A microelectrode element for being implanted into tissues such as a neural tissue or for being brought into contact with a single cell, and its manufacturing method

Abstract

A microelectrode element (A) for being implanted into tissues such as neural tissues or for being brought into contact with a single cell, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell, wherein the microelectrode element comprises a first fibrous layer (1), an electrically conductive layer (2), a second fibrous layer (3), and a polymer compound layer (4), wherein each of the layers (1, 2, 3, 4) extends in an xy-plane being perpendicular to a z-direction and has a thickness in the z-direction, wherein the polymer compound layer (4), the first fibrous layer (1) and the electrically conductive layer (2) are stacked in this order in the z-direction, and wherein the second fibrous layer (3) is formed on at least a part of a surface of the electrically conductive layer (2), wherein each of the first fibrous layer (1) and the second fibrous layer (3) comprises nanofibers and / or microfibers, wherein the polymer compound layer (4) is capable to form a hydrogel.

Description

DESCRIPTION TITLE A microelectrode element for being implanted into tissues such as a neural tissue or for being brought into contact with a single cell, and its manufacturing method TECHNICAL FIELD

[0001] The present disclosure relates to a microelectrode element for being implanted intotissues such as a neural tissue or for being brought into contact with a single cell, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell, and a method for manufacturing the microelectrode. BACKGROUND

[0002] In current practice, implantable microelectrodes are used for neuroscience, for alleviatingsymptoms of neurological diseases, such as Parkinson’s disease or epilepsy, or for restoring body functions after injury. Implantable microelectrodes are brought into the body of the patient via surgery and are used for recording from or stimulating the neural target tissue and nerves. Examples of implantable microelectrodes are stimulation electrodes that are used for deep brain stimulation. Other examples are cuff electrodes used for interfacing nerves and electrodes used for electrocorticography.

[0003] US2021 / 0270764 A1 discloses a microelectrode having a layered structure, including alayer containing a polymer compound having an aromatic ring (polymer compound layer) and a layer containing a conductive material (conductive layer), and the microelectrode has a three-dimensional curved shape. Such a microelectrode having a small dimension (nano- and micro-meter range) would be generally difficult to handle during an operation, for example, when implanting the microelectrode into a tissue. A further challenge may be accurate fabrications of such three-dimensional structures of the microelectrode. SUMMARY OF INVENTION

[0004] The technical problem to be solved may be formulated to provide a microelectrode elementfor being implanted into tissues such as a neural tissue or for being brought into contact with a single cell, which provides a stable, reliable, and controlled structure and may realize more accurate handling by a user.

[0005] A microelectrode element (A) according to the present disclosure is for being implantedinto tissues such as a neural tissue or for being brought into contact with a single cell, in order to apply and / or record an electrical signal to and / or from the tissue or the single cell. The microelectrode element according to the present disclosure may be for example a cuff electrode that is able to interface nerves with a diameter of about 50 to 500 µm. The microelectrode element may be used, for example, for recordingsignals from the nerve tissue in the mV range or V range, or for stimulation of the nerve tissue with current pulses in the µA to mA range or with voltage pulses in the mV or V range.

[0006] The microelectrode element (A) comprises a first fibrous layer (1), an electricallyconductive layer (2) (and / or a semiconductive layer), a second fibrous layer (3), and a polymer compound layer (4). Each of the layers (1, 2, 3, 4) extends in an xy-plane (i.e. in an x-direction and a y-direction). The xy-plane is perpendicular to a z-direction. Each of the layers has a thickness in a z-direction. The x-, y-, z- axes and / or directions are of the three-dimensional rectangular coordinate system, which are orthogonalto each other. Each of the layers (1, 2, 3, 4) has a first surface and a second surface extending in the x-direction and the y-direction (and / or in an xy-plane being perpendicular to the z-direction), and the second surface is opposite to the first surface in the z-direction. The polymer compound layer (4), the first fibrous layer (1) and the electrically conductive layer (2) are stacked in this order in the z-direction. The second fibrous layer (3) and the electrically conductive layer (2) are stacked in the z-direction and the second fibrous layer (3) is formed on (and / or covers) at least a part of a surface of the electrically conductive layer (2). The polymer compound layer (4), the first fibrous layer (1), the electrically conductive layer (2) and the second fibrous layer (3) may be stacked in this order in the z-direction. The first surface of the polymer compound layer (4), the second surface of the polymer compound layer (4), the first surface of the first fibrous layer (1), the second surface of the first fibrous layer (1), the first surface of the electrically conductive layer (2), the second surface of the electrically conductive layer (2), the first surface of the second fibrous layer (3), the second surface of the second fibrous layer (2) are arranged in this order in the z- direction. The second fibrous layer (3) may only partly cover the second surface of the electrically conductive layer (2), or the second fibrous layer (3) may cover the entire second surface of the electrically conductive layer (2). Each of the layers may be in direct contact with its adjacent layer or an additional sub- layer may be formed between the two layers. For example, the second fibrous layer (3) may be formed directly on the electrically conductive layer (2), and / or the electrically conductive layer (2) may be formed directly on the first fibrous layer (1), and / or the first fibrous layer (1) may be formed directly on the polymer compound layer (4).

[0007] Each of the first fibrous layer (1) and the second fibrous layer (3) comprises nanofibersand / or microfibers. The nanofibers have preferably a diameter between 1 nm to 1 µm, more preferably between 1 to 100 nm, more preferably between 5 to 30 nm. The microfibers have preferably a diameter in a micrometer range. The nanofibers and / microfibers may comprise or consist of fiber segments and / or fiber fragments. Each of the nanofibers and / or microfibers (and / or fiber segments and / or fiber fragments thereof) preferably have a length of 1 µm to 1 mm, more preferably between 1.5 µm to 950 µm. A nanofiber may be a fiber with a diameter less than 100 nm and an aspect ratio exceeding 100. The first fibrous layer (1) and the second fibrous layer (3) may consist of the same components. Preferably, the main component of each of the first fibrous layer (1) and the second fibrous layer (3) is nanofibers and / or microfibers. Preferably, the volumetric percentage of nanofibers and / or microfibers of each of the first fibrous layer (1) and the second fibrous layer (3) is more than 50 %, more preferably, more than 70 %, more preferably, more than 90 %. Alternatively, each of the first fibrous layer (1) and the second fibrous layer (3) consists of nanofibers and / or microfibers.

