A microelectrode element for being implanted into tissues and its manufacturing methods

Abstract

A microelectrode element (A) for being implanted into tissues such as a nerve or for being brought into contact with a single cell or cells, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell or the cells, wherein the microelectrode element (A) comprises a base layer (1) extending in a xy-plane being perpendicular to a z-direction, the base layer (1) being electrically insulative, and an electrode portion (2) comprising a pillar portion (2-1) protruding from an upper surface of the base layer (1) along the z-direction and comprising a polymer compound, an electrically conductive layer (2-2) covering at least a side surface of the pillar portion (2-1), and a first insulating layer (2-3) at least partly covering the electrically conductive layer (2-2), wherein a part of the electrically conductive layer (2-2) is contactable.

Description

DESCRIPTIONTITLEA microelectrode element for being implanted into tissues and its manufacturing methodsTECHNICAL FIELD[OOOl]The present disclosure relates to a microelectrode element for being implanted into tissues such as a nerve and / or for being brought into contact with a single cell or cells, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell or the cells, and its manufacturing methods. More specifically, the microelectrode element according to the present disclosure is for microelectrode arrays for clinical and / or biosensing applications.BACKGROUND

[0002] In current practice, implantable microelectrodes are used for neuroscience, for alleviating symptoms of neurological diseases, such as Parkinson’s disease or epilepsy, or for restoring body functions after injuiy. 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. One popular tool for measuring electrophysiological activity within networks is the microelectrode array (MEA).

[0003] A document “Clinical applications of penetrating neural interfaces and Utah Electrode Array technologies” (Normann et. al, Journal of Neural Engineering 13, 2016) discloses a silicon-based three-dimensional MEA having a plurality of microelectrode elements being microneedles. This silicon-based MEA however requires a rigid silicon substrate, and the height of the microelectrode element is restricted to the thickness of the substrate. Additionally, the complex manufacturing process and high costs of silicon-based 3D MEAs can be prohibitive for some applications.SUMMARY OF THE INVENTION

[0004] The technical problem to be solved can be formulated to provide a microelectrode element with more accurate microstructures and to provide its manufacturing method, which provide more accurate microstructures of the microelectrode element and more efficient manufacturing in low costs.

[0005] A microelectrode element (A) according to the present disclosure is for being implanted into tissues such as a nerve or for being brought into contact with a single cell or cells, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell or the cells. The microelectrode element (A) may be for invasive and / or non-invasive clinical applications. The microelectrode element (A) may be a part of microelectrode array, in particular one ofmicroelectrodes of a microelectrode array and / or a microelectrode and / or neural electrode and / or implantable electrode and / or neural probe, which is capable of measuring and / or recording voltage changes or electrical currents within tissue and / or a single biological cell. The microelectrode element (A) is preferably used for delivering an electrical stimulus to a tissue and / or cell e.g. in a (human) brain. A targeted tissue and / or cell may have typically a diameter and / or size in a range of one to several tens of micrometres. Such a tissue and / or a single biological cell may be for example a neuron or a nerve fibre having a dimeter in a range of several hundred nanometres to ten micrometres. The microelectrode element (A) is preferably capable of measuring t LIV to several mV, and / or the electrode element (A) is preferably capable of applying stimulation currents to a biological sample in the regime of to LIA to several mA, and / or the electrode element (A) is preferably capable of measuring currents from a biological sample, e.g. for measuring neurotransmitter release, from 500 fA to too LIA.

[0006] The microelectrode element (A) according to the present disclosure comprises a base layer (1) extending in a xy-plane being perpendicular to a z-direction. The base layer (1) is electrically insulative. The base layer (1) extends in the x-direction and the y-direction or and has a thickness in the z-direction. The x-, y-, z- axes and / or directions are of the three-dimensional rectangular coordinate system, which are orthogonal to each other. The base layer (1) has an upper surface and a lower surface. The lower surface is different from the upper surface and is opposite the upper surface in the z-direction. Each of the upper and lower surfaces extends in the x-direction and the y-direction or in a xy-plane being perpendicular to the z-direction.

