Nanostructured Biomimetic Neuromorphic Systems

Abstract

The present invention relates to a nanostructured biomimetic neuromorphic system comprising a solution containing reverse micelles (14) that are in contact with each other and form a lipid bilayer (13) at the contact points, at least a portion of the contact points containing at least one ion transport membrane protein (15) that allows the transport of at least one ion from one reverse micelle (14) to another reverse micelle (14) by back exchange of at least one ion, the solution containing the reverse micelles (14) being a compressed emulsion (10). Such a system is capable of generating an ion gradient that can generate a voltage.

Description

[Technical Field]

[0001] The present invention relates to nanostructured biomimetic neuromorphic systems based on biological components such as reverse micelles. [Background technology]

[0002] State-of-the-art electrical neuromorphic systems aim to replicate the full functionality of synapses using electronic components. These devices have some limitations in achieving typical synaptic processes (e.g., activity-dependent synaptic plasticity) that underlie learning in biological systems. To mimic synaptic plasticity, state-of-the-art electronic devices are presumed to be able to change their resistance (synaptic strength or weight) upon receiving appropriate electrical stimuli (during synaptic activity) and exhibit several stable resistance states (analogous behavior) throughout a dynamic range. Furthermore, these electronic systems aim to implement spike-timing-dependent plasticity (STDP), a learning rule for associative homosynaptic plasticity that attempts to mimic biological neural function by utilizing the time delay between multiple firing neurons connected to a synapse to influence the learning response of the output signal from the connected synapse. For example, Covi et al. (2016) demonstrated that analog, rather than binary, memristive synaptic elements in small-scale spiking neuromorphic networks can perform unsupervised learning of feature recognition.

[0003] Memristors provide complementary metal-oxide semiconductor-based electronic building blocks for such cutting-edge electronic devices capable of pattern learning and recognition (Ziegler et al., 2015; Li et al., 2015; Saighi et al., 2015). In 2018, significant progress was made in forming the foundation for building next-generation computing systems using multiple memristive devices. Boybat et al. (2018) demonstrated an efficient spiking neural network capable of unsupervised learning, particularly time-correlated learning, using over one million phase-change memristive devices in a neuromorphic system. Furthermore, this device represented a significant step toward building large-scale, energy-efficient neuromorphic computing systems.

[0004] Because memristors are primarily targeted for future high-density nanoscale arrays, complementary metal-oxide semiconductor (CMOS) driver circuits must scale to these dimensions as well. However, developing suitable, scaled driver circuits for memristive synapses without significant overhead presents a significant challenge to cutting-edge research in designing CMOS-based systems. This is especially true for systems utilizing passive crossbar integration. Such circuit topologies are particularly attractive to neuromorphic engineers because they provide a direct equivalent to highly parallel, highly integrated neuron / synapse circuits, where a single device couples one synapse between two neurons (an input line and an output column). However, such topologies introduce circuit challenges (e.g., crosstalk, sneak paths, or impedance mismatch) that must be overcome (Saighi et al., 2015).

[0005] Despite the considerable advantages of electronic design for developing memristors, these electronic devices still consume significantly more energy to perform neuromorphic computing functions and remain significantly larger than biological neurons and synapses. Furthermore, to mimic brain functions with neuromorphic systems, artificial electronic synapses must be constructed in three dimensions. However, the fabrication complexity of complementary metal-oxide semiconductor structures prevents the achievement of three-dimensional interconnectivity. The use of field-programmable gate arrays (FPGAs) offers specific hardware technology and, because FPGAs are reprogrammable, also offers reconfigurable sensor systems. Therefore, the corresponding circuits can be modified to adapt their functionality to perform different tasks (Garcia et al., 2014). Nevertheless, systems designed using FPGAs remain large and require additional complex digital signal processors (DSPs), VLSI chips, or microcontrollers (Shimonouura et al., 2008).

[0006] Attempts have been made to solve this problem using flexible three-dimensional artificial chemical synapse networks, in which two-terminal memristive devices are connected by vertically stacked 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 on either slow ion diffusion rates through the polymer to maintain their state or charge storage in metal nanoparticles, which inherently limit their performance and stability.

[0007] Recently, van de Burgt et al. (2017) described the ENODe device, a novel organic electronic device that functions as an artificial synapse and is constructed from inexpensive, commercially available polymers. The ENODe artificial synapse exhibits numerous non-volatile and reproducible states (over 500 cycles) and operates at very low voltages. The ENODe device utilizes two poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) / poly(ethyleneimine) (PEI) electrodes ("presynaptic" and "postsynaptic") separated by an electrolyte, from which cations permeate the postsynaptic electrode and protons permeate the presynaptic electrode. Charging the presynaptic electrode stimulates ion diffusion within the PEDOT:PSS / PEI electrode, thereby endowing the artificial synapse with switching capabilities and the ability to retain charge at each electrode. The lack of volatility is due to the separate electrolyte, which is not an electronic conductor. The ENODe mechanism is fundamentally different from that of existing organic electronic neuromorphic devices. Summary of the Invention

[0008] Therefore, the object of the present invention is to overcome the above-mentioned drawbacks by proposing a nanostructured biomimetic neuromorphic system comprising a solution containing reverse micelles that are in contact with each other and form a lipid bilayer at the contact points, at least a portion of the contact points containing at least one ion transport membrane protein that allows the transport of at least one ion from one reverse micelle to another by reverse exchange of at least one ion, and wherein the solution containing the reverse micelles is a compressed emulsion.

[0009] The present invention advantageously allows for the generation of electrical voltages or ionic signaling pathways that are recognizable by living cells or body tissues.

[0010] Preferably, the ion transport membrane protein transports protons H by back exchange of at least one ion. +from one reverse micelle to another. Such electrogenic antiporters are suitable for use in generating voltage and electricity.

[0011] In a preferred embodiment, the ion transport membrane protein transports calcium ions, Ca, by back exchange of at least one ion. 2+ from one reverse micelle to another. Such electrogenic antiporters are suitable for use in generating ion signaling pathways that can be recognized by cells or tissues in the body.

[0012] Ion transport membrane proteins are proteins that are configured to be embedded in a cell membrane or lipid bilayer, which is substantially impermeable to ions. Ion transport membrane proteins typically allow the transport of ions across the membrane or lipid bilayer to control the uptake and export of ions in cells.

[0013] Electrogenic antiporters are ion-transporting membrane proteins that cause ion exchange across membranes or lipid bilayers. Electrogenic antiporters typically exchange one or more inwardly transported ions with one or more outwardly transported ions. This exchange is electrogenic due to the imbalance in the number of inwardly transported ions compared to the outwardly transported ions. This electrogenic exchange results in the generation of a gradient of these ions across the membrane or lipid bilayer in which the electrogenic antiporter is incorporated.