[0008] The polymer compound layer (4) is capable to (or able to) form a hydrogel. Morespecifically, the polymer compound layer (4) is capable to form a hydrogel, when contacting liquid, forexample liquid containing water molecules, and after having absorbed components of the liquid, for example water molecules. The polymer compound layer (4) is capable to volumetric swell and / or volumetric expands by absorbing components of liquid. A hydrogel(s) represent(s) a cross-linked hydrophilic polymer network(s) that absorb components (e.g. water molecules) from liquid including water molecules but do not dissolve in the liquid when brought into contact with the liquid including water molecules.

[0009] The microelectrode element may be able to curve and / or fold itself when the polymercompound layer (4) contacts liquid and is swelled by the liquid, more specifically, is swelled by absorbing a component of the liquid, e.g. the liquid including water molecules. When contacting the liquid, the microelectrode element may be able to curve such that the second surface of the second fibrous layer is concave (and / or an inner surface of the microelectrode) and the first surface of the polymer compound layer is convex (and / or an outer surface of the microelectrode element). Upon contacting the liquid and / or being dipped and / or submerged in the liquid, the microelectrode element preferably curves and / or bends and / or folds itself, such that a radius of curvature of the folded or curved microelectrode element is defined along the z-direction, i.e. along the direction being perpendicular to the second surface of the second fibrous layer (3). A radius of curvature of the folded or curved microelectrode element is preferably 50 to 1000 µm, more preferably 50 to 150 µm.

[0010] The microelectrode element according to the present disclosure may have one or more ofthe following technical effects / advantages:

[0011] As the polymer compound layer (4) is capable to form a hydrogel, the polymer compoundlayer (4) functions as a liquid absorbing layer, while a swelling ratio of each of the first fibrous layer (1) and the second fibrous layer (3) (as well as the electrically conductive layer (2)) is negligible compared to a swelling ratio of the polymer compound layer (4). This structure allows deformations of the microelectrode element being the stack of the layers (1, 2, 3, 4), when the microelectrode element (or at least a part of the polymer compound layer (4)) contacts or is submerged in liquid, e.g. liquid including water molecules. The polymer compound layer (4) expands volumetrically by absorbing components of the liquid, e.g. water molecules, and forming a hydrogel. This creates a stress against other layers, i.e. the first / second fibrous layers and the electrically conductive layer, and the stress is released by curving or folding of the microelectrode element. Each of the first and second fibrous layers is flexible and / or elastic in the sense of that it is be capable of being folded and / or curved without breaking. Since the folding is induced by contact of the microelectrode element and / or the polymer compound layer to liquid, the microelectrode element provides a stable and reliable mechanism of self-folding or self-assembling that is controllable by an external stimulus.

[0012] This characteristic may be further advantageous in that, in case the microelectrode elementis used for an implantable microelectrode such as a microelectrode or cuff electrode for bioelectronics, the implantable microelectrode can wrap around a biological sample such as a nerve tissue via self-folding during implantation without any further measures that otherwise would have to be carried out manually by the operating personnel. The microelectrode element of the present disclosure may be further advantageous in that the folding or curving is reversible. More precisely, if the microelectrode element (or at least the polymer compound layer) in the folded-state is allowed to dry after having contacted liquid including water molecules, the microelectrode element would revert to an unfolded or uncurved state.

[0013] Furthermore, the structure of the microelectrode element in which the electricallyconductive layer is sandwiched by the first and second fibrous layers may be further advantageous in that the electrically conductive layer (2) would be structurally stabilized. For example, the electrically conductive layer (2) may be hydrophobic, which may be negatively affect for a formation of the polymer compound layer (4), and the first and second fibrous layer being more hydrophilic than the electrically conductive layer (3) may facilitate a uniform formation of the polymer compound layer (4) and further biocompatibilities.

[0014] The microelectrode element according to the present disclosure may be furtheradvantageous in that its thin structure in a range of several tens micrometers would be achieved by non- complicated manufacturing processes: The multi-layered structure of the microelectrode element can be obtained by filtering and / or drying liquid suspensions including each of fibers and conductive materials, and forming the polymer compound layer by applying a polymer mixture on the first surface of the first fibrous layer (1) through a polymerization process. These steps may be performed with non-expensive equipment, for example, a filter and a vacuum pump.

[0015] Preferably, the microelectrode element has a length (l) in the x-direction and a width (w)in the y-direction, and the length (l) is longer than the width (w). This structure may be further advantageous in that a direction of folding (or curving) of the microelectrode element stimulated by liquid can be more accurately controlled, such that the axis of curvature (or curving or folding) is defined in or along the x-direction, and the curving radius is defined in a yz-plane being perpendicular to the x-direction. Namely, the microelectrode element would be self-folded (or self-curved) along the length (l) of the microelectrode element, i.e. the longer direction (the main longitudinal direction) of the microelectrode element. Preferably, the ratio (l / w) of the length (l) to the width (w) is more than 2, more preferably more than 4, more preferably more than 6, which may be further advantageous to control the self-folding direction of the microelectrode element.