[0007] The microelectrode element (A) according to the present disclosure comprises an electrode portion (2) to be brought into (direct) contact with a biological sample to be measured and / or delivered an electrical stimulus. The electrode portion (2) comprises a pillar portion (2-1) (or rod portion or needle portion) protruding (or extending or projecting) from the upper surface of the base layer (1) along (and / or in) the z-direction and comprising (or consisting of) a polymer compound (or polymer compounds). The pillar portion (2-1) has a side surface extending along the z-direction and a top surface or a (distal) end surface. The top surface of the pillar portion (2-1) crosses the z-direction. The pillar portion may have a cylinder form or a truncated conical form having one continuous side surface around its axis extending in the z-direction. The pillar portion may have a prism form or a truncated pyramid form having a plurality of side surfaces. The electrode portion (2) further comprises an electrically conductive layer (2-2) covering at least the side surface(s) of the pillar portion (2-1) and / or covering the pillar portion around an axis of the pillar portion, the axis being along / in the z-direction. The electrically conductive layer (2-2) may entirely cover the side surface of the pillar portion and / or entirely cover all of the side surfaces of the pillar portion. The electrically conductive layer (2-2) may comprise (or consist of) conductive polymer(s) and / or metal(s) such as Au and / or Pt and / or Cr and / or Ag and / or Cu and / or W and / or Al and / or ITO and / or MgO and / or NbOx. The electrode portion (2) may accordingly have a pillar shape with a side surface(s) extending along the z-direction and a top surface or a (distal) end surface. The top surface of the electrode portion crosses the z-direction.[ooo8]The electrode portion (2) further comprises a first insulating layer (2-3) at least partly covering the electrically conductive layer (2-2). A part (i.e. the other part) of the electrically conductive layer (2-2) is (electrically and / or physically and / or mechanically) contactable (from the z-direction). The first insulating layer (2-3) may cover only a part of the surface of the electrically conductive layer (2- 2) extending in the z-direction. The other part of the electrically conductive layer (2-2) may be exposed on the top surface of the electrode portion (2-2) and uncovered by the first insulating layer (2-3). The first insulating layer (2-3) may comprise polymers such as parylene and / or polydimethylsiloxane (PDMS).

[0009] The microelectrode element according to the present disclosure may have one of more of following technical advantages: Since the pillar portion comprises and / or is composed of a polymer compound, it would be possible to manufacture the microelectrode element in lower costs than conventional silicon -based microelectrodes. In particular, the polymer-based pillar portion enables to finely time mechanical properties such as hardness and dimensions of the electrode portion to be brought into contact with biological targets, according to the application. For example, a polymer compound with higher hardness can be used for microelectrodes for invasive clinical applications, while a flexible and soft polymer compound can be used for observing biological samples without damaging the biological samples. The polymer-based pillar portion further can be more efficiently manufactured without any expensive equipment, while the structure of the pillar portion can be more accurately formed in the micrometre range.

[0010] Preferably, the polymer compound of the pillar portion (2-1) is obtained by (obtained by polymerizing and / or is formed by and / or comprises and / or consists of and / or is based on and / or is polymerized from) a photoresistive polymer(s) and / or photoresist(s), wherein preferably the pillar portion (2-1) is obtained by (and / or is obtained / formed from and / or comprises and / or consists of and / or is based on and / or is polymerized from) an epoxy-based polymer and / or negative photoresist. A photoresistive polymer and / or a photoresist is a light-sensitive material used typically in photolithography to form a patterned coating on a surface. A photoresistive polymer and / or a photo resist may be a negative photoresist, which is capable to be polymerized or cross-linked by light, and a developer can dissolve away only a region which has not been exposed to light, while the other region which has been exposed to light remains. An example of the negative photoresist is an epoxy-based negative photoresist such as SU-8.

[0011] The epoxy-based photoresist may be especially advantageous in that it is possible to obtain structures with a high aspect ratio (typically 20 to 40) via photolithography processes, and it is therefore especially advantageous to form pillar structures in the micrometre range, which would not be achieved by conventional techniques such as 3D polymer printing. It is further advantageous in that the polymerized epoxy-based photoresist would typically have a hardness higher than flexible polymer compounds such as elastomers and PDMS, and the electrode portion comprising the pillarportion formed by the epoxy-based photoresist would be especially advantageous in invasive clinical / biosensing applications, for example for a brain and / or nerve fibres.