[0014] Reverse micelles are spherical aggregates of amphiphilic molecules, i.e., molecules with hydrophilic polar heads facing the inside of the sphere and hydrophobic chains facing the outside of the sphere, i.e., toward the aliphatic solvent. The term "oil phase" is also used to refer to this aliphatic solvent. Thus, a solution containing reverse micelles is considered an emulsion. An emulsion contains an aqueous solution (also called the aqueous phase of the emulsion) inside the reverse micelles and a lipid solution (also called the hydrophobic or oil phase of the emulsion) outside the reverse micelles.

[0015] Reverse micelles that contain at least one ion transport membrane protein and are in contact with each other at their contact points are considered interconnected. The ion transport membrane protein transports protons, H + When an ion specifically exchangeable for γ reaches the outer edge of the solution containing the reverse micelles, an ion gradient of that ion is created across the reverse micelles in the solution. Preferably, when such an ion gradient is created, protons H + Movement (H + A gradient of ions is created, generating a voltage.

[0016] A compressed emulsion is an emulsion in which reverse micelles are compressed together, increasing the surface area of ​​the lipid bilayer in the emulsion. The surface area of ​​the lipid bilayer in the emulsion corresponds to the contact area between the reverse micelles. The compressed state of the emulsion means that the outer edge of the emulsion is compressed along with the inside of the emulsion, and the inside and outer edge of the compressed emulsion share the same shape / appearance. The contact area between the reverse micelles at the inside and outer edge of the compressed emulsion is larger than the contact area between the reverse micelles in the uncompressed emulsion, which we may call a "free emulsion." In the uncompressed state (free emulsion) of an emulsion containing reverse micelles, the reverse micelles are in contact with each other, but the contact area between them is smaller than in the compressed state. The outer edge of the free emulsion has a different shape / appearance from the inside of the free emulsion, and the contact area between the reverse micelles is smaller than the contact area inside the free emulsion, resulting in a smaller surface area of ​​the lipid bilayer.

[0017] Therefore, the compressed state of the emulsion makes it possible to favorably obtain a large surface area of ​​the lipid bilayer, which allows more ion transport membrane proteins to be accommodated in the compressed emulsion than in the free emulsion, thereby allowing for a greater capacity for ion exchange between the reverse micelles.

[0018] Depending on the compression state of the emulsion, 1 mL of compressed emulsion may produce 1.5 m 2 More than 1.75m, preferably 2 / mL or more, preferably 1.9m 2 / mL or more, preferably 2m 2 Total bilayer areas of 1000µg / mL or more can be achieved. These significant bilayer area values ​​advantageously allow the incorporation of more ion transport membrane proteins than in free emulsions.

[0019] The emulsion is compressed to 100 molecules / μm 2 More than 120 molecules / μm, preferably 2 More preferably, 140 molecules / μm 2 Emulsion 1μm or more 2 It is possible to reach the number of ion transport membrane protein molecules per 1 μm. 2 This number of molecules of ion transport membrane proteins per microemulsion allows for a 1 μm pore size, which is unattainable in free emulsions. 2 This allows for favorable ion transport capability between the reverse micelles.

[0020] Preferably, the compressed emulsion comprises more than 90% aqueous solution and less than 10% lipid solution. In particular embodiments, the compressed emulsion comprises at least 92% aqueous solution and no more than 8% lipid solution. In particular embodiments, the compressed emulsion comprises at least 94% aqueous solution and no more than 6% lipid solution.

[0021] A compressed emulsion can be achieved by centrifuging the emulsion and removing the supernatant (mostly the oil phase). Furthermore, a compressed emulsion can be achieved by including protein or peptide anchor molecules in the solution containing the reverse micelles. These protein or peptide anchor molecules are introduced into the lipid bilayer, allowing the reverse micelles to remain compressed against each other.

[0022] The nanostructured biomimetic neuromorphic systems of the present invention utilize biological components to construct neuromorphic systems smaller in size than is achievable using state-of-the-art electrical neuromorphic engineering designs. Furthermore, the ion-transporting membrane proteins (ion channels) of the present invention possess memristive functions, and arrays of reverse micelles enable the assembly of very large three-dimensional arrays ranging from nanoscale to microscale to infinite scale. Indeed, a large number of reverse micelles can be assembled in three dimensions to produce the nanostructured biomimetic neuromorphic systems of the present invention.

[0023] Ion transport membrane proteins control ion diffusion through a nanostructured biomimetic neuromorphic system of interconnected reverse micelles, resulting in a system capable of generating voltage. By utilizing biological components, this biological nanostructured biomimetic neuromorphic system operates with extremely low input power requirements compared to state-of-the-art electrical neuromorphic engineering systems.

[0024] In particular embodiments, the invention further satisfies the following characteristics, implemented individually or by any combination of these technical operations:

[0025] Preferably, for example, H + ions (especially those that create pH differences), or Ca 2+ The efficiency of the present invention in generating large ion gradients and maintaining such large gradients at steady state is improved by increasing the number of reverse micelles that are in contact with one another in the nanostructured biomimetic neuromorphic system. Increasing the number of contacting reverse micelles increases the number of compartments in the nanostructured biomimetic neuromorphic system where the transport process can occur (reverse micelles are identified as compartments). This increased compartmentalization increases the number of ions (e.g., H) that can be transported. + or Ca 2+The amount of ions (e.g., H) increases, and the number of membranes containing more ion transport membrane proteins also increases to achieve increased ion transport. Such compartmentalization allows for a larger gradient of ions, e.g., H, than in the case of a system containing only a single reverse micelle with the same number of ion transport membrane proteins as in the multiple reverse micelle compartmentalized system. + ions (larger pH gradient), or Ca 2+ A nanostructured biomimetic neuromorphic system of the present invention is created that is capable of generating ions.

[0026] In a preferred embodiment, the nanostructured biomimetic neuromorphic system comprises at least 0.7 nL, preferably 1 nL, of a solution containing reverse micelles. This small volume allows the device or electrode to contain small clusters of reverse micelles contained on or within the device or electrode, creating a local pH gradient.

[0027] In a preferred embodiment, the concentration of the ion transport membrane protein in the solution containing the reverse micelles is at least 10 nM.

[0028] In one embodiment of the system defined above, the system comprises at least 15 reverse micelles contacted to form a reverse micelle chain, each contact point between the reverse micelles containing at least one electrogenic antiporter. A series of 15 reverse micelles produces greater than 90% of the maximum attainable response to either a pH gradient or a voltage gradient. The system will function with fewer than 15 reverse micelles contacted to form a reverse micelle chain, but the percentage of the maximum attainable output power will decrease. Conversely, the system will function with more than 15 reverse micelles contacted to form a reverse micelle chain, but the output power will approach the maximum attainable output power.

[0029] In another embodiment of the system defined above, at least one ion that can be transported by the ion transport membrane protein in the proton exchange is sodium (Na + ), lithium (Li +) is selected.