[0016] Preferably, the second fibrous layer (3) covers the entire surface of the electricallyconductive layer (2) and / or the entire second surface of the electrically conductive layer (2). This feature may be further advantageous in that the microelectrode element would have more stable structure. The first fibrous layer (1) and / or the second fibrous layer (2) may be conductive when the first fibrous layer (1) and / or the second fibrous layer (2) is wet, i.e. when the first fibrous layer (1) and / or the second fibrous layer (2) contain(s) water molecules, and an electrical contact between the electrically conductive layer and a targeted tissue (or an external device) could be established via the first fibrous layer (1) and / or the second fibrous layer (2).

[0017] Preferably, the second fibrous layer (3) covers only a part of the surface of the electricallyconductive layer (2), such that another part of the surface of the electrically conductive layer (2) is contactable. This feature may be further advantageous in that an electrical contact between the electrically conductive layer the electrically conductive layer and a targeted tissue (or an external device) may be established via the other part of the surface of the electrically conductive layer, the other part of the surface being exposed and not covered by the second fibrous layer (2).

[0018] Preferably, the nanofibers and / or the microfibers comprise a thermoset polymer and / or athermoplastic polymer and / or an elastomer and / or a polysaccharide such as cellulose, chitosan, chiton and / or combinations thereof, and / or wherein the nanofibers and / or the microfibers are obtained by an electrospinning method.

[0019] Preferably, the electrically conductive layer (2) comprises (electrically) conductivemacromolecules. The conductive macromolecules preferably comprise carbon nanotubes, wherein preferably the carbon nanotubes are multi-walled, and / or carbon rods, and / or metal nanoparticles, silver nanowires (AgNW) and / or gold nanowires (AuNW) and / or grafene and / or (electrically) conductive polymers, wherein the conductive polymers preferably comprise a conductive polymer mixture of at least two ionomers, such as poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS).

[0020] Preferably, the electrically conductive layer (2) comprises carbon nanotubes, wherein asurface density of the carbon nanotubes is in a range of 1 µg / cm2to 50 µg / cm-2, more preferably 10 µg / cm- 2to 20 µg / cm-2. This range may be especially advantageous to provide an optically transparent electrically conductive layer (2), while maintaining sufficient electrical conductivity.

[0021] Preferably, the polymer compound layer (4) is based on a first component and a secondcomponent. The first component being a hydrophilic monomer and / or a hydrophilic oligomer and / or a hydrophilic polymer and / or a hydrophilic copolymer, the second component being a cross-linker and / or initiator and / or quencher, wherein the first component includes a first functional group, and the first component is capable to be polymerized through the first functional group and the second component. The polymer compound layer (4) is (has been) accordingly polymerized from and / or is obtained by a process of polymerization of a mixture comprising the first component and the second component. The first component may be acrylamide related monomer, such as N-Isopropylacrylamide (NIPAM), and / ornanofiber such as carboxymethyl cellulose (CMC). The second component may be N,N'-Methylenebisacrylamide (MBAA), and / or ammonium persulfate (APS), and / ortetramethylethylenediamine (TEMED).

[0022] Preferably, the polymer compound layer (4) has a swelling ratio in a range of between 1 and10, more preferably between 2 to 3. This range may be especially advantageous in more efficient deformations of the microelectrode element, when the polymer compound layer (4) expands volumetrically by absorbing components of liquid, e.g. water molecules, and forming a hydrogel. A swelling ratio of the polymer compound layer (4) may be defined as a fractional increase in the weight and / or the volume and / or the surface area (of the first surface) of the polymer compound layer (4) due to water absorption. A swelling ratio may be defined as (Ws-Wd) / Wd, wherein Ws represents the weight and / or volume and / or surface area (of the first surface) of the polymer compound layer (4) after swelling in aqueous solution such as deionized water (i.e. saturated by components of aqueous solution), and Wd represents the weight and / or volume and / or surface area (of the first surface) of the polymer compound layer (4) at a dried state (i.e. before swelling and being submerged in aqueous solution). The swelling ratio of the polymer compound layer (4) may be defined by a mixture of the first and second components after polymerization.

[0023] Preferably, the microelectrode element further comprises an insulating layer (5) coveringat least a part of a surface of the microelectrode element (A).

[0024] Preferably, the thickness of the first fibrous layer (1) is in a range of between 1 µm and100 µm, more preferably between 1 µm and 10 µm. Preferably, the thickness of the electrically conductive layer (2) is in a range of between 1 µm and 100 µm, more preferably between 1 µm and 10 µm, more preferably between 1 µm and 3 µm. Preferably, the thickness of the second fibrous layer (3) is in a range of between 1 µm and 100 µm, more preferably between 1 µm and 10 µm. Preferably, the thickness of the polymer compound layer (4) is in a range of 1 um to 1 mm.

[0025] A microelectrode according to the present disclosure includes a microelectrode element (A)according to the present disclosure. The microelectrode comprises an edge portion (B) being attached to at least a part of an outer side surface of the microelectrode element (A), the outer side surface including the electrically conductive layer (2) and extending in the z-direction, wherein, preferably, the edge portion (B) is integrally formed with the microelectrode element (A).

[0026] Preferably, the microelectrode comprises an insulating layer (5) covering at least a part ofa surface of the edge part (B).

[0027] A microelectrode system according to the present disclosure comprises a plurality ofmicroelectrodes according to the present disclosure. The microelectrode system comprises a joint portion (C) connecting each of the plurality of the microelectrodes.

[0028] According to the present disclosure, a method for manufacturing a microelectrode element(A) comprises a step of forming the first fibrous layer (1) by filtering a first liquid suspension (1S) through a filter (F), the first liquid suspension (1S) comprising nanofibers and / or microfibers and a first dispersion medium, the first dispersion medium being deionized water and / or organic solvent. The method further comprises a step of forming the electrically conductive layer (2) on the first fibrous layer (1) by filtering a second liquid suspension (2S) through the first fibrous layer (1) and the filter (F), the second liquid suspension (2S) comprising conductive macromolecules and a second dispersion medium, the second dispersion medium being deionized water and / or organic solvent, a step of forming the second fibrous layer (3) on the electrically conductive layer (2) by filtering a third liquid suspension (3S) through the electrically conductive layer (2), the second fibrous layer (3) and the filter (F), the third liquid suspension (3S) comprising nanofibers and / or microfibers and a third dispersion medium, the third dispersion medium being deionized water and / or organic solvent, a step of forming the polymer compound layer (4) on a surface of the first fibrous layer (1).