[0012] Alternatively, the polymer compound of the pillar portion (2-1) preferably comprises (and / or consists of) a flexible polymer compound(s), wherein the flexible polymer compound(s) is elastomer and / or polydimethylsiloxane (PDMS) and / or hydrogel and / or natural rubber and / or styrene butadiene rubber and / or chloroblen rubber and / or aciylonitrile rubber and / or butyl rubber and / or ethylene propylene rubber and / or urethane rubber and / or silicone rubber and / or fluorine rubber.

[0013] The pillar portion (2-1) formed by a flexible (and soft) polymer compound(s) would be especially advantageous e.g. for in vivo signal measurements and / or stimulations to cells or tissues in motion such as cardiomyocytes, as the flexible and soft pillar portion would not restrict movements of targeted cells or tissues when it is brought into contact with or implanted into cells or tissues.

[0014] Preferably, the electrically conductive layer (2-2) covers or entirely covers the top surface of the pillar portion (2-1). Alternatively, a top surface of the electrode portion (2) preferably comprises a part of the electrically conductive layer (2-2) being ring-formed when viewing from the top of the electrode portion. The top surface of the electrode portion (2) includes the top surface of the pillar potion (2-1).

[0015] Preferably, the microelectrode element (A) comprises a lead line (3) being electrically conductive and being stacked on a part of the upper surface of the base layer (1) in the z-direction and being connected to the electrically conductive layer (2-2), and a second insulating layer (4) being stacked at least on a part of the lead line (3) in the z-direction.

[0016] Preferably, the microelectrode element (A) comprises an adhesive layer between the electrically conductive layer (2-2) and the side surface of the pillar portion (2-1), and / or an adhesive layer between the lead line (3) and the upper surface of the base layer (1). For example, Cr and / or Ti can be used as an adhesive layer for Pt and / or Au as an electrically conductive layer.

[0017] Preferably, a cross-sectional area of the pillar portion (2-1) (and / or the electrode portion) in a xy-plane being perpendicular to the z-direction is constant at least 90 % of the extension of the pillar portion (2-1) (and / or the electrode portion) in the z-direction. Preferably, the pillar portion (2-1) (and / or the electrode portion) has a cylindrical form and a cross-section of the pillar portion in the xy-plane has a circular form or an oval form, or preferably, the pillar portion (2-1) (and / or the electrode portion) has a prism form and a cross-section of the pillar portion (2-1) (and / or the electrode portion) in the xy-plane has a polygonal form.

[0018] Preferably, a cross-sectional area of the pillar portion (2-1) (and / or the electrode portion) in a xy-plane being perpendicular to the z-direction becomes smaller in the z-direction from the upper surface of the base layer (1) to the upper surface of the pillar portion (2-1). Preferably, the pillarportion (2-1) (and / or the electrode portion) has a truncated conical shape and a cross-section of the pillar portion (2-1) (and / or the electrode portion) in a xy-plane has a circular form or an oval form, or preferably, the pillar portion (2-1) (and / or the electrode portion) has a truncated pyramid shape and a cross-section of the pillar portion (2-1) (and / or the electrode portion) in the xy-plane has a polygonal form.

[0019] Preferably, the pillar portion (2-1) and the base layer (1) are integrally formed and comprise the same polymer compound(s). Preferably, the first insulating layer and the second insulating layer are integrally formed and comprise the same polymer compound(s), for example parylene or PDMS. Preferably, the first insulating layer and the second insulating layer comprise (or consist of) PDMS, which would be further advantageous for cell culturing, and cells would more stably attach to the microelectrode element.

[0020] Preferably, the base layer (1) is obtained by (and / or is obtained / formed from and / or comprises and / or consists of and / or is based on and / or is polymerized from) a photoresistive polymer, wherein preferably the photoresistive polymer comprises an epoxy-based polymer and / or negative photoresist. Alternatively, the base layer (1) preferably comprises a flexible polymer compound, wherein the flexible polymer compound is elastomer and / or polydimethylsiloxane and / or hydrogel and / or natural rubber and / or styrene butadiene rubber and / or chloroblen rubber and / or acrylonitrile rubber and / or butyl rubber and / or ethylene propylene rubber and / or urethane rubber and / or silicone rubber and / or fluorine rubber.

[0021] Preferably, a contacting portion being electrically conductive, wherein the contacting portion is attached to the top surface of the pillar portion (2-1) and being (physically and / or mechanically and / or electrically) connected to the electrically conductive layer (2-2).