[0030] In another embodiment of the system defined above, the ion transport membrane protein is the transmembrane protein NhaA. The protein NhaA is an electrogenic antiporter. In a specific embodiment, the ion transport membrane protein is the protein NhaA from Escherichia coli. NhaA orthologues of E. coli, such as NhaA proteins from Helicobacter pylori, Catenulispora acidiphia, Salinispora arenicola, Deferibacter desulfuricans, Acidithiobacillus ferivorans, or Halorubrum baclolactam, can be used in the system of the present invention. Organisms that produce NhaA proteins survive under different salinity, pH, and temperature conditions. Using these NhaA proteins should allow the system of the present invention to be adapted to less stringent conditions, for example, to function over a wider pH range.

[0031] In another embodiment of the system defined above, the ion transport membrane protein is the A167P mutant of the E. coli NhaA protein. This mutation corresponds to the substitution of alanine (A) at position 167 of the NhaA protein sequence with proline (P). This mutant allows for the addition of 7.5 H + 1 Li for + This allows for advantageous exchange of NhaA, thereby generating a stronger charge gradient. The sequence of the NhaA protein from E. coli is known and is available in the NCBI GenBank database under accession numbers NC_000913 and NC_000913.3 (nucleotide sequence) and NP_414560 and NP_414560.1 (peptide sequence). The sequence of the A167P mutant of the E. coli NhaA protein is set forth in the sequence listing submitted with this patent application and is SEQ ID NO: 1.

[0032] In another embodiment, the system defined above comprises at least one ion, e.g., H + ions or Ca 2+The present invention relates to an ion exchange system, comprising a first tank configured to supply ions that can be transported by the ion transport membrane protein and a second tank configured to sequester ions that can be transported by the ion transport membrane protein, the first tank and the second tank being disposed on either side of a solution containing reverse micelles. The presence of the first and second tanks creates an ion gradient in the solution containing the reverse micelles, and thus, for example, in the presence of such a gradient, protons H + One of the reasons why voltage can be generated is due to ion transport membrane proteins that exchange ions with the membrane.

[0033] In certain embodiments, the ions provided by the first tank and sequestered by the second tank are sodium ions (Na + ) or lithium ion (Li + ) Na + or Li + These first and second tanks, which supply and isolate protons H, make it possible to generate a gradient of sodium or lithium ions in the solution containing the reverse micelles, and therefore, in the presence of such a gradient, protons H + Ion transport membrane proteins, such as the NhaA antiporter, which exchange ions with sodium or lithium ions, enable the generation of voltage.

[0034] In another embodiment of the system defined above, the system comprises a cathode and an anode, which are capacitive or electrochemical oxidation / reduction electrodes that allow the generated ion gradient to be converted into an electric current.

[0035] The systems of the present invention are composed of biological components that use biological signaling mechanisms that allow for the self-assembly of synaptic connections between the systems and living cells, thereby allowing for the formation of stable and biocompatible connections to living cells (such as nerves and muscles) for implanted use. Thus, under another aspect, the present invention relates to the use of the nanostructured biomimetic neuromorphic systems of the present invention as voltage sources for implantable medical devices, non-implantable medical devices, or nomadic consumer electronic devices.

[0036] In certain embodiments, the present invention relates to the use of nanostructured biomimetic neuromorphic systems in contact with living mammalian cells and body tissues to sense ionic and chemical responses of the cells and tissues or to provide ionic signals to affect the biological responses of the cells and tissues.

[0037] Under another aspect, the present invention provides a method for producing a nanostructured biomimetic neuromorphic system of the present invention, comprising: -preparation of a lipid solution; - adding dropwise an aqueous solution containing an ion transport membrane protein to a lipid solution, in a lipid solution / aqueous solution volume ratio of 2, to produce a solution containing reverse micelles, which is an emulsion; - fragmenting the reverse micelles to reduce their size and increase the number of reverse micelles and the homogeneity of the emulsion; - compressing the reverse micelles together to increase the surface of the lipid bilayer produced; The present invention relates to a method, including:

[0038] When aqueous solution is added dropwise to a lipid solution, the aqueous droplets are immediately surrounded by bipolar lipids, thereby forming reverse micelles that remain discrete. Reverse micelles can therefore be defined as vesicles containing aqueous solution (known as the aqueous phase of the emulsion) that are present in a lipid solution (known as the hydrophobic phase of the emulsion) and are bounded by lipid layers.

[0039] The lipid solution can be prepared by adding lipids to an oil such as mineral oil. In one example, the lipid is provided by a solution of asolectin dissolved in hexadecane. Examples of aqueous solutions include Tris-HCl buffer (25 mM) and Tris-KCl buffer (250 mM) containing 10-30 μg / mL of an ion transport membrane protein. The aqueous solution preferably contains a surfactant. The surfactant advantageously stabilizes the emulsion. Preferably, a surfactant with a hydrophilic-lipophilic balance (HLB) value of 1-9 is used. In a specific embodiment, the surfactant used is n-dodecyl-β-D-maltoside (DDM) (20-30 μM). Emulsion stability can also be achieved by using a high concentration of salt (e.g., 250 mM KCl) and by the presence of MgCl or glycerol. The Pickering effect can also be used to improve emulsion stability.

[0040] The fragmentation of reverse micelles can be achieved by agitating the solution. For example, mechanical agitation can be applied, such as flushing with a pipette. The fragmentation process allows the size of the reverse micelles to be reduced, increasing the number of reverse micelles and the homogeneity of the emulsion. The resulting reverse micelles are highly heterogeneous in size, on the order of several hundred micrometers.

[0041] To obtain emulsions containing reverse micelles with a surface area polydispersity in the range of 0.015 μm to 150 μm, preferably 0.020 μm to 150 μm, more preferably 1 to 150 μm, a second comminution step can be applied. This second step of comminution can be, for example, mechanical comminution, such as by rapidly transferring the solution containing the reverse micelles between two syringes.

[0042] By compressing reverse micelles together, the number of lipid bilayers between the micelles can be increased, and therefore the total lipid bilayer surface area in a solution containing reverse micelles can be increased. Increasing the number of ion transport membrane proteins located in the lipid bilayer also allows for better circulation of protons between the micelles. In a preferred embodiment, 1.5 ml of ion transport membrane proteins are used per mL of compressed emulsion. 2 More than 1.75m, preferably 2 / mL or more, preferably 1.9m 2 / mL or more, preferably 2m 2 A step of compressing the reverse micelles together is carried out to reach a total bilayer area equal to 1 / mL.

[0043] According to a method of implementation, the compression step can be achieved by centrifuging the emulsion, for example at 13,400 RPM for 3 minutes, after which the supernatant (mostly lipid solution) is removed.

[0044] Under another embodiment, the present invention provides a method for generating voltage using the nanostructured biomimetic neuromorphic system of the present invention, wherein a solution containing reverse micelles is charged with protons H by an ion transport membrane protein present in the solution. + and applying an ion gradient of ions in a solution containing reverse micelles.