[0029] Preferably, the step of forming the electrically conductive layer (2) comprises a step ofcovering a surface of the first fibrous layer (1) by a first mask (M1), a step of applying the first liquid suspension (1S) on the first mask (M1), a step of filtering the first liquid suspension through the first mask (M1), the second fibrous layer (3) and the filter (F), so that the electrically conductive layer (2) being patterned by the first mask (M1) is formed on the second fibrous layer (3), and a step of removing the first mask (M1) after the step of filtering the second liquid suspension (2S).

[0030] Preferably, the step of forming the second fibrous layer (3) comprises a step of covering asurface of the electrically conductive layer (2) by a second mask (M2), a step of applying the third liquid suspension (3S) on the second mask (M2), a step of filtering the third liquid suspension (3S) through the second mask (M2), the electrically conductive layer (2), the second fibrous layer (3) and the filter (F), so that the second fibrous layer (3) being patterned by the first mask (M1) is formed on the electrically conductive layer (2), and a step of removing the second mask (M2) after the step of filtering the third liquid suspension (3S).

[0031] Preferably, the step of forming the polymer compound layer (4) comprises a step ofpreparing a mixture by mixing the first component, the second component and a polymerization agent, and a step of applying the mixture (4S) of the first component, the second component and the polymerization agent on the surface of the first fibrous layer (1). The polymerization agent may include monomer(s) and / or cross-linker(s) and / or initiator(s).BRIEF DESCRIPTION OF THE DRAWING

[0032] Figs. 1A, 2A: Schematic illustrations of a perspective view and a top view of a microelectrode element (A) according to an embodiment, Fig.1B, 2B: Schematic illustrations of a perspective view and a top view of a microelectrode element (A) according to a variation of the embodiment, Figs.3A and 3B: Schematic illustrations of a perspective view and a top view of a microelectrode system including a plurality of the microelectrode elements (A) according to the embodiment, Figs.4(a) to 7(b): Schematic illustrations of steps of a manufacturing method of the microelectrode element as well as the microelectrode system according to the present embodiment, Fig.8: Photos of self-folding of a sample microelectrode element of an example, Fig.9: A photo a sample microelectrode element of an example, Fig.10: Results of conductivity measurements performed with sample microelectrode elements of an example, Fig.11: A cyclic voltammogram performed with a sample microelectrode element of an example, Fig.12: A plot of average curling radii against average swollen thicknesses of sample microelectrode elements of an example. DETAILED DESCRIPTION OF EMBODIMENTS

[0033] Figs. 1A and 2A show schematic illustrations of a perspective view and a top view of themicroelectrode element (A) according to the present embodiment, respectively. The microelectrode element (A) according to the present embodiment comprises a first fibrous layer (1), an electrically conductive layer (2), a second fibrous layer (3), and a polymer compound layer (4). The polymer compound layer (4), the first fibrous layer (1) and the electrically conductive layer (2) are stacked in this order in the z-direction. The electrically conductive layer (2) is accordingly sandwiched by two fibrous layers. The microelectrode element has a length (l) in the x-direction and a width (w) in the y-direction, and according to this embodiment, the length (l) is longer than the width (w), and the microelectrode element (A) has a stripe structure.

[0034] Each of the first fibrous layer (1) and the second fibrous layer (3) comprises nanofibersand / or microfibers, and these fibrous layers are so-called paper-based layers. The first fibrous layer (1) and the second fibrous layer (3) may comprise the same components. For example, the main component (i.e. more than 50 % of the volumetric percentage) of each the first fibrous layer (1) and the second fibrous layer (2) may be the same nanofibers such as polysaccharide. In particular, the first fibrous layer (1) comprising chitin as the main component may be especially advantageous in forming a uniform polymer layer onto asurface of the first fibrous layer (1), as chitin would have a better water holding capacity than other polysaccharide such as chitosan, cellulose and silk.

[0035] The electrically conductive layer (2) according to the present embodiment is formed byconductive macromolecules, such as carbon nanotubes, metal nanowires and conductive polymers. Preferably, the electrically conductive layer (2) comprises carbon nanotubes as the main component. In case the electrically conductive layer (2) is hydrophobic, this structure in which the electrically conductive layer (2) being sandwiched by two fibrous layers may be especially advantageous in forming better contact between the microelectrode element (A) and a surface of a biological sample.

[0036] The polymer compound layer (4) is capable to form a hydrogel. The polymer compoundlayer (4) is polymerized from hydrophilic components and is capable to volumetric expand by absorbing components of liquid, for example water molecules.

[0037] The microelectrode element (A) is able to fold itself (self-fold) when at least the polymercompound layer (4) contacts liquid and is swelled by the liquid, e.g. the liquid including water molecules. This self-folding process can be thus induced only by bringing the microelectrode element (A) (or at least the polymer compound layer) into contact with liquid. The polymer compound layer (2) would volumetric expand by absorbing components of the liquid, e.g. water molecules and the microelectrode element (A) folds such that an outer surface (i.e. the second surface opposite to the electrically conductive layer) of the second fibrous layer (3) is concave (and / or an inner surface of the microelectrode element) and an outer surface (i.e. the first surface opposite to the first fibrous layer) of the polymer compound layer is convex (and / or an outer surface of the microelectrode element). The structure in which the length (l) of the microelectrode element is the width (w) would be especially advantageous in accurate controlling of folding directions. The microelectrode element (A) folds itself, such that its folding axis is along the y-direction and a radius of folding curvature is defined along the z-direction and an xz-plane being perpendicular to the y- direction. The microelectrode element would accordingly folds up in or along the x-direction, i.e. the longer side of the microelectrode element.