[0022] A microelectrode array (B) according to the present disclosure comprises a plurality of microelectrode elements (A) according to any one of the above-mentioned microelectrode elements. The number of the microelectrode elements (A) (i.e. the number of the electrode portions) may be for example 16 or 64 or 128 or 256 or 512 or 1024, and the microelectrode array (B) has a dimension preferably in a range of 2.5 cm X2.5 cm to 5 cm x 5 cm.

[0023] According to the present disclosure, a first method for manufacturing any one of the above- mentioned microelectrode elements comprises a step of applying (and / or laminating and / or coating and / or pouring) a pre-polymer mixture onto and / or into a mold (S) and polymerizing (and / or curing) the mixture. The polymerized mixture patterned by the mold (S) is obtained after the polymerization (and / or curing) process in the mold. The pre-polymer mixture comprises monomers and / or oligomers and / or polymers and / or copolymers, each of which being capable to polymerization by reactive groups. The pre-polymer mixture preferably comprises a polymerizationinitiator(s) and / or polymerization agent(s) and / or a cross-linker(s). The polymerizing (and / or curing) may be performed by a heat-treatment and / or by radiating light such as UV light.

[0024] The first method further comprises a step of detaching (and / or removing) the polymerized mixture from the mold (S). The polymerized mixture comprises the base layer (1) and a pillar structure(s) (P) protruding from the upper surface of the base layer (1) along the z-direction. The base layer (1) and the pillar structure(s) (P) are integrally formed and comprising (or consisting of) the polymerized mixture. At least a part of the pillar structure will be formed into the pillar portion (2-2) of the electrode portion (2).

[0025] The first method further comprises a step of applying (and / or depositing and / or laminating and / or covering) an electrically conductive material (M) on only a part of the surface or the entire surface of the pillar structure(s) (P). The step of applying the electrically conductive material (M) may be performed by (metal) sputtering and / or (metal) electroplating and / or spin coating (for e.g. conductive polymers). The electrically conductive material (M) applied on the pillar structure will be formed into the electrically conductive layer (2-2) and may comprise conductive polymer(s) and / or metal(s) such as Au and / or Pt and / or Cr and / or Ag and / or Cu and / or W and / or Al and / or ITO and / or MgO and / or NbOx.

[0026] The first method further comprises a step of applying (and / or depositing and / or laminating and / or covering) an insulating material (I) on only a part of the surface or the entire surface of the electrically conductive material (M) being on the surface of the pillar structure (P). The insulating material (I) applied on the electrically conductive material (M) will be formed into the first insulating layer (2-3). The insulating material (I) may be spin-coated on the surface of the electrically conductive material (M) and may comprise polymers such as parylene and / or polydimethylsiloxane (PDMS).

[0027] The first method further comprises a step of removing at least a part of the insulating material (I) from a tip portion of the pillar structure (P), so that a part of the electrically conductive layer (2- 2) is contactable and / or is exposed on the top surface of the electrode portion (2).

[0028] The first method according to the present disclosure may have one or more of following technical advantages: According to the first method, the pillar structure(s) and the base portion are manufactured by pouring a pre-polymer solution into a mold and polymerizing the pre-polymer solution, and the method would be especially suitable for mass production, as it is possible to make multiple pillar structures at simultaneously in quick and uncomplicated manufacturing steps. The size and shape of the pillar structures can be controlled by changing the trench of the mold, which can be formed into any cross-sectional shape by any known etching process, such as (conventional) reactive ion etching. The size / shape of the pillar structure would be therefore not limited by the manufacturing process, which is further advantageous over conventional manufacturing method forMEAs. In addition, the pillar structures and the base portion can be manufactured without any expensive machinery.

[0029] According to the present disclosure, a second method is alternative to the first method and is for manufacturing a method for manufacturing any one of the above-mentioned microelectrode elements. The second method comprises a step of attaching (and / or applying and / or laminating and / or depositing) a photoresistive polymer layer (2’) (and / or photoresist layer) to the upper surface of the base layer (1) . The photoresistive polymer layer (2’) may be a photoresist film and the step of attaching may include a step of rolling the photoresist film onto the upper surface of the base layer (1).