[0045] In a particular embodiment of the method for generating voltage, the ion transport membrane protein contained in the nanostructured biomimetic neuromorphic system is the transmembrane protein NhaA from Escherichia coli or the A167P mutant of the E. coli NhaA protein, and the ions supplied to the solution containing the reverse micelles are sodium ions or lithium ions, respectively, resulting in a sodium gradient or lithium gradient in the 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 may also be referred to as a "battery" in common usage.

[0047] Such batteries can also be used as voltage sources for implantable medical devices, non-implantable medical devices, or nomadic consumer electronic devices. [Brief explanation of the drawings]

[0048] The invention will be better understood from reading the following description, given by way of non-limiting example, and with reference to the following figures: [Figure 1] FIG. 1 shows diagram A of an emulsion obtained after the fragmentation step of the method for producing a nanostructured biomimetic neuromorphic system of the present invention; diagram B of a compressed emulsion obtained after the compression step of the method for producing a nanostructured biomimetic neuromorphic system of the present invention (ion transport membrane proteins not shown); diagram C of a compressed emulsion obtained after the compression step of the method for producing a nanostructured biomimetic neuromorphic system of the present invention (non-activated ion transport membrane proteins shown as black squares); and diagram D of a compressed emulsion obtained after the compression step of the method for producing a nanostructured biomimetic neuromorphic system of the present invention, in which non-activated (black squares) and activated (white squares) ion transport membrane proteins are shown in the presence of an ion gradient of ions that can be specifically exchanged by the ion transport membrane proteins for protons H+. [Figure 2]Figure 2 shows diagram A of one embodiment of the present invention in which the compressed emulsion of the nanostructured biomimetic neuromorphic system of the present invention is placed between a first tank and a second tank, the first tank supplies an ion (Na+) that can be exchanged for protons and transported by an ion transport membrane protein (NhaA), and the second tank is configured to sequester the ion; and diagram B of the same embodiment as in (A) in which an ion gradient (Na+ gradient) exists at an appropriate position in the compressed emulsion by an ion transport membrane protein (NhaA protein) present in the compressed emulsion, transporting Na+ from the first tank to the second tank. [Figure 3] Figure 3A shows an image of a compressed emulsion taken with a confocal microscope equipped with a 63x objective (micelle diameter within the microscope field is 1-5 μm), depicting the movement of protons 2H+ and Na+ ions by the electrogenic antiporter (NhaA protein) in the polarized lipid bilayer between two reverse micelles, where Na+ ions are supplied to the compressed emulsion by the cathode (acting as the first reservoir) and sequestered by the anode (acting as the second reservoir). Figure 3B shows an image of a compressed emulsion taken with a confocal microscope equipped with a 63x objective (micelle diameter within the emulsion is 1-5 μm), depicting the movement of protons 2H+ and Na+ ions. Figure 3C shows an image of the compressed emulsion taken with a confocal microscope equipped with a 63x objective, depicting the movement of protons 2H+ and Na+ ions. Figure 3D shows a schematic diagram of the compressed emulsion at a smaller magnification than in C, depicting the movement of protons 2H+ and Na+ ions and where Na+ ions are supplied to the compressed emulsion by the cathode and sequestered by the anode. [Figure 4] FIG. 4 shows a longitudinal section of a fuel cell (A) comprising a hermetically sealed envelope enclosing the nanostructured biomimetic neuromorphic system of the present invention in the form of a compressed emulsion, a cathode, and an anode, and a cross section of the battery (B). [Figure 5] FIG. 5 shows a diagram illustrating a mathematical model of ion transport by NhaA protein as an ion transport membrane protein incorporated into the lipid bilayer between two reverse micelles. [Figure 6] Figure 6 shows a graphical representation of the steady-state H + ion gradient (ΔpH) with respect to the number of contacting reverse micelles in a series derived from a model of a nanostructured biomimetic neuromorphic system. [Figure 7] FIG. 7 shows a graphical representation of the steady-state voltage gradient (ΔVm) with respect to the number of contacting micelles in a series from a model of a nanostructured biomimetic neuromorphic system. [Figure 8] FIG. 8 shows a graphical representation of the distribution of cross-sectional areas of micelles in a nanostructured biomimetic neuromorphic system of the present invention.

[0049] In these drawings, references with the same numbers from one drawing to another indicate the same or similar elements. Furthermore, for reasons of clarity, the drawings are not drawn to scale unless otherwise noted. DETAILED DESCRIPTION OF THE INVENTION

[0050] The following description details the fabrication of a nanostructured biomimetic neuromorphic system in which the solution containing reverse micelles is an emulsion. The procedure described in the following description is a preferred procedure for disclosing the present invention. In the detailed description below, the described embodiment is a system in which an ion transport membrane protein transports protons H by back-exchanging at least one ion. + This is an embodiment of the present invention, which is an electrogenic antiporter that allows the transport of from one reverse micelle to another.

[0051] [Procedure for obtaining isolated NhaA protein from E. coli] Protein production followed the procedure 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 E. coli strain C43(DE3) using the plasmid vector pET15b (Novagen®) for overexpression. The cells were cultured in ampicillin-containing LB medium at 37°C until the optical density at 600 nm (OD600) reached 0.4. Next, 200 μM IPTG (isopropyl-β-D-1-thiogalactopyranoside) was added to the medium to induce overexpression of the NhaA gene, followed by further culture for 5 hours. The resulting cells were collected by centrifugation at 8000 rpm for 5 minutes to form a pellet.

[0052] For purification, the pellet was first incubated for 30 minutes with binding buffer containing 20 mM Tris, 500 mM KCl, 10 mM imidazole, and 12.6% (v / v) glycerol, plus lysozyme and benzonase nuclease. The incubated cells were disrupted in a French pressure cell and subsequently centrifuged at 14,000 rpm for 20 minutes, followed by ultracentrifugation at 36,000 rpm for 2 hours to separate the membrane fraction from the soluble components. The membrane fraction was then resuspended in binding buffer containing an additional 20 mM DDM and incubated overnight. Purification was performed using an immobilized metal affinity chromatography column (Ni-NTA agarose, Qiagen). The column was first equilibrated with binding buffer, and the membrane fraction was then incubated for 2 hours. After rinsing with wash buffer, purified NhaA was recovered in an elution buffer of 20 mM Tris, 500 mM KCl, 300 mM imidazole, 12.6% (w / v) glycerol, and 225 μM DDM at a concentration of 0.5–2 mg / mL, preferably 0.75 mg / mL.

[0053] [Procedure for obtaining lipid solution] The lipid solution is composed of lipids, which are first dissolved in hexadecane at a concentration of 350 mg / mL to 1 g / L, e.g., 500 mg / mL. The lipids used can be standard mixtures of lipids such as asolectins (phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC)), pure lipids, or mixtures of lipids at different concentrations (e.g., PC / cardiolipin (CL)). Alternatively, plant lipids such as monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) can be used. Alternatively, lipids derived from archaea (diphytanyl lipids, ether lipids), or synthetic lipids can be used. Archaeal lipids advantageously provide greater stability for lipid solutions and emulsions. Asolectins are emulsifiers.