[0038] The second fibrous layer (3) may cover only a part of a surface of the electrically conductivelayer (2), so that a part of the electrically conductive layer (2) is exposed and directly contactable, as illustrated in Fig. 1B (a perspective view) and Fig. 2B (a top view). The uncovered part of the electrically conductive layer (2) may be used as a portion for establishing contact with a biological sample. However, even when the electrically conductive layer (2) does not include such an exposed part as illustrated in Figs. 1A and 2A, when the second fibrous layer (3) is wet, namely, when the second fibrous layer (3) contains (sufficient) water molecules between the nanofibers, it is possible to establish electrical contact between the electrically conductive layer (2) and an external object (e.g. a biological sample) through the second fibrous layer (3), as the second fibrous layer (3) have typically a thickness of a micrometer range, e.g. between 1 µm and 100 µm. At least the second fibrous layer (3) may be therefore partly covered by an additional insulating material.

[0039] The thickness of each of the layers of the microelectrode element (A) is typically severalmicrometers, and the total thickness of the microelectrode element is typically between 5 µm to 500 µm. The microelectrode element according to the present embodiment would have a folding curvature typically between 50 µm and 1 mm, which can be controlled for example by a thickness of each of the layers. The microelectrode element (A) is therefore suitable for being implanted into tissues such as a neural tissue orfor being brought into contact with a single cell, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell, for example as a cuff electrode that is able to interface nerves with a diameter of about 50 to 500 µm.

[0040] Figs. 3A and 3B schematically show a perspective view and a top view of a microelectrodesystem including a plurality of the microelectrode elements (A) being integrally formed. Edge parts (B) are attached to each of side surfaces of the microelectrode elements (A). Each of the side surfaces extend along the x-direction and the z-direction (i.e. in an xz-plane being perpendicular to the y-direction). Each of the edge parts (B) comprises a third fibrous layer and a second polymer compound layer. The third fibrous layer may be continuous with the first and second fibrous layers and may be formed integrally and manufactured simultaneously with the second fibrous layer (3). The second polymer compound layer is continuous with the polymer compound layer (4) of the microelectrode element and may be formed integrally and manufactured simultaneously with the polymer compound layer (4).

[0041] A microelectrode comprises one microelectrode element (A) and two edge parts (B), and aplurality of the microelectrodes are further joined by joint portions (C). Two microelectrodes are connected by one joint portion (C) and the two microelectrodes are separated along the y-direction. In this embodiment, the microelectrodes are aligned parallel to each other. The joint portion (C) comprises a fourth fibrous layer and a third polymer layer. The third fibrous layer may be continuous with the first and second fibrous layers and may be formed integrally and manufactured simultaneously with the second fibrous layer (3). The third polymer compound layer is continuous with the second polymer compound layer of the edge part (B) and may be formed integrally and manufactured simultaneously with the edge part (B), i.e. with the second fibrous layer (3). The third polymer compound layer is continuous with the second polymer layer, i.e. with the polymer compound layer (C) of the microelectrode element and may be formed integrally and manufactured simultaneously with the second polymer layer and the polymer compound layer (4). The joint portion (C) can be cut e.g. manually by scissors, and each of the microelectrode may be separated and / or cut into a preferable form to suit intended uses. <Manufacturing method>

[0042] Figs. 4 to 7 schematically show steps of the manufacturing method of the microelectrodeelement as well as the microelectrode system according to the present embodiment.

[0043] Nanofibers and / or microfibers are dispersed in dispersion medium to prepare a first liquidsuspension (1S) and a third liquid suspension (3S). A second liquid suspension (2S) is prepared by dispersing conductive macromolecules (such as carbon nanotubes) into / through a second dispersion medium.

[0044] At first, the first liquid suspension (1S) is poured onto a membrane filter (F) and filteredfor example by applying negative pressures (P) (Fig.4(a)), thereby forming the first fibrous layer (1) (as well as the fibrous layer of the edge parts and the joint portions) (Fig.4(b)). Subsequently, a first mask (M1) is placed (directly) on the first fibrous layer (1), and the second liquid suspension (2S) was poured onto / over the first mask (M1) (Fig.5(a)). The second liquid suspension (2S) was filtered through the first mask (M1), the first fibrous layer (1) and the filter (F), in order to remove the dispersion medium by applying negative pressures (P), thereby forming the electrically conductive layers (2) patterned by the first mask (M1) (Fig. 5(b)). The first mask (M1) is then removed, and a second mask (M2) is placed (directly) on the electricallyconductive layer (2). The third liquid suspension (3S) is poured onto / over the second mask (M2) and filtered through the second mask (M2), the electrically conductive layer (2), the first nanofiber layer (1) and the filter (F), in order to remove the dispersion medium by applying negative pressures (P) (Fig. 6(a)), thereby forming the second nanofiber layers (2) patterned by the second mask (M2) (Fig.6(b)). Although not illustrated, the edge parts covering the side surface of the electrically conductive layers can be formed simultaneously with the second nanofiber layers (2). The first liquid suspension (1S) and the third liquid suspension (3S) may be different from each other or may be identical.