[0030] The method further comprises a step of exposing the photoresistive polymer layer (2’) to a pattern of light and a step of developing the photoresistive polymer layer (2’) having been exposed to the pattern of light to remove only a part of the photoresistive polymer layer (2’), thereby forming a pillar structure(s) (P) protruding from the upper surface of the base layer (1) along the z-direction. The pillar structure (P) is accordingly formed by another part of the photoresistive polymer layer (2’) having been remained attached on the upper surface of the base part after the step of developing. The step of developing is preferably performed by rinsing the photoresistive polymer layer (2’) in developer solution.

[0031] The second method further comprises a step of applying (and / or depositing and / or laminating and / or covering) an electrically conductive material (M) on only a part of the surface or the entire surface of the pillar structure(s) (P), thereby forming the electrically conductive layer. The step of applying the electrically conductive material (M) may be performed by (metal) sputtering and / or (metal) electroplating and / or spin coating (for e.g. conductive polymers). The electrically conductive material (M) applied on the pillar structure will be formed into the electrically conductive layer (2- 2) and may comprise conductive polymer(s) and / or metal(s) such as Au and / or Pt and / or Cr and / or Ag and / or Cu and / or W and / or Al and / or ITO and / or MgO and / or NbOx.

[0032] The second method further comprises a step of applying (and / or depositing and / or laminating and / or covering) an insulating material (I) on only a part of the surface or the entire surface of the electrically conductive material (M) being on the surface of the pillar structure (P), thereby forming the first insulating layer. The insulating material (I) applied on the electrically conductive material (M) will be formed into the first insulating layer (2-3) . The insulating material (I) may be spin-coated on the surface of the electrically conductive material (M) and may comprise polymers such as parylene and / or polydimethylsiloxane (PDMS).

[0033] The second method further comprises a step of removing at least a part of the insulating material (I) from a tip portion (and / or a top portion and / or a distal portion) of the pillar structure (P) (or each of the pillar structures), so that a part of the electrically conductive layer (2-2) is contactable and / or is exposed on the top surface of the electrode portion (2).

[0034] The second method according to the present disclosure may have one or more of following technical advantages: As the pillar structures are formed by the photoresist via a photolithography process, accurate structures in the micrometer range can be obtained. In particular, the epoxy-based photoresist may be especially advantageous in that it is possible to obtain structures with a high aspect ratio (typically 20 to 40), which would not be achieved by conventional techniques such as 3D polymer printing. It is further advantageous in that the polymerized epoxy-based photoresist would typically have a hardness higher than flexible polymer compounds such as elastomers and PDMS, and the electrode portion comprising the pillar portion formed by the epoxy-based photoresist would be especially advantageous in invasive clinical / biosensing applications, for example for a brain and / or nerve fibres.

[0035] Preferably, according to the first method or the second method, the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) includes (and / or is performed by) a step of cutting off (and / or removing) the tip portion of the pillar structure (P) together with a part of the electrically conductive material (M) covering the tip portion and a part of the insulating material (I) on the tip portion, preferably thereby obtaining the electrode portion (2) comprising the pillar portion (2-1), the electrically conductive layer (2-2) and the first insulating layer (2-3). The tip portion of the pillar structure (P) includes the top surface of the pillar structure (P), which is a top end of the pillar structure (P) and is for example 1 % to 10 % of the extension of the pillar structure (P) in the z-direction.

[0036] Alternative to the step of cutting off the tip portion, the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) may include a step of removing a part of the insulating material (I) only from the tip portion of the pillar structure (P), while the electrically conductive material (M) remains attached on the tip portion, preferably thereby obtaining the electrode portion (2) comprising the pillar portion (2-1), the electrically conductive layer (2-2) and the first insulating layer (2-3). The electrically conductive layer (2-2) accordingly covers a top surface of the pillar portion (2-1) (i.e. the pillar structure).

[0037] Preferably, the first and / or second method further comprises a step of depositing the adhesive layer directly on the surface of the pillar structure (P), prior to the step of applying the electrically conductive material (M). The step of depositing the adhesive layer may be performed by (metal) sputtering and / or (metal) electroplating and / or spin coating (for e.g. polymers).