[0054] This first solution is then dissolved 1 / 100 in mineral oil, which contains a mixture of saturated alkanes (C7-C40) to obtain a lipid concentration of at least 3.5 mg / mL, typically 5 mg / mL.

[0055] [Procedure for obtaining aqueous solution] To obtain an aqueous solution, add 10–60 µL of NhaA protein (ideally 40 µL) derived from 0.75 mg / mL NhaA protein in elution buffer obtained in the purification step to a previously prepared 500 µL solution containing:

[0056] Tris (25 mM, pH 7) (Na, although other types of buffers, such as potassium phosphate buffer or Hepes, can be used) + (This prevents the formation of NaOH and the pH from balancing.) -KCl (250 mM) (150-300 mM, Na + or Li + (Other salts can be used except for Na.) Above 300 mM there is a risk of lipid instability, and below 150 mM + When adding 100 mg of ethanol, there is a risk that the overall osmolarity will change. -Glycerol (3%) (0-3%) -MgSO4(1%)(0-1%)

[0057] [Preparation of nanostructured biomimetic neuromorphic systems in which solutions containing reverse micelles are compressed emulsions] First, add 1 mL of the lipid solution prepared as described above to the tube. This lipid solution represents the hydrophobic phase of the ongoing emulsion.

[0058] Next, 500 μL of the aqueous solution containing NhaA protein (an ion transport membrane protein) prepared as described above is added dropwise to the tube using a pipette. The aqueous solution represents the aqueous phase of the ongoing emulsion. The aqueous phase is the discontinuous phase of the emulsion. A volume ratio of lipid solution / aqueous solution equal to 2 is important.

[0059] The aqueous droplets are immediately surrounded by bipolar lipids and remain individualized at the bottom of the tube. This process leads to the formation of reverse micelles in the solution, which can be called emulsions. In the presence of DDM surfactant, the NhaA electrogenic protein present in the aqueous solution spontaneously localizes in lipid bilayers formed at the contact points between the reverse micelles. The NhaA electrogenic protein is randomly oriented.

[0060] The next step corresponds to the first fragmentation step, which fragments the reverse micelles, reducing their size and increasing the number of reverse micelles and the homogeneity of the emulsion. To achieve this step, the solution containing the reverse micelles is mechanically stirred using a pipette flushing technique. Approximately 20 flushes are performed with the pipette. The emulsion takes on a milky / milky appearance. The resulting reverse micelles are very heterogeneous in size, on the order of several hundred microns. The emulsion is now homogeneous.

[0061] To obtain an emulsion with a polydispersity ranging from 1 to 150 μm and a bell curve shape with a maximum value around 1 μm, a second break-up step is applied. This second break-up is achieved mechanically by rapidly transferring the emulsion between two syringes.

[0062] The resulting emulsion 10 (FIG. 1A) contains twice the amount of lipid solution 11 (33% aqueous solution and 66% lipid solution) as aqueous solution 12.

[0063] Next, the reverse micelles are compressed together to increase the number of lipid bilayers 13 across the entire surface of the emulsion 10 and the number of ion transport membrane proteins 15 located within the lipid bilayers (improving proton circulation between the reverse micelles 14). This process is accomplished by centrifuging the emulsion in a tube at 13,400 RPM for 3 minutes. The supernatant (mostly lipid solution) is then discarded. The tube is left with a compressed emulsion 10 (Figures 1B and 1C) with the ion transport membrane proteins 15 located within the lipid bilayers 13. This compressed emulsion 10 contains 92% aqueous solution 12 and 8% lipid solution 11. In the compressed emulsion 10, the compressed reverse micelles 14 have a polyhedral shape. The logarithmic distribution of the surface area of ​​the reverse micelles can be obtained by analyzing the emulsion image (Figure 8).

[0064] [Generation of voltage by compressed emulsion of the present invention] As described above, the lipid solution 11 was almost completely removed. Only 8% of the lipid solution 11 remained in the compressed emulsion 10, revealing the hydrophobic phase of the lipid solution. The hydrophobic phase is revealed by the interfaces between the reverse micelles. These interfaces are lipid bilayers 13 connected in a network. Because the hydrophobic phase is insulating, and the conductive aqueous phase revealed by the aqueous solution 12 is sequestered in the reverse micelles 14, the nanostructured biomimetic neuromorphic system in the form of the compressed emulsion 10 is also insulating.

[0065] Ion transport membrane proteins 15 provided by the aqueous solution are localized in the lipid bilayer 13 formed at the contact points between the reverse micelles 14. On either side of the lipid bilayer 13, L + or Na + And, H + When there is no ion difference between the two, no ion movement occurs, and ion transport membrane proteins 15 are not activated (black squares in Figure 1C).

[0066] Ion transport membrane protein 15 inhibits H + ions specifically exchangeable for, e.g., Na in the NhaA protein + or Li + When lipid bilayer 13 (Figure 1D) is supplied to the compressed emulsion 10, ion transport membrane proteins 15 are activated only if there is an ion difference on either side of the lipid bilayer 13 (white squares in Figure 1D). Ion transport membrane proteins 15 located on the side of the lipid bilayer 13 separating two regions of equal ion concentration remain inactive (black squares in Figure 1D). If present, a difference in ion concentration creates directional ion movement, thus generating an ion gradient and voltage.

[0067] In a particular embodiment, to generate a voltage by applying an ion gradient to a solution containing reverse micelles (here, compressed emulsion 10), the nanostructured biomimetic neuromorphic system of the present invention transports protons H via ion transport membrane proteins 15 present in the compressed emulsion 10. + The emulsion 10 includes a first tank 16 configured to supply exchangeable ions specifically to the emulsion 10 and a second tank 17 configured to sequester the ions (FIG. 2A), with the first tank 16 and the second tank 17 each being disposed on one side of the compressed emulsion 10. Of course, the first tank 16 is in contact with the compressed emulsion 10, allowing ions supplied by the first tank 16 to pass through the first tank 16 into the compressed emulsion 10. The second tank 17 is also in contact with the compressed emulsion 10, allowing ions that have passed through the compressed emulsion 10 via the ion transport membrane protein 15 to be captured in the second tank 17.

[0068] The ions supplied by the first tank 16 and sequestered by the second tank 17 are transported by the ion transport membrane protein 15, which is an NhaA protein, or Na + Or Li + Another ion transport membrane protein15 transports protons during the exchange of ions, e.g., Na + ions or Li + In the embodiment shown in Figures 2A and 2B, the ions that are delivered and sequestered are Na + It is an ion.

[0069] As shown in FIG. 2B, sodium Na is transported by ion transport membrane proteins 15 located within the lipid bilayer 13 and by the difference in ion concentration, first between the first reservoir 16 and the reverse micelles 14, and then between the different reverse micelles 14. + leaves the first tank 16, passes through the compressed emulsion 10 and is isolated in the second tank 17, thereby creating a sodium gradient in the compressed emulsion 10.