[0045] The stack of the first fibrous layer (1), the electrically conductive layer (2) and the secondfibrous layer (2) is then detached from the filter (F) and is attached to a substrate (G) such as a glass, such that the first surface of the first fibrous layer (1) is a top of the stack and the second surface of the second fibrous layer (3) is (directly) attached to the substrate (G) (Fig.7(a)). A polymer mixture is then applied on the first surface of the first fibrous layer (1) and polymerized to form the polymer compound layer (4) (as well as the polymer compound layers of the edge parts and the joint portions). The prepared microelectrode is then prepared by cutting the microelectrode system e.g. by scissors and by removing the joint portions. The microelectrode element may be formed by removing the edge parts (B) e.g. by scissors or the edge parts (B) may be not formed during the manufacturing process. EXAMPLES

[0046] Examples of a microelectrode element / microelectrode / microelectrode system accordingto the embodiment and its manufacturing method are explained by the following examples. The components and conditions used are only examples. Through these examples, we demonstrated that microelectrode elements according to the present disclosure would be suitable for e.g. implantable applications, especially for wrapping around cells having micrometer-order structures, such as nerve fibers. <Manufacturing>

[0047] Nanofibers were dispersed in distilled water (0.1 % w / w) and left to sonicate in an iced bathand the nanofiber liquid suspension was prepared, which is an example of the first liquid suspension (1S) and the third liquid suspension (3S). In the examples, different nanofibers (each of chitin, chitosan, cellulose, silk) were tested. Multi-walled carbon nanotubes (CNT) were used as an example of conductive macromolecules.14.3 mg of CNT (CM-250 from Hanwha Nanotech) with 200 mg of sodium dodecyl sulfate (SDS), a surfactant (polyionic or nonionic surfactant), were mixed in 100 ml of distilled water and the MWCNT was dispersed by using a wet jet milling device (Star Burst Mini, Sugino). The mixture was diluted with a 200 g / l SDS - distilled water solution at a 1:9 ratio to produce the CNT liquid suspension, which is an example of the second liquid suspension (2S).

[0048] At first, the nanofiber liquid suspension (5 to 20 ml) was poured onto a PVDF membranefilter (0.1 µm, 47 mm diameter, an example of the filter) and filtered using a Büchner flask to remove the dispersion medium by applying negative pressures with a vacuum pump, thereby forming a first nanofiber layer, which is an example of the first fibrous layer. Subsequently, a patterned first polyimide (PI) mask (which is an example of the first mask) was placed on the nanofiber layer, and the CNT liquid suspension (5.8 ml) was poured onto / over the first PI mask. The CNT liquid suspension was then filtered through the first PI mask, the first nanofiber layer and the filter, in order to remove the dispersion medium by applyingnegative pressures with the vacuum pump, thereby forming a CNT layer (patterned by the first PI mask), which is an example of the electrically conductive layer. The first PI mask was then removed, and a patterned second polyimide (PI) mask (which is an example of the second mask) was placed on the CNT layer, and the nanofiber liquid suspension (3.4 ml) was poured onto / over the second PI mask. The nanofiber liquid suspension was then filtered through the second PI mask, the CNT layer, the first nanofiber layer and the filter, in order to remove the dispersion medium by applying negative pressures with the vacuum pump, thereby forming a second nanofiber layer (patterned by the second PI mask), which is an example of the second fibrous layer. In the examples, the first nanofiber layer and the second nanofiber layer are formed by filtering the same nanofiber liquid suspension, and therefore the first nanofiber layer and the second nanofiber layer have the same substances.

[0049] After a drying-up process, the stack of the first nanofiber layer, the CNT layer and thesecond nanofiber layer was detached from the filter and was then attached to a glass substrate using distilled water, such that the surface of the second nanofiber layer faces the surface of the glass substrate and the surface of the first nanofiber layer was exposed. The surroundings of the stack were manually covered by parafilm. Subsequently, a polymer mixture of 500 ml 5 M Acrylamide (AAM) with 0.8% (w / w) CMC (carboxymethylcellulose) (being an example of the first component), 5 µL of 100 mM N,N’- Methylenbisacrylamid (MBAA) (being an example of the second component), 5 µl of 1 M Ammonium persulfate (APS), 0.75 µl of 1 M Tetramethylethylenediamine (TEMED) was prepared. The APS, MBAA and TEMED are examples of the polymerization agent. The polymer mixture was then applied on the surface of the first nanofiber layer and polymerized to form the PAAM layer, which is an example of the polymer compound layer. After the polymerization process, the parafilm and the glass substrate were manually removed from the prepared sample (an example for the microelectrode system) being the stack of the first nanofiber layer, the CNT layer, the second nanofiber layer and the polymer layer. A sample of the microelectrode was then prepared by cutting the stack by scissors in a rectangular shape (1 mm X 25 mm).

[0050] Samples 1 to 8 were prepared with various nanofibers as described above. The usednanofiber, the layer thickness of the nanofiber of each of Samples 1 to 8 are summarized in Table 1. An average swollen thickness was further measured for each of Samples 1 and 2. The samples were submerged in deionized water and saturated with water, and an average swollen thickness was measured by drawing lines across the innermost and outermost layer of an image of the folded sample. Table 1:<Characterization of the polymer compound layer and the CNT layer>

[0051] A swelling ratio of a polyacrylamide (PAAM) layer was measured, which would be utilizedas an example of the polymer compound layer. A polymer mixture of 500 ml 5 M Acrylamide (AAM) with 0.8% (w / w) CMC (carboxymethylcellulose) (being an example of the first component), 5 µL of 100 mM N,N’-Methylenbisacrylamid (MBAA) (being an example of the second component), 5 µl of 1 M Ammonium persulfate (APS), 0.75 µl of 1 M Tetramethylethylenediamine (TEMED) (being examples of the polymerization agent) was prepared. The polymer mixture (10 ml) was then polymerized in a container (85 mm diameter), and then was cut into different sizes. The swelling ratio was determined by a slope of data points plotting Ss versus SD, wherein Ss is a surface area of the PAAM layer (i.e. an upper layer extending in an xy-plane being perpendicular to the z-direction) being saturated with deionized water and SD is a surface area of the PAAM layer in a dried state, i.e. before being submerged in deionized water. The swelling ratio has been determined as 1.88. The surface density of the CNT surface was measured by using laser microscopy and determined as 12.5 µg / cm2. <Self-folding behavior>

[0052] Fig. 8 shows photos of the self-folding process of Sample 1. Sample 1 was submerged indeionized water at t = 0. The scale bar in the photo indicates 1 mm. The self-folding of Sample 1 was observed every 10 seconds. We observed that Sample 1 folded along its longer side (i.e. the length), and the folding axis was along its width. The self-folding process was completed at t = 50 sec.

[0053] Fig. 9 shows a photo of Sample 2, in which one end of the Sample 2 (i.e. a lower part of theSample 1) was wrapped around a 100 µm diameter polylactic acid (PLA) wire. The upper end of the Sample 2 was clamped with a crip and the lower end of the Sample 2 was positioned beneath the PLA wire, such that the second fibrous layer faced to the PLA wire (i.e. then second fibrous layer was positioned nearer to the PLA wire than the polymer compound layer). Sufficient amount of deionized water was applied to the lower end of the Sample 2, and it was observed that the lower end of the Sample 2 self-folded, such that the second fibrous layer was an internal side and the polymer compound layer was an outer side, and the lower end the Sample 2 successfully wrapped around the 100 µm diameter PLA wire. The self-folding process was completed ca.150 sec after applying deionized water.

[0054] <Conductivity measurements>

[0055] The conductivity measurements were performed on Samples 3 to 6 and Fig. 10 shows theresults. The conductive measurements were performed in a dried state. Samples 3 to 6 show similar conductance in a range of 0.04 mS to 0.07 mS, in which Sample 3 with chitin and Sample 4 with cellulose show relatively higher conductance than those of Sample 5 with chitosan and Sample 6 with silk. These discrepancies may be induced by the difference in each material’s molecular structure – such as chitin being polyanion, whereas chitosan is polycation. <Electrochemical performance>

[0056] Cyclic voltammetry (CV) was on Sample 7. The main setup used was a three-electrode cellconfiguration, with the nanofiber-hydrogel electrodes as the working electrode, an Ag / AgCl (NaCl 3 M) reference electrode, and a platinum wire counter electrode. All three were submerged in a small petri dish containing Gibco’s Dulbecco’s Phosphate-Buffered Saline, no calcium, no magnesium (DPBS or PBS) to simulate an organic environment. The measured Cyclic Voltammogram was shown in Fig. 11. The graph exhibits water oxidation and reduction thresholds at approximately +2 V and -2 V, respectively, along with a significant water window. This broad window provides a crucial advantage, enabling the electrode to be polarized to high potentials without initiating irreversible faradaic reactions, thereby mitigating the risk of tissue damage. The electrochemical behavior is stable and substantially the same results were obtained with 10 cycles. <Folding radius>

[0057] Nineteen samples with 4 µm thickness of chitin were prepared (Sample 8), and their foldingradii were plotted against their average swollen thicknesses (Fig. 12, circles). The experimental error margins are indicated by solid lines. Each of samples had various thickness of the polymer compound layer, which results in various swollen thicknesses. Each of Samples 8 was submerged in deionized water and was left in the water until the polymer compound layer formed hydrogel and was saturated. The folding radius was determined by measuring the inner most curve of each sample in the folded-state in the cross-section along an xz-plane being perpendicular to the y-direction. Furthermore, an average thickness of the polymer compound layer of the folded-state (i.e. an average thickness of the polymer compound layer in the z- direction after swelling) was measured. A theoretical model of a folding radius (ρ) was calculated by the Timoshenko Beam Model:wherein(^^^^is an average swollen thickness of a polymer compound layer, ^^^^is an elastic modulus of a nanofiberlayer, ^^^^is an elastic modulus of a polymer compound layer, ^^^^ is a thickness of a nanofiber layer, ^^^^ is aswelling ratio of the polymer compound layer, ^^^^ is a swelling ration of the nanofiber layer being negligiblesmall). The physical properties necessary to the calculation, ^^^^ , ^^^^ , ^^^^ were determined by separatemeasurements (not shown). The theoretical calculation is shown in Fig.12 with a dashed line. The results show that the folding behavior of sample microelectrodes matches the theoretical model. <List of reference signs>

[0058] 1: First fibrous layer, 2: Electrically conductive layer, 3: Second fibrous layer, 4: Polymer compound layer, A: Microelectrode element, B: Edge part(s), C: Joint portion(s), 1S: First liquid suspension, 2S: Second liquid suspension, 3S: Third liquid suspension, F: Filter, M1: First mask, M2: Second mask, G: Substrate.

Claims

Claims1. A microelectrode element (A) for being implanted into tissues such as a neural tissue or forbeing brought into contact with a single cell, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell, wherein the microelectrode element comprises a first fibrous layer (1), an electrically conductive layer (2), a second fibrous layer (3), and a polymer compound layer (4), wherein each of the layers (1, 2, 3, 4) extends in an xy-plane being perpendicular to a z- direction and has a thickness in the z-direction, wherein the polymer compound layer (4), the first fibrous layer (1) and the electrically conductive layer (2) are stacked in this order in the z-direction, and wherein the second fibrous layer (3) is formed on at least a part of a surface of the electrically conductive layer (2), wherein each of the first fibrous layer (1) and the second fibrous layer (3) comprises nanofibers and / or microfibers, wherein the polymer compound layer (4) is capable to form a hydrogel.

2. The microelectrode element (A) according to claim 1,wherein the microelectrode element has a length (l) in the x-direction and a width (w) in the y- direction, and the length (l) is longer than the width (w), wherein, preferably, the ratio (l / w) of the length (l) to the width (w) is more than 2, more preferably more than 4, more preferably more than 6.

3. The microelectrode element (A) according to any one of claims 1 or 2,wherein the second fibrous layer (3) covers the entire surface of the electrically conductive layer (2).

4. The microelectrode element (A) according to any one of claims 1 to 3,wherein the second fibrous layer (3) covers only a part of the surface of the electrically conductive layer (2), such that another part of the surface of the electrically conductive layer (2) is contactable.

5. The microelectrode element (A) according to any one of claims 1 to 4,1wherein the nanofibers and / or the microfibers comprise a thermoset polymer and / or a thermoplastic polymer and / or an elastomer and / or a polysaccharide such as cellulose, chitosan, chiton and / or combinations thereof, and / or wherein the nanofibers and / or the microfibers are obtained by an electrospinning method.

6. The microelectrode element (A) according to any one of claims 1 to 5,wherein the electrically conductive layer (2) comprises conductive macromolecules, wherein the conductive macromolecules comprise -carbon nanotubes, wherein preferably the carbon nanotubes are multi-walled,and / or -carbon rods,and / or -metal nanoparticlesand / or -conductive polymers, wherein the conductive polymers preferably comprise aconductive polymer mixture of at least two ionomers, such as poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS).

7. The microelectrode element (A) according to any one of claims 1 to 6,wherein the electrically conductive layer (2) comprises carbon nanotubes, wherein a surface density of the carbon nanotubes is in a range of 1 µg / cm2to 50 µg / cm-2, more preferably 10 µg / cm-2to 20 µg / cm-2.

8. The microelectrode element (A) according to any one of claims 1 to 7,wherein the polymer compound layer (4) is based on a first component being a hydrophilic monomer and / or a hydrophilic oligomer and / or a hydrophilic polymer and / or a hydrophilic copolymer, and a second component being a cross-linker, wherein the first component includes a first functional group, and the first component is capable to be polymerized through the first functional group and the second component.

9. The microelectrode element (A) according to any one of claims 1 to 8,wherein the polymer compound layer (4) has a swelling ratio in a range of between 1 and 10, more preferably between 2 to 3.

210. The microelectrode element (A) according to any one of claims 1 to 9 comprisingan insulating layer (5) covering at least a part of a surface of the microelectrode element (A).

11. The microelectrode element (A) according to any one of claims 1 to 10,wherein the thickness of the first fibrous layer (1) is in a range of between 1 µm and 500 µm, more preferably between 1µm to 100 µm, more preferably between 1 µm and 10 µm, and / or wherein the thickness of the electrically conductive layer (2) is in a range of between 1 µm and 100 µm, more preferably between 1 µm and 10 µm, more preferably between 1 µm and 3 µm, and / or wherein the thickness of the second fibrous layer (3) is in a range of between 1 µm and 100 µm, more preferably between 1 µm and 10 µm, and / or wherein the thickness of the polymer compound layer (4) is in a range of 1 µm to 1 mm.

12. A microelectrode including a microelectrode element (A) according to any one of 1 to 11,wherein the microelectrode comprises an edge portion (B) being attached to at least a part of an outer side surface of the microelectrode element (A), the outer side surface including the electrically conductive layer (2) and extending in the z-direction, wherein, preferably, the edge portion (B) is integrally formed with the microelectrode element (A).

13. The microelectrode according to claim 12 comprisingan insulating layer (5) covering at least a part of a surface of the edge part (B).

14. A microelectrode system comprising a plurality of microelectrodes according to claim 12 or 13,wherein the microelectrode system comprises a joint portion (C) connecting each of the plurality of the microelectrodes.

15. A microelectrode comprising a plurality of the microelectrode elements (A) according to anyone of claims 1 to 11, wherein the electrode system comprises a joint portion (C) connecting each of the plurality of the microelectrode elements (A).

316. A method for manufacturing a microelectrode element (A) according to any one of 1 to 11 comprising a step of forming the first fibrous layer (1) by filtering a first liquid suspension (1S) through a filter (F), the first liquid suspension (1S) comprising nanofibers and / or microfibers and a first dispersion medium, the first dispersion medium being deionized water and / or organic solvent, a step of forming the electrically conductive layer (2) on the first fibrous layer (1) by filtering a second liquid suspension (2S) through the first fibrous layer (1) and the filter (F), the second liquid suspension (2S) comprising conductive macromolecules and a second dispersion medium, the second dispersion medium being deionized water and / or organic solvent, a step of forming the second fibrous layer (3) on the electrically conductive layer (2) by filtering a third liquid suspension (3S) through the electrically conductive layer (2), the second fibrous layer (3) and the filter (F), the third liquid suspension (3S) comprising nanofibers and / or microfibers and a third dispersion medium, the third dispersion medium being deionized water and / or organic solvent, a step of forming the polymer compound layer (4) on a surface of the first fibrous layer (1).

17. The method according to claim 16,wherein the step of forming the electrically conductive layer (2) comprises a step of covering a surface of the first fibrous layer (1) by a first mask (M1), a step of applying the first liquid suspension (1S) on the first mask (M1), a step of filtering the first liquid suspension through the first mask (M1), the second fibrous layer (3) and the filter (F), so that the electrically conductive layer (2) being patterned by the first mask (M1) is formed on the second fibrous layer (3), and a step of removing the first mask (M1) after the step of filtering the second liquid suspension (2S).

18. The method according to claim 16 or 17, wherein the step of forming the second fibrous layer (3) comprises a step of covering a surface of the electrically conductive layer (2) by a second mask (M2), a step of applying the third liquid suspension (3S) on the second mask (M2), a step of filtering the third liquid suspension (3S) through the second mask (M2), the electrically conductive layer (2), the second fibrous layer (3) and the filter (F), so that 4the second fibrous layer (3) being patterned by the first mask (M1) is formed on the electrically conductive layer (2), and a step of removing the second mask (M2) after the step of filtering the third liquid suspension (3S).

19. The method according to any one of claims 16 to 18,wherein the step of forming the polymer compound layer (4) comprises a step of preparing a mixture (4S) by mixing the first component, the second component and a polymerization agent, and a step of applying the mixture (4S) of the first component, the second component and the polymerization agent on the surface of the first fibrous layer (1). 5

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