[0038] Preferably, the first and / or second method further comprises a step of applying the electrically conductive material (M) on the upper surface of the base layer (1), wherein preferably the method further comprises a step of depositing the adhesive layer directly on the upper surface of the base layer (1), prior to the step of applying the electrically conductive material (M). The first and / or second method preferably further comprises a step of removing a part of the electrically conductive material (M) and / or the adhesive layer from a part of the upper surface of the base layer (1), therebyforming (and / or outlining) the lead line (3) on the upper surface of the base layer (1), and a step of applying the insulating material (I) on a surface of the electrically conductive material (M) being on the upper surface of the base layer (1), thereby forming the second insulating layer (4). Preferably, the step of applying the electrically conductive material (M) on the upper surface of the base layer (1) and the step of applying an electrically conductive material (M) on the surface of the pillar structure (P) are simultaneously performed preferably by the same step, and / or the step of applying the insulating material (I) on the surface of the electrically conductive material (M) being on the upper surface of the base layer (1) and the step of applying the insulating material (I) on the surface of the electrically conductive material (M) being on the surface of the pillar structure (P) are simultaneously performed preferably by the same step. The step of applying the electrically conductive material (M) may be performed by (metal) sputtering and / or (metal) electroplating and / or spin coating (for e.g. conductive polymers). The electrically conductive material (M) may comprise conductive polymer(s) and / or metal(s) such as Au and / or Pt and / or Cr and / or Ag and / or Cu and / or W and / or Al and / or ITO and / or MgO and / or NbOx. The insulating material (I) may be spin-coated on the surface of the electrically conductive material (M) and may comprise polymers such as parylene and / or polydimethylsiloxane (PDMS).

[0039] Preferably, the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) is performed by laser- ablation, and / or the step of cutting off the tip portion of the pillar structure (P) is performed by laser- ablation, and / or the step of removing only the part of the insulating material (I) from a tip portion of the pillar (P) is performed by laser-ablation. The laser power for the laser-ablation step is preferably 0.2 to 0.8 W. The laser power for the step of removing only the part of the insulating material is lower than the laser power for the step of cutting off the tip portion of the pillar structure.

[0040] Alternatively, the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) may be performed by a spinning process, thereby removing the insulating material (I) only from the tip portion by centrifugal forces. More specifically, a (intermediate) product obtained after or directly after the step of applying the insulating material (I) on the surface of the electrically conductive material (M) is made to spin (and / or rotated) preferably by a spin coater around an axis being perpendicular to the upper surface of the base layer (1), before the insulating material (I) is cured and / or hardened. For example, the insulating material (M) is a pre-polymer mixture, for example PDMS, and the spinning process is applied to the (intermediate) product before the pre-polymer mixture is (completely) polymerized. The spinning process is preferably performed at a high speed, for example 8000 to 9000 rpm, only for a short time period, for example for 1 to 2 sec.

[0041] Preferably, the step of removing a part of the electrically conductive material (M) and / or the adhesive layer from a part of the upper surface of the base layer (1) is performed by laser-ablation.

[0042] The second method preferably further comprises a step of forming a coating layer on a surface of the mold (S) prior to the step of applying the pre-polymer mixture, wherein the coating layer is preferably paiylene. The coating layer may be especially advantageous to facilitate the step of removing the polymerized mixture from the mold (S).

[0043] The second method preferably further comprises a step of preparing the mold (S) by applying reactive ion etching on an upper surface of a silicon wafer (S), thereby forming a trench extending from the upper surface of the silicon wafer (S) in a thickness direction of the silicon wafer (S), the trench being for forming the pillar structure (P) and the upper surface of the silicon wafer (S) being for forming the base layer (1).

[0044] The second method preferably further comprises a step of transferring the polymerized mixture to a support (G), such that a lower surface of the base layer (1) is attached to a surface of the support (G), subsequent to the step of removing the polymerized mixture from the mold (S) and prior to the step of applying the electrically conductive material (M) on the surface of the pillar structure (P). Preferably the step of transferring comprises a step of applying organic solvent such as alcohol and / or acetone and / or ethanol between the support and the polymerized mixture and subsequently drying the organic solvent (E). The organic solvent being applied between the polymerized mixture and the support may be especially advantageous to tightlyy adhere the lower surface of the base layer to the support without any air bubbles in between, and it would be possible to form the electrically conductive layer and / or the first / second insulating layer homogeneously without any undesired substructures.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figs. 1 and 2: schematic perspective and top views of a microelectrode element according to the first embodiment;Fig. 3: a cross -section of the microelectrode element according to the first embodiment;Figs. 4 and 5: schematic perspective and side views of a microelectrode array comprising a plurality of the microelectrode elements according to the first embodiment;Figs. 6 and 7: schematic perspective and top views of a microelectrode element according to a variation of the first embodiment;Fig. 8: a cross-section of the microelectrode element according to the variation of the first embodiment;Figs. 9 (a) to (j): manufacturing steps of the microelectrode element and the microelectrode array according to the first embodiment;Figs. 10 and 11: schematic perspective and top views of a microelectrode element according to the second embodiment;Fig. 12: a cross-section of the microelectrode element according to the second embodiment;Figs. 13 (a) to (h): manufacturing steps of the microelectrode element and the microelectrode array according to the second embodiment;Figs. 14 (a) to (f): SEM images of multiple samples according to the first example;Fig. 15: impedance measurements performed with multiple samples according to the first example;Figs. 16 (a) and (b): SEM images of pillar structures according to the second example;Figs. 16 (c) and (d): SEM images of the microelectrode array and the electrode portion according to the second example;Fig. 17(a): average absolute impedance and phase recorded from eight microelectrode elements according to the second example;Figs. 17(b) to (d): extracellular signals from HL-1 cells observed with the microelectrode elements according to the second example.DETAILED DESCRIPTION OF EMBODIMENTSFirst Embodiment

[0046] Fig. 1 schematically show a perspective view, a top view and a cross-section of a microelectrode element (A) according to the first embodiment, respectively.

[0047] The microelectrode element (A) comprises an electrically insulative base layer (1) extending in a xy-plane and an electrode portion (2) having a pillar and / or needle structure protruding from the upper surface of the base layer (1). The top surface of the electrode portion (2), i.e. the tip end portion of the electrode portion (2), is for bringing into contact with targeted biological samples. The electrode portion (2) comprises a pillar portion (2-1), which forms a core of the electrode portion (2) and extends through the center part of the electrode part (2) in the z-direction.

[0048] In the first embodiment, the pillar portion (2-1) and the base layer (1) are integrally formed into one body and consist of the same polymer compound comprising PDMS (preferably only PDMS). The material of the polymer compound is however not limited to PDMS and may be replaced by flexible and soft polymer compounds, such as elastomer and / or hydrogel. The pillar portion (2-1) formed by flexible and soft polymer compound (here PDMS) would be especially advantageous for in vivo signal measurements and / or stimulations to cells or tissues in motion such as cardiomyocytes, as the flexible and soft pillar portion would not restrict movements of targeted cells or tissues when it is brought into contact with or implanted into cells or tissues.

[0049] The electrode portion (2) further comprises an electrically conductive layer (2-2), and according to the first embodiment, the electrically conductive layer (2) covers the entire side surface of the pillar portion (2-1). However, a part of the side surface of the pillar portion (2-1) maybe uncovered by the electrically conductive layer (2-2). As can be seen in the top view of the electrode portion (2) shown in Fig. 2, the electrically conductive layer (2-2) is exposed on the top surface (the tip end portion) of the electrode part (2), so that the electrically conductive layer (2-2) is physically and / or mechanically contactable on the top surface of the electrode portion (2). The electrically conductivelayer (2-2) has a ring-form when viewing from the top of the electrode portion (2), which surrounds the pillar portion (2-2) around the axis in the z-direction of the pillar portion (2-2) and is surrounded by the first insulating layer (2-3) around the axis. According to the first embodiment, the electrically conductive layer (2-2) comprises a metal material such as gold (Au). Optionally, an adhesive layer such as chromium (Cr) or titan (Ti) may be formed between the electrically conductive layer (2-2) and the pillar portion (2-1).

[0050] The electrode portion further includes a first insulating layer (2-3) covering the electrically conductive layer (2-2) except the part being exposed on the top surface of the electrode part (2). According to the first embodiment, the first insulating layer (2-3) consists of the same polymer compound of the base layer (1) and the pillar portion (2-1).

[0051] On the upper surface of the base layer (1), a lead line (3) is formed and connected to the electrically conductive layer (2-2) of the electrode portion (2). In this embodiment, the lead line (3) is formed by the same metal material (e.g. Au) of the electrically conductive layer (2-2) , which would be advantageous in that the electrically conductive layer (2-2) and the lead line (3) can be formed simultaneously. A part of the lead line (3) is covered by a second insulating layer (4), which may be the same insulating material of the first insulating layer (2-3). Another part of the lead line (3), which is opposite to the part being connected to the electrically conductive layer (2-2), is exposed and uncovered by the second insulating layer (4), so that an external device can be further connected to the lead line (3). Similar to the electrode portion (2), an adhesive layer (e.g. Cr or Ti) may be formed between the lead line (3) and the base layer (1).

[0052] According to the first embodiment, the pillar portion (2-1) has a cylindrical form, and therefore the electrode portion (2) has a cylindrical form. However, the pillar portion (2-1) (therefore also the electrode portion (2)) may be formed into a prism, such as a square prism. The electrode portion (2).

[0053] The base layer (1) has a thickness preferably in a range of 5 pm to 10 mm, more preferably 300 pm to 1 mm, which may be more advantageous in the step of detaching the polymerized mixture from the mold, which will be described later. The pillar portion (2-1) has a height in the z-direction preferably in a range of 1 pm to 5 mm, more preferably 500 pm to 1 mm. A cross-section of the pillar portion (2-1) in a xy-plane has a maximum width in the x-direction or in the y-direction preferably in a range of 2 pm to 750 pm, more preferably 10 pm to 500 pm. The electrically conductive layer (2-2) has a thickness (i.e. in a direction perpendicular to the side surface of the pillar portion) preferably in a range of 10 nm to 3 pm, more preferably 30 nm to too nm. The first insulating layer (2-3) has a thickness (i.e. in the direction perpendicular to the side surface of the pillar portion) preferably in a range of 50 nm to 3 pm, more preferably 1 pm to 3 pm. The lead line (3) has a thickness (i.e. in the z-direction) preferably in a range of 10 nm to 3 pm, more preferably 30 nm to too nm. The lead line (3) extends from the pillar portion in the y-direction to a part being exposed and has a width in the x direction. The width of the lead line (3) is preferably 10 pm to 1 mm,preferably 20 to 50 pm. The second insulating layer (4) has a thickness (i.e. in the z-direction) preferably in a range of 50 nm to 3 pm, more preferably 1 pm to 3 pm.

[0054] Fig. 4 illustrates a perspective view of a microelectrode array (B) formed by a plurality of the microelectrode elements (A) according to the first embodiment, and Fig. 5 shows a side view thereof. The microelectrode elements (A) are connected by joint portions being integrally formed with the base layer (1) and the second insulating layer (2) of the microelectrode elements. The plurality of the microelectrode elements (B) and the joint portions between the microelectrode elements (B) are therefore manufactured simultaneously.

[0055] The electrically conductive layer (2-2) shown in Figs. 1 to 5 has a ring-form when viewing from the top of the electrode portion (2). An alternative microelectrode element is illustrated in Figs. 6 to 8 as a variation of the first embodiment. According to this variation, the electrode portion (2) further comprise a contacting portion, which covers the top surface of the pillar portion (2-1). According to this variation, the contact portion is continuous to and integrally formed with the electrically conductive layer (2-2) and consists of the same material of the electrically conductive layer (2-2). Alternatively, a separate contact portion, such as a metal cap, may be attached to the top surface of the electrode portion (2) and connected to the electrically conductive layer (2-2). The contacting portion may be further advantageous to increase a contacting area for being brought into contact with targeted biological samples.

[0056] < Manufacturing of the Microelectrode Element>Figs. 9 (a) to (j) schematically illustrates steps of the manufacturing process of the microelectrode element (A) as well as the microelectrode array (B) according to the first embodiment. Figs. 9 (a) to (j) illustrates a case where a plurality of the microelectrode elements is formed simultaneously, however, the number of the microelectrode elements shown in Fig. 9 is only an example and not limited thereto.

[0057] A mold (S) for forming the base layer (1) and the pillar portion (2-1) is prepared by applying reactive ion etching on an upper surface of a silicon wafer (S) (Fig. 9(a)). The silicon wafer (S) has two main surfaces (i.e. an upper and lower surface), each of which extends in an xy-plane, and has a thickness in the z-direction. The mold (S) comprises a plurality of trenches, each of which extends from the upper surface of the silicon wafