane proteins 15 can be collected in the second tank 17.

[0080] The ions supplied by the first tank 16 and sequestered by the second tank 17 may be for example Na⁺ ions or Li⁺ ions when the ion transporting membrane proteins 15 is NhaA protein or another ion transporting membrane protein 15 that transport protons in exchange of Na⁺ or Li⁺ ions. In the embodiment presented in figures 2A and 2B the ions supplied and sequestered are Na⁺ ions.

[0081] As shown in figure 2B, due to the ion transporting membrane proteins 15 located in the lipid bilayers 13 and the difference in ionic concentration first between the first tank 16 and inverted micelles 14, then between different inverted micelles 14, the sodium Na⁺ leave the first tank 16, go through the compacted emulsion 10 and is sequestered in the second tank 17, thus creating a sodium gradient in the compacted emulsion 10.

[0082] In the embodiment presented in figure 3, the ion transporting membrane proteins 15 are NhaA proteins and the ionic gradient used is a sodium (Na⁺) gradient (Figure 3A). As shown in figures 3A, 3B, 3C and 3D, Na⁺ goes to the right and H⁺ to the left of lipid bilayers 13 (oriented ionic transfers).

[0083] In a particular embodiment the nanostructured biomimetic neuromorphic system comprises capacitive or electrochemical oxidation/reduction electrodes that will play the role of first and second tanks (16, 17): a cathode 18 (plays the role of first tank 16) and an anode 19 (plays the role of second tank 17) (Figure 3D). These electrodes permit to translate the ionic gradient produced into electronic current. Thus, advantageously, one of the aspects of the present invention is a fuel cell 20 (also commonly called battery) comprising the nanostructured biomimetic neuromorphic system in the form of a compacted emulsion 10, a cathode 18 and an anode 19 (Figure 4A, 4B). Advantageously said fuel-cell 20 is implantable into a mammal so as to power an implanted medical device.

[0084] Realization of a fuel-cell comprising the nanostructured biomimetic neuromorphic system in the form of an emulsion: the implantable fuel-cell

[0085] In a special embodiment, the fuel-cell 20 comprises a hermetically sealed envelope 21 enclosing the nanostructured biomimetic neuromorphic system in the form of a compacted emulsion 10, a cathode 18 and an anode 19.

[0086] The envelope 21 is permeable to ions and molecules. Said envelope 21 gives the advantage to the fuel-cell 20 to be implantable in the body of a mammal by avoiding contact of other elements of the fuel-cell 20 than the envelope 21 with the mammal’s body. As an example, the envelope 21 is made of a polyvinyl alcohol (PVA) hydrogel. The PVA hydrogel is permeable to ions and molecules. Preferably, the envelope 21 has the form of a sealed tube (figure 4).

[0087] The cathode 18 comprises an inert support 22 covered with a conductive material 23 and impregnated with a capacitive material 24 (ensures ionic and electrical conduction), and a first collector 25 which is located in the thickness of the inert support 22. [0088] In the present embodiment, the inert support 22 has a hollow cylinder shape and is made of a cross-linked polyurethane foam. The polyurethane foam is covered with the conductive material 23 which is a conductive porous carbon layer, and is impregnated with the capacitive material 24 which is poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). At least one but preferably six gold wires act as collector 25 located in the thickness of the inert support 22. Instead of gold, the wire can be made of tinned copper for example.

[0089] The first collector 25 extends from the inert support 22 through the envelope 21 to the outside of the fuel-cell 20. The first collector 25 is sheathed at least on its part extending from the inert support 22 to the outside of the fuel-cell 20. The first collector 25 sheath 26 is preferably made of polyester.

[0090] The cathode 18 having a large developed surface area is in intimate contact with a large number of polarized lipid bilayers 13 of the compacted emulsion 10.

[0091] The anode 19 comprises, an ion-permeable cover 27 enclosing a microporous conductive material 28 of elongated shape and a second collector 29 which is located in the thickness of said microporous conductive material 28.

[0092] Preferably the ion-permeable cover 27 is made of a PVA hydrogel.

[0093] In the present embodiment, the microporous conductive material 28 comprises a mixture of microporous carbon (between 20% and 80%), molybdenum disulfide (MoS2) (between 20% and 80%), reduced graphene oxide (rGO) (between 20% and 80%) and polyacrylic acid (PAA) (between 5 and 40 %).

[0094] The molybdenum disulfide in the anode in a form associated to the microporous carbon and the reduced graphene oxide allows to sequester the sodium ions.

[0095] At least one but preferably six gold wires act as second collector 29 located in the thickness of the microporous conductive material 28. Instead of gold, the wire can be made of tinned copper for example.

[0096] The second collector 29 passes through the cover 27 and extends from the cover 27 through the envelope 21 to the outside of the fuel-cell 20. The second collector 29 is sheathed at least on its part extending from the cover 27 to the outside of the fuel-cell 20. The second collector 29 sheath 30 is preferably made polytetrafluoroethylene (PTFE) also called teflon.

[0097] The anode 19 has its surface area in intimate contact with a large number of polarized lipid bilayers 13 of the compacted emulsion 10 and is placed approximately in the center of the hollow cylinder-shaped inert support 22 of the cathode 18, so that there is compacted emulsion 10 between the cathode 18 and the anode 19.

[0098] Manufacture of the cathode 18

[0099] The cathode 18 is preferably formed by taking an inert support 22 which is a porous material, here polyurethane foam that is rendered conductive by impregnating with a conductive material 23, here conducting ink. The conducting ink is made by combining activated carbon (between 100 mg and 300 mg), M0S2 (200 mg), rGO (40 mg), and PEDOT:PSS (100 mI_). The PEDOT:PSS is a polymer mixture of the two ionomers poly(3,4- ethylenedioxythiophene) (PEDOT) and polystyrene sulfonate) (PSS). Advantageously, the impregnation of the polyurethane foam is assisted by adding a detergent such as Tween^® 20 at a dilution of between 0.8% and 20% in distilled water. Alternatively, the cathode 18 can be constructed using a polyurethane foam as inert support 22 that is already rendered to be conductive from the existing inclusion of activated carbon with the polyurethane, such as for the foam designed to be used as an activated carbon filter and available from electronics supply companies that are known to a person skilled in the art. In this alternative method of construction, the conductive polyurethane foam is coated with a solution that comprises PEDOT:PSS, ethylene glycol (200 mI_) and detergent Tween^® 20 (1 % dilution in distilled water).

[00100] An unsheathed part of the first collector 25 is then inserted into the polyurethane foam.

[00101 ] Manufacture of the anode 19

[00102] The anode is made from several components to provide the microporous conductive material 28 in the form of a paste. These components to make the paste include activated carbon (between 100 mg and 300 mg), M0S2 (200 mg), rGO (40 mg), PAA (200 mg) and distilled water (4 ml_). Advantageously, the conductive properties of the paste can be enhanced by adding to the paste 100 mI_ of PEDOT:PSS solution, which is polymer mixture of the two ionomers poly(3,4-ethylenedioxythiophene) (PEDOT) and polystyrene sulfonate) (PSS).

[00103] When the microporous conductive material 28 is ready we make it take an elongated shape and make a groove along its length. An unsheathed part of the second collector 29 is then inserted into the groove and the microporous conductive material 28 is closed on itself with a fold along its length so as to completely cover the part of the second collector 29 positioned in the groove.

[00104] The microporous conductive material 28 is then enclosed in the cover 27 of PVA hydrogel which is hermetically sealed by self-healing of the PVA hydrogel. A sheathed part of the second collector 29 passes through the cover 27 and extends outside of it.

[00105] Manufacture of the fuel-cell

[00106] The anode 19 is inserted in the middle of the hollow cylinder-shaped foam of the cathode 18.

[00107] Then both the cathode 18 and the anode 19 are inserted into the tube-shape envelope 21 in PVA hydrogel. Then the envelope 21 is filed up with compacted emulsion 10 and is hermetically sealed by auto-healing of the PVA hydrogel. The cathode 18 and the anode 19 are positioned so that there is compacted emulsion 10 between them (Figures 4A, 4B).

[00108] A sheathed part of the first collector 25 and a sheathed part of the second collector 29 pass through the envelope 21 and extend outside of it. These sheathed part of the first collector 25 and second collector 29 can for example be connected to an implanted or non-implanted medical device.

[00109] Using the said nanostructured biomimetic neuromorphic system in the form of a fuel-cell generate for the cathode 18 to capture H⁺ ions and the anode 19 to capture Na⁺ ions. Indeed, when the fuel-cell 20 is working Na⁺ ions will be brought in the fuel-cell 20 (inside the envelope 21 ) from the outside of the envelope 21 (from any source of Na⁺ ions) by going through the envelope 21 since it is permeable to ions and molecules. These Na⁺ ions entering the fuel-cell 20 will go through the compacted emulsion 10 and will be captured by the anode 19, thus forming a sodium ions gradient in the compacted emulsion 10. The creation of the sodium ions gradient will generate a H⁺ ions gradient in the opposite way of the sodium ions gradient through the compacted emulsion 10, the cathode 18 capturing the H⁺ ions. The cathode 18 and anode 19 will translate the ionic Na⁺ and H⁺ gradients produced into electronic current. Thus, an electronic device can be powered by the fuel-cell 20 if said electronic device is linked to the first collector 25 and the second collector 29.

[00110] Calculating the optimum number of inverted micelles in nanostructured biomimetic neuromomhic system

[00111] The characteristics for the optimum number of inverted micelles in the said nanostructured biomimetic neuromorphic system is determined according to an ion-transport modelling of the biomimetic neuromorphic system (Figure 5), with the relationship between the number of inverted micelles and the steady-state gradient of H⁺ ions (pH) shown in Figure 6. The pH gradient also corresponds directly to the voltage generated across the membrane (Figure 7).

[00112] The inventors developed a mathematical model of ions transport by NhaA protein as an ion transporting membrane protein placed in a lipid bilayer membrane between two inverted micelles.

[00113] As shown in Figure 5, Na⁺ and H⁺ ions compete inside the inverted micelle 1 to bind the active site C12 of the NhaA ion transporting membrane protein with binding rate a and c, respectively. At this stage, NhaA antiporter has the active site on the inverted micelle 1 and the inactive site on the inverted micelle 2. To simplify, we imagine that H⁺i represents the two protons and Na⁺i is the sodium ion in inverted micelle 1. The NhaA antiporter works by following an alternate access principle. Once an ion binds the single substrate binding site, creating either the complex HC12 or NaC-12, a thermodynamically favored conformational change moves both the ion and the active site to the other side of the membrane with rates k-12 and f2i, respectively. Such mechanism of active site translocation prevents any further ion-leaking. In the inverted micelle 2, the complexes HC21 and NaC2i dissociate with a rate b and d, respectively, into the active site C21 and ions H⁺2 and Na⁺2. Since the pH measure the amount of H⁺ ions, it was shown that the Na⁺ and H⁺ ion competition suffices to explain the pH dependence of the NhaA antiporters without invoking the presence of an active pH- sensing mechanism.

[00114] Inventors first studied the case of transport across a single membrane, then along a simple line of inverted micelles and finally in complex 3-D geometries. In the model, the inverted micelles in a line are all supposed to have the same volume. Generally, the volume of the inverted micelle may be much larger than the average diffusion distance of the ions, therefore inventors also included the possibility that ions are diffusing in the inverted micelle bulk where are not available to react with the active site of the ion transporting membrane protein (i.e. ions can move far away from the membrane where the ion transporting membrane proteins are located). The transition rate between the ions at the surface H⁺, Na⁺ and the one diffusing in the bulk H^+*, Na^+* is l and y, respectively.

[00115] The system of differential equations describing the time evolution of the concentration (measured in [nM]) of the components in inverted micelle 1 (ODE System 1 ) is:

[Math. 1] where a and c are measured in [1/(s nM)] and b, d, k and f in [1/s]. [00116] Table 1 reports all their values: [Table 1]

[00117] Addition of new inverted micelles in the line requests to add to the ordinary differential equations system (ODE system 1 ) similar equations with indexes corresponding to the inverted micelle's number.

[00118] The pH value in each inverted micelle is given by the concentration of H⁺ ions by:

[Math. 2] where the factor 2 comes from the fact that H⁺ correspond to two protons, 10^'9 converts [nM] to [M] and [M] at the denominator makes the argument of the base-ten logarithm dimensionless.

[00119] In computing the pH we include only the ions available (in bulk and surface of the inverted micelle) and not the one bound in complexes with the ion transporting membrane proteins, because these are not able to flow.

[00120] Figure 6 illustrates a graphic representation of the gradient of H⁺ ions (DrH) that is achieved at a steady-state regarding the number of contacting inverted micelles in a line from the model of the nanostructured biomimetic neuromorphic system of the present invention. The system starts with a pH of 7 (at time t=0) and a starting concentration of Na⁺ ions of 0.1 mM. The concentration of NhaA protein in each micelle is 0.01 mM.

[00121] With a patch-clamp experiment is possible to measure the membrane voltage, namely the voltage that can exploit to generate a voltage difference and induce a current. The membrane voltage between two inverted micelles is given by the Goldman equation:

[Math. 3]

[00122] Figure 7 illustrates a graphic representation of the gradient of voltage (AVm) that is achieved at a steady-state regarding the number of contacting inverted micelles in a line from the model of the nanostructured biomimetic neuromorphic system of the present invention. The system starts with the same parameters (pH (at time t=0), concentration of Na⁺ ions and concentration of NhaA proteins in each micelle) as stated above for figure 6.

[00123] The ODE system 1 represents the functional unit that is repeated several times in the inverted micelle line. The number of variables and of equations increases by increasing the number N of micelle in a line as 10N - 6. It is clearly impossible to write each time a new ordinary differential equations system.

[00124] Calculating the total bilaver area in the compacted emulsion

[00125] We can suppose that during the process of compression, the formation of bilayers dries the liquid in excess among the inverted micelles. In this case, the emulsion should resemble a dry foam. For a dry foam, the most favorable conditions at equilibrium follows the Plateau’s laws, so that the micelles organize with three bilayers meeting at each planar section, forming angles on 120° in 2D, and vertex angles in 3D of 109,5°.

[00126] If we suppose that during the compacting step, few inverted micelles coalesce, starting from a volume distribution that follow the uncompressed observation, each final compressed micelle can have a different volume and surface area. This resembles a polydispersed foam. Thus, each inverted micelle shares with the neighboring inverted micelles surface areas that are of different dimensions and can lodge for a different amount of ion transporting membrane protein. To stay simple, we suppose each inverted micelle to have an average volume V_m and an average surface area Am. We introduce the control parameter C, that modulates the degree of inverted micelle compacting.

[00127] For example, a homogeneous free micelle assumes a spherical conformation by minimizing its energy state and has a volume:

[Math. 4]

Vm=4w R³/3

[00128] If we fill a box of volume VB with inverted micelles, the minimal conformation whereby the space is filled by spheres contacting on every side other spheres, is when such micelles are inscribed into a cube, so that at least six point of the sphere have formed several points bilayer. We assume this to be the minimal case to have for sure a continuous transport of ions. Other less ordered configurations with less contact points are feasible. Thus, the volume occupied by an inverted micelle is :

[Math. 5] = (4p R³/3)/(2R)³ = TT /6 where Vc is the volume of the cube.

[00129] We can think to compress the micelle, change its shape, and adapt it to the cube, keeping the volume constant. In such case, the occupancy is:

[Math. 6]

V_m / V_c = 1

[00130] Generally, we can suppose to have N_m equal inverted micelles in a box of volume Vb, so that:

[Math. 7]

N_m V_m/ V_b = C

Where C is equal to 0,1 and is the control parameter that describes the compacting ratio.

[00131] Given the packing ratio C, a fix box can allocate a different number of inverted micelles depending on the compacting ratio C,

[Math. 8]

N_m = C V_b/ V_m [00132] It was shown that the highest average ratio density (greatest fraction of volume) occupied by the sphere in a dense compacting is C=0.74 of the total volume. By deforming the micelle, the packing ratio C can be larger, up to 1.

[00133] Once compressed, we can suppose that the total volume of the inverted micelle stays the same, but since the shape is not a sphere anymore, the surface area differs from usual formula to calculate the area of a sphere. It has been shown that most commonly 3D shape observed in foam are irregular polyhedrons with 13 faces. To simplify, we can suppose that there are only regular shapes. From computational models, we know that on average their surface area is:

[Math. 9]

A_m = b V_m™ where b is equal to 5,3 and is a constant.

[00134] For a sphere b is equal to 4,8.

[00135] Thinking that the volume during compacting step is preserved and the area changed, so that the excess area is redistributed among the rest of the solution, an equivalent way to formulate the relation is as a function of uncompacted free spherical inverted micelles:

[Math. 10]

A_m / V_m = c / R_mUN where RmUN is the radius of the equivalent free micelle with the same volume of the compressed one,

[Math. 11]

Rm_UN = (3 V_m / 4TT)¹'³ and c is a constant equal to 3,3.

[00136] This is a convenient way since RmUN is accessible experimentally by measuring the average inverted micelle radius before compacting the emulsion (supposing no coalescence occurs during the process).

[00137] According to preceding formulas (Math. 4 and Math. 10), then the area of a non-spherical inverted micelle is:

[Math. 12] A_m = 4tt c R_mUN ²/3 [00138] For a sphere c is equal to 3.

[00139] Then during compacting step some compacting of the inverted micelles happens and, in the process, c becomes superior to 3:

[Math. 13] c— [3,c_max] where in principle Cmax can be larger than 3,3 for some shapes.

[00140] Then we know that:

[Math. 14]

A_m = c V_m/R_mUN N_m V_m/ V_b = C

[00141] We can suppose that for C inferior to TT/6 there is no contact full among the micelles and they are still spheres, so that c is equal to 3. For C superior to TT/6 but inferior to 1 , there is an increasing compacting and c is superior to 3 but inferior to Cmax. There, we can suppose a linear relation such as [Math. 15] c (C) = m C +q where q = (3 - c_max p/6) / (1 - p/6)

And m = e_max - q

[00142] The total area of all the inverted micelles is N_m x Am. The inverted micelles start forming a bilayer from when they get in contact, that is when C is superior to TT/6. For C inferior to TT/6 no bilayer is formed, for C superior to TT/6 and inferior to 1 the total bilayer area is:

[Math. 16]

A_Bim ⁼ h N_m A_m/2 since the bilayer has 2 sheets, and h is the fraction of total area in the bilayer.

[00143] Although h must have a complex relation with C, we can suppose that h is linear with C in the range where C is superior to TT/6 and inferior to 1 , with: [Math. 17] h (C) = m C +q where q = TT/6 / (TT/6 - 1 )

And m = 1 - q

[00144] Then we have:

[Math. 18]

A_Bim = h N_m A_m/2 = (h c C V_b ) / (2 R_mU[\i)

[00145] It is then possible to realize plots that show c, h, and ABIm as function of the compression factor C. For a box of 1 ml_, the total bilayer area at maximal compacting is around 2m².

[00146] More generally, it should be noted that the modes of implementation and realization of the invention considered above have been described as non- exhaustive examples and that other variants are therefore conceivable.

SEQUENCE LISTING

<110> UNIVERSITE GRENOBLE ALPES

INSTITUT POLYTECHNIQUE DE GRENOBLE

CENTRE HOSPITALIER UNIVERSITAIRE DE GRENOBLE

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE

<120> Nanostructured biomimetic neuromorphic system

<130> 33950 EP

<160> 1

<170> Patentln version 3.5

<210> 1 <211> 388

<212> PRT

<213> Escherichia coli <400> 1

Met Lys His Leu His Arg Phe Phe Ser Ser Asp Ala Ser Gly Gly lie 1 5 10 15 lie Leu lie lie Ala Ala lie Leu Ala Met lie Met Ala Asn Ser Gly 20 25 30

Ala Thr Ser Gly Trp Tyr His Asp Phe Leu Glu Thr Pro Val Gin Leu 35 40 45

Arg Val Gly Ser Leu Glu lie Asn Lys Asn Met Leu Leu Trp lie Asn 50 55 60

Asp Ala Leu Met Ala Val Phe Phe Leu Leu Val Gly Leu Glu Val Lys 65 70 75 80

Arg Glu Leu Met Gin Gly Ser Leu Ala Ser Leu Arg Gin Ala Ala Phe 85 90 95

Pro Val lie Ala Ala lie Gly Gly Met lie Val Pro Ala Leu Leu Tyr 100 105 110

Leu Ala Phe Asn Tyr Ala Asp Pro lie Thr Arg Glu Gly Trp Ala lie 115 120 125

Pro Ala Ala Thr Asp lie Ala Phe Ala Leu Gly Val Leu Ala Leu Leu 130 135 140

Gly Ser Arg Val Pro Leu Ala Leu Lys lie Phe Leu Met Ala Leu Ala 145 150 155 160 lie lie Asp Asp Leu Gly Pro lie lie lie lie Ala Leu Phe Tyr Thr 165 170 175

Asn Asp Leu Ser Met Ala Ser Leu Gly Val Ala Ala Val Ala lie Ala 180 185 190

SHEET INCORPORATED BY REFERENCE (RULE 20.6) Val Leu Ala Val Leu Asn Leu Cys Gly Ala Arg Arg Thr Gly Val Tyr 195 200 205 lie Leu Val Gly Val Val Leu Trp Thr Ala Val Leu Lys Ser Gly Val 210 215 220

His Ala Thr Leu Ala Gly Val lie Val Gly Phe Phe lie Pro Leu Lys 225 230 235 240

Glu Lys His Gly Arg Ser Pro Ala Lys Arg Leu Glu His Val Leu His 245 250 255

Pro Trp Val Ala Tyr Leu lie Leu Pro Leu Phe Ala Phe Ala Asn Ala 260 265 270

Gly Val Ser Leu Gin Gly Val Thr Leu Asp Gly Leu Thr Ser lie Leu 275 280 285

Pro Leu Gly lie lie Ala Gly Leu Leu lie Gly Lys Pro Leu Gly lie 290 295 300

Ser Leu Phe Cys Trp Leu Ala Leu Arg Leu Lys Leu Ala His Leu Pro 305 310 315 320

Glu Gly Thr Thr Tyr Gin Gin lie Met Val Val Gly lie Leu Cys Gly 325 330 335 lie Gly Phe Thr Met Ser lie Phe lie Ala Ser Leu Ala Phe Gly Ser 340 345 350

Val Asp Pro Glu Leu lie Asn Trp Ala Lys Leu Gly lie Leu Val Gly 355 360 365

Ser lie Ser Ser Ala Val lie Gly Tyr Ser Trp Leu Arg Val Arg Leu 370 375 380

Arg Pro Ser Val 385

SHEET INCORPORATED BY REFERENCE (RULE 20.6)

Claims

Claims

1. Nanostructured biomimetic neuromorphic system including a solution comprising inverted micelles (14) in contact with each other so as to form lipid bilayers (13) at contact points, at least part of said contact points comprising at least one ion transporting membrane protein (15) allowing at least one ion to transport from an inverted micelle (14) to another in return for a reverse exchange of at least one ion, wherein said solution comprising inverted micelles (14) is a compacted emulsion.

2. Nanostructured biomimetic neuromorphic system according to claim 1, comprising at least 15 inverted micelles (14) in contact so as to form a line of inverted micelles (14), each contact points between inverted micelles (14) comprising at least one ion transporting membrane protein (15).

3. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 2, wherein the ion transporting membrane protein (15) is an electrogenic antiporter.

4. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 3, wherein the ion transporting membrane protein (15) is the transmembrane protein NhaA.

5. Nanostructured biomimetic neuromorphic system according to claim 4, wherein the ion transporting membrane protein (15) is the transmembrane protein NhaA from Escherichia coli or A167P mutant of Escherichia col is NhaA protein.

6. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 5, comprising a first tank (16) supplying ions that can be transported by the ion transporting membrane protein (15) in exchange of at least one ion and a second tank (17) sequestering said ions that can be transported by the ion transporting membrane protein (15), said first tank (16) and said second tank (17) being positioned on either side of the solution comprising the inverted micelles (14).

7. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 6, comprising a cathode (18) and an anode (19).

8. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 7, wherein the ion transporting membrane protein (15) is an electrogenic antiporter allowing calcium ions Ca²⁺ to transport from an inverted micelle to another in return for a reverse exchange of at least one ion.

9. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 7, wherein the ion transporting membrane protein (15) is an electrogenic antiporter allowing protons H⁺ to transport from an inverted micelle to another in return for a reverse exchange of at least one ion.

10. Nanostructured biomimetic neuromorphic system according to claim 9, wherein said at least one ion that can be transported by the ion transporting membrane protein (15) in exchange of protons is chosen from sodium (Na⁺), and lithium (Li⁺).

11. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 10, for its use as voltage source for an implanted medical device, a non- implanted medical device, or a nomadic consumer electronic device.

12. Nanostructured biomimetic neuromorphic system according to any one of claims 1 to 10, for its use to contact living cells and tissues of a mammal body so as to sense the ionic and chemical responses of cells and tissues, or to provide an ionic signal so as to influence the biological responses of cells and tissues.

13. Method for manufacturing the nanostructured biomimetic neuromorphic system according to any one of claims 1 to 10, comprising the following steps of:

- preparing a lipidic solution (11 );

- adding drop by drop an aqueous solution (12) comprising ion transporting membrane proteins (15) into the lipidic solution wherein the volume ratio lipidic solution/aqueous solution is 2, so as to create a solution comprising inverted micelles (14) that is an emulsion (10);

- fragmenting the inverted micelles (14) so as to reduce the size of inverted micelles (14), to increase their number and the emulsion (10) homogeneity; - compacting the inverted micelles (14) against each other so as to increase the surface of lipid bilayers (13) created.

14. Method for generating a voltage using the nanostructured biomimetic neuromorphic system according to any one of claims 9 to 10, comprising the step of supplying to the solution comprising inverted micelles (14), ions that can specifically be exchanged against a proton H⁺ by the ion transporting membrane protein (15) present in said solution so as to apply an ionic gradient of said ions in the solution comprising inverted micelles (14).

15. Method for generating a voltage according to claim 14, wherein the ion transporting membrane protein (15) comprised in the nanostructured biomimetic neuromorphic system is the transmembrane protein NhaA from Escherichia coli or A167P mutant of Escherichia coli s NhaA protein, and the ions supplied in the solution comprising inverted micelles (14) are sodium ions or lithium ions so as to apply respectively a sodium gradient or a lithium gradient in said solution.

16. Fuel-cell (20) comprising the nanostructured biomimetic neuromorphic system according to any of claims 1 to 10.

17. Fuel-cell (20) according to claim 16, for its use as voltage source for an implanted medical device, a non-implanted medical device, or a nomadic consumer electronic device.