[0070] In the embodiment depicted in FIG. 3, the ion transport membrane protein 15 is the NhaA protein, and the ion gradient used is sodium (Na + ) gradient (Figure 3A). As shown in Figures 3A, 3B, 3C, and 3D, Na + is on the right side of the lipid bilayer 13, and H + moves to the left (ion movement in a certain direction).

[0071] In certain embodiments, the nanostructured biomimetic neuromorphic system includes capacitive or electrochemical oxidation / reduction electrodes serving as first and second reservoirs (16, 17), a cathode 18 (serving as first reservoir 16), and an anode 19 (serving as second reservoir 17) (FIG. 3D). These electrodes allow the generated ion gradient to be converted into an electric current. Accordingly, one aspect of the present invention is preferably a fuel cell 20 (also commonly referred to as a battery) containing the nanostructured biomimetic neuromorphic system in the form of compressed emulsion 10, cathode 18, and cathode 19 (FIGS. 4A, 4B). Preferably, fuel cell 20 is implantable in a mammal for powering an implantable medical device.

[0072] [Fuel cells containing nanostructured biomimetic neuromorphic systems in the form of emulsions: realization of implantable fuel cells] In a particular embodiment, the fuel cell 20 includes a hermetically sealed envelope 21 that encapsulates the nanostructured biomimetic neuromorphic system in the form of a compressed emulsion 10 , a cathode 18 , and an anode 19 .

[0073] The envelope 21 is permeable to ions and molecules. The envelope 21 provides the advantage of preventing other components of the fuel cell 20 from coming into contact with the mammalian body, thereby making the fuel cell 20 implantable within the mammalian body. By way of example, the envelope 21 is made of polyvinyl alcohol (PVA) hydrogel. PVA hydrogel is permeable to ions and molecules. The envelope 21 preferably takes the form of a sealed tube (FIG. 4).

[0074] The cathode 18 includes an inert support 22 (which provides ion and electrical conduction) covered with a conductive material 23 and impregnated with a capacitive material 24, and a first collector 25 located within the thickness of the inert support 22.

[0075] In this embodiment, the passive support 22 has a hollow cylindrical shape and is made of cross-linked polyurethane foam. The polyurethane foam is covered with a conductive material 23, which is a conductive porous carbon layer, and is impregnated with a capacitive material 24, which is poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). At least one, and preferably six, gold wires act as collectors 25 located within the thickness of the passive support 22. Instead of gold, the wires could be made of, for example, tinned copper.

[0076] The first collector 25 extends from the passive support 22, through the envelope 21, to the outside of the fuel cell 20. The first collector 25 is covered at least over the portion that extends from the passive support 22 to the outside of the fuel cell 20. The outer casing 26 of the first collector 25 is preferably made of polyester.

[0077] The increased surface area cathode 18 is in intimate contact with a number of polarized lipid bilayers 13 of the compressed emulsion 10 .

[0078] The anode 19 includes an ion-permeable cover 27 enclosing an elongated strip of microporous conductive material 28 and a second collector 29 positioned within the thickness of the microporous conductive material 28 .

[0079] The ion-permeable cover 27 is preferably made from PVA hydrogel.

[0080] In this embodiment, the microporous conductive material 28 comprises a mixture of microporous carbon (20%-80%), molybdenum disulfide (MoS) (20%-80%), reduced graphene oxide (rGO) (20%-80%), and polyacrylic acid (PAA) (5-40%).

[0081] Sodium ions can be sequestered by molybdenum disulfide in the anode in combination with microporous carbon and reduced graphene oxide.

[0082] At least one, and preferably six, gold wires act as second collectors 29 located within the thickness of the microporous conductive material 28. Instead of gold, the wires can be made of, for example, tinned copper.

[0083] The second collector 29 extends through the cover 27, from the cover 27 through the envelope 21, and to the outside of the fuel cell 20. The second collector 29 is covered at least in part from the cover 27 to the outside of the fuel cell 20. The outer casing 30 of the second collector 29 is preferably made of polytetrafluoroethylene (PTFE), also known as Teflon.

[0084] The anode 19, with its surface area in intimate contact with the numerous polarized lipid bilayers 13 of the compressed emulsion 10, is positioned approximately at the center of the hollow cylindrical inert support 22 of the cathode 18, with the compressed emulsion 10 between the cathode 18 and the anode 19.

[0085] [Cathode 18 Production] The cathode 18 is preferably formed by utilizing an inert support 22, a porous material, here a polyurethane foam, rendered conductive by impregnation with a conductive material 23 (here, a conductive ink). The conductive ink is made by combining activated carbon (100 mg to 300 mg), MoS2 (200 mg), rGO (40 mg), and PEDOT:PSS (100 μL). PEDOT:PSS is a polymer blend of two ionomers, poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(styrenesulfonate) (PSS). Preferably, impregnation of the polyurethane foam is aided by the addition of a detergent, such as Tween® 20, diluted to 0.8% to 20% in distilled water. Alternatively, cathode 18 can be constructed using polyurethane foam as inert support 22, such as foams designed for use as activated carbon filters and available from electronics suppliers known to those skilled in the art, which already contain activated carbon, thereby rendering the polyurethane conductive. In this alternative construction method, the conductive polyurethane foam is coated with a solution containing PEDOT:PSS, ethylene glycol (200 μL), and the detergent Tween® 20 (1% diluted in distilled water).

[0086] The uncovered portion of the first collector 25 is then inserted into the polyurethane foam.

[0087] [Production of Anode 19] The anode is fabricated with several components to provide a microporous conductive material 28 in paste form. These components for forming the paste include activated carbon (100 mg-300 mg), MoS2 (200 mg), rGO (40 mg), PAA (200 mg), and distilled water (4 mL). Advantageously, the conductivity of the paste can be enhanced by adding to the paste a solution (100 μL) of PEDOT:PSS, a polymer blend of two ionomers, poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(styrenesulfonate) (PSS).

[0088] Once the microporous conductive material 28 is prepared, it is stripped and grooved along its length, the uncovered portion of the second collector 29 is then inserted into the groove, and the microporous conductive material 28 is pleated over itself along its length to completely cover the portion of the second collector 29 located within the groove.

[0089] The microporous conductive material 28 is then sealed within a PVA hydrogel cover 27. The material is hermetically sealed by the self-healing properties of the PVA hydrogel. The covered portion of the second collector 29 extends through the cover 27 to the outside of the cover 27.

[0090] [Fuel cell manufacturing] The anode 19 is inserted into the center of the hollow cylindrical foam cathode 18 .

[0091] Next, both the cathode 18 and anode 19 are inserted into a tubular envelope 21 within the PVA hydrogel. The envelope 21 is then filled with the compressed emulsion 10 and hermetically sealed by the self-healing PVA hydrogel. The cathode 18 and anode 19 are positioned such that the compressed emulsion 10 is between them (FIGS. 4A and 4B).

[0092] The covered portions of the first collector 25 and the second collector 29 extend through the envelope 21 to the outside of the envelope 21. The covered portions of the first collector 25 and the second collector 29 can be connected to, for example, an implantable or non-implantable medical device.

[0093] Using a nanostructured biomimetic neuromorphic system in the form of a fuel cell, the cathode 18 is H + ions, and the anode 19 captures Na + In fact, when the fuel cell 20 is operating, Na +Since the ions are permeable to ions and molecules, by passing through the envelope 21, they can be absorbed from the outside of the envelope 21 (any Na + These Na ions entering the fuel cell 20 are brought into the fuel cell 20 (inside the envelope 21) from the ion source. + The ions pass through the compressed emulsion 10 and are captured by the anode 19, creating a sodium ion gradient within the compressed emulsion 10. The sodium ion gradient creates a flow of H + An ion gradient is generated and the cathode 18 is charged with H + The cathode 18 and the anode 19 capture the generated ions Na + and H + The gradient is converted into a current. Thus, an electronic device can be powered by the fuel cell 20 when the electronic device is connected to the first collector 25 and the second collector 29.

[0094] [Calculation of the optimal number of reverse micelles in nanostructured biomimetic neuromorphic systems] The optimal number of reverse micelles in nanostructured biomimetic neuromorphic systems was characterized by ion transport modeling of biomimetic neurophilic systems (Figure 5), which showed that the number of reverse micelles and the H + The relationship with the steady-state gradient of ions (pH) is shown in Figure 6. The pH gradient also corresponds directly to the voltage generated across the membrane (Figure 7).

[0095] The present inventors developed a mathematical model of ion transport by NhaA protein as an ion transport membrane protein located in the lipid bilayer membrane between two reverse micelles.

[0096] As shown in Figure 5, Na + and H + Ions compete with each other in reverse micelle 1 with binding rates a and c, respectively, to bind to the active site C of the NhaA ion transport membrane protein. 12At this stage, the NhaA antiporter has an active site in reverse micelle 1 and an inactive site in reverse micelle 2. For simplicity, we will use H + 1 represents two protons, Na + Let 1 be a sodium ion in the reverse micelle 1. The NhaA antiporter operates according to the principle of alternating access. An ion binds to a single substrate binding site and forms the complex HC 12 or NaC 12 When either of these is generated, both the ion and the active site undergo a thermodynamically favorable conformational change, respectively. 12 and f 21 This mechanism of active site transfer prevents any further ion leakage. In reverse micelle 2, the complex HC 21 and NaC 21 At the rates of b and d, respectively, the active site C 21 , and ion H + 2 and Na + Dissociates into 2. pH is H + To measure the amount of ions, Na + and H + Ion competition has been shown to be sufficient to explain the pH dependence of the NhaA antiporter without invoking the presence of an active pH-sensing mechanism.

[0097] We first investigated the case of transport through a single membrane, then through a series of simple reverse micelles, and finally through complex 3D geometries. In this model, all reverse micelles in the series are assumed to have the same volume. In general, the volume of a reverse micelle can be much larger than the average diffusion distance of ions. Therefore, we included the possibility that ions can diffuse even in the bulk of the reverse micelle, where they cannot react with the active sites of the ion transport membrane proteins (i.e., ions can move far away from the membrane where the ion transport membrane proteins are located). Surface H + , Na + ions in the bulk and H +* , Na +*The velocity of the ions diffusing at the axial direction is λ and γ, respectively.

[0098] The system of differential equations (ODE system 1) describing the time evolution of the concentrations of the components (measured in [nM]) in reverse micelle 1 is as follows:

number

[0099] Table 1 shows all these values: [Table 1] Adding a series of new reverse micelles requires adding a similar equation to the conventional differential equation system (ODE system 1) with an exponent corresponding to the number of reverse micelles.

[0100] The pH value of each reverse micelle is calculated by the following formula: + It is indicated by the concentration of ions:

number

[0101] In calculating pH, only available ions (in the bulk and on the surface of the reverse micelles) are included, not ions found in complex with ion transport membrane proteins (because they cannot move).

[0102] Figure 6 shows the steady-state activated H with respect to the number of contacting reverse micelles in a series derived from a model of the nanostructured biomimetic neuromorphic system of the present invention. + A graphical representation of the ionic gradient (ΔpH) is shown. The system starts at pH 7 (at time t=0) and+ The starting concentration of ions is 0.1 mM. The concentration of NhaA protein in each micelle is 0.01 mM.

[0103] Using patch clamp experiments, it is possible to measure the membrane voltage, i.e., the voltage across which a voltage difference can be created to induce and harness a current. The membrane voltage between two reverse micelles is given by the Goldmann equation:

number

[0104] Figure 7 shows a graphical representation of the steady-state activated voltage gradient (ΔVm) with respect to the number of contacting reverse micelles in a series derived from a model of a nanostructured biomimetic neuromorphic system of the present invention. This system is modeled after the same parameters as shown above with respect to Figure 6 (pH (at t = 0), Na in each micelle, + The starting concentration is 0.05% (the concentration of ions and the concentration of NhaA protein).

[0105] The ODE system 1 represents a functional unit that is repeated several times in the reverse micelle series. The number of variables and equations increases by 10N-6 as the number N of micelles in the series increases. It is clearly impossible to write a new conventional differential equation system every time.

[0106] [Calculation of total bilayer area in compressed emulsions] It can be assumed that during the compression process, the excess liquid in the reverse micelles dries out by forming bilayers. In this case, the emulsion should resemble a dry foam. For a dry foam, the most favorable conditions at equilibrium follow Plateau's law, so the micelles organize with three bilayers meeting at each planar section, forming a vertex angle of 120° in two dimensions and 109.5° in the third dimension.

[0107] If we start with a volume distribution according to the uncompressed observations and assume that the reverse micelles coalesce little during the compression process, then the final compressed micelles can each have a different volume and surface area. This resembles a polydisperse foam. Thus, each reverse micelle shares surface area with neighboring reverse micelles of different dimensions and can accommodate different amounts of ion transport membrane proteins. For simplicity's sake, we will assume that each reverse micelle has an average volume, V m and average surface area A m A control parameter C is introduced to control the degree of compression of the reverse micelles.

[0108] For example, a homogeneous free micelle minimizes its energy state, adopting a spherical conformation and having a volume of:

number

[0109] The volume is V B If we imagine a box filled with reverse micelles, the smallest conformation in which the space is filled by spheres contacting other spheres on each side is when such a micelle is inscribed in a cube, so that several bilayers form at least six points on the spheres. We estimate this to be the minimum case that ensures continuous ion transport. Other structures with fewer contact points, orders of magnitude smaller, are also possible. Thus, the volume occupied by a reverse micelle is:

number

[0110] We can imagine compressing a micelle, changing its shape to fit a cube and keeping the volume constant. In such a case, the occupancy is:

number

[0111] Generally, Nm is the volume V b can be estimated to be equivalent to an inverse micelle in a box with

number

[0112] Considering the packing ratio C, a different number of reverse micelles can be assigned to a certain number of boxes depending on the compression ratio C.

number

[0113] The maximum average fractional density (largest fraction of volume) occupied by the spheres in high-density compression was shown to be C = 0.74 of the total volume. By deforming the micelles, the packing fraction C can be made larger, up to 1.

[0114] When compressed, the total volume of the reverse micelles remains the same, but because the shape is no longer spherical, the surface area can be estimated differently from the usual formula for calculating the area of ​​a sphere. The most common 3D shape observed in foams has been shown to be an irregular polyhedron with 13 sides. For simplicity, it can be assumed that only regular shapes exist. From computational modeling, it is known that the average surface area is:

number

[0115] In a sphere, b is equal to 4.8.

[0116] Considering that the volume remains constant during the compression process, the area changes, and the excess area is redistributed among the rest of the solution, an equivalent way to formulate the relationship is as a function of the uncompressed free spherical reverse micelle:

number

number

[0117] By measuring the average reverse micelle radius before compressing the emulsion, R mUN This is a convenient method since σ can be obtained by experiment (assuming no coalescence occurs during this process).

[0118] According to the above equations (Equation 4 and Equation 10), the area of ​​a non-spherical reverse micelle is:

number

[0119] In a sphere, c is equal to 3.

[0120] Then, during the compression step, compression of some of the reverse micelles occurs, and in this process c becomes greater than 3:

number

[0121] Next, we know that:

number

[0122] For C less than π / 6, we can infer that c is equal to 3, since the micelles are not in full contact with each other and are still spherical. For C greater than π / 6 but less than 1, the compression increases, so c is greater than 3, but c max is smaller than . Here, we can estimate the following linear relationship:

number

[0123] The total area of ​​all reverse micelles is Nm × Am. When reverse micelles come into contact, i.e., when C is greater than π / 6, they begin to form a bilayer. If C is less than π / 6, no bilayer is formed; if C is greater than π / 6 but less than 1, the total area of ​​the bilayer is:

number

[0124] Although h must have a complex relationship with C, it can be estimated that h is linear with C in the range where C is greater than π / 6 and less than 1.

number

[0125] Next, we have the following formula:

number

[0126] It is then possible to realize plots showing c, h, and ABIm as a function of the compression factor C. In a 1 mL box, at maximum compression, the total area of ​​the bilayer is approximately 2 m 2 is.

[0127] More generally, it should be noted that the modes of implementation and realization of the invention considered above are described as non-limiting examples, and that other variations are therefore possible.

Claims

1. A nanostructured biomimetic neuromorphic system comprising a solution containing reverse micelles (14) that contact each other to form a lipid bilayer (13) at the contact points, at least a portion of the contact points containing at least one ion transport membrane protein (15) that enables the transport of at least one ion from one reverse micelle (14) to another reverse micelle (14) by reverse exchange of at least one ion, and the solution containing the reverse micelles (14) is a compressed emulsion.

2. The nanostructured biomimetic neuromorphic system of claim 1, comprising at least 15 reverse micelles (14) that contact to form a series of reverse micelles (14), each contact point between the reverse micelles (14) comprising at least one ion transport membrane protein (15).

3. 3. The nanostructured biomimetic neuromorphic system of claim 1 or 2, wherein the ion transport membrane protein (15) is an electrogenic antiporter.

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

5. 5. The nanostructured biomimetic neuromorphic system of claim 4, wherein the ion transport membrane protein (15) is the transmembrane protein NhaA from Escherichia coli or the A167P mutant of the E. coli NhaA protein.

6. A nanostructured biomimetic neuromorphic system according to any one of claims 1 to 5, comprising a first tank (16) that supplies ions transportable by the ion transport membrane protein (15) that exchanges at least one ion, and a second tank (17) that segregates the ions transportable by the ion transport membrane protein (15), wherein the first tank (16) and the second tank (17) are each disposed in one of the solutions containing the reverse micelles (14).

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

8. The ion transport membrane protein (15) transports calcium ions Ca by back-exchange of at least one ion. 2+ 8. The nanostructured biomimetic neuromorphic system according to claim 1, wherein the nanostructured biomimetic neuromorphic system is an electrogenic antiporter that allows the transport of a molecule from one reverse micelle to another.

9. The ion transport membrane protein (15) transports protons H by back-exchange of at least one ion. + 8. The nanostructured biomimetic neuromorphic system according to claim 1, wherein the nanostructured biomimetic neuromorphic system is an electrogenic antiporter that allows the transport of a molecule from one reverse micelle to another.

10. The at least one ion that can be transported by the ion transport membrane protein (15) in exchange for a proton is sodium (Na + ) and lithium (Li + 10. The nanostructured biomimetic neuromorphic system of claim 9, wherein the nanostructured biomimetic neuromorphic system is selected from the group consisting of:

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

12. 11. The nanostructured biomimetic neuromorphic system of any one of claims 1 to 10, which is in contact with living cells and mammalian body tissues and used to sense ionic and chemical responses of cells and tissues or to provide ionic signals to affect biological responses of cells and tissues.

13. - preparing a lipid solution (11); - adding dropwise to the lipid solution an aqueous solution (12) containing an ion transport membrane protein (15), in a lipid solution / aqueous solution volume ratio of 2, thereby preparing a solution containing reverse micelles (14), which is an emulsion (10); - fragmenting the reverse micelles (14) to reduce the size of the reverse micelles (14) and increase the number of the reverse micelles (14) and the homogeneity of the emulsion (10); - compressing said reverse micelles (14) together to increase the surface of the lipid bilayer (13) produced; A method for manufacturing a nanostructured biomimetic neuromorphic system according to any one of claims 1 to 10, comprising:

14. The solution containing the reverse micelles (14) is charged with protons H by the ion transport membrane protein (15) present in the solution. + 11. A method for generating voltage using the nanostructured biomimetic neuromorphic system of claim 9 or 10, comprising the step of supplying exchangeable ions specifically to the reverse micelles (14) and applying an ion gradient of the ions in a solution containing the reverse micelles (14).

15. 15. The method for generating a voltage according to claim 14, wherein the ion transport membrane protein (15) contained in the nanostructured biomimetic neuromorphic system is the transmembrane protein NhaA from Escherichia coli or the A167P mutant of the E. coli NhaA protein, and the ions supplied to the solution containing the reverse micelles (14) are sodium ions or lithium ions, respectively, resulting in a sodium gradient or lithium gradient in the solution.

16. A fuel cell (20) comprising a nanostructured biomimetic neuromorphic system according to any one of claims 1 to 10.

17. 17. The fuel cell (20) of claim 16 for use as a voltage source for an implantable medical device, a non-implantable medical device, or a nomadic consumer electronic device.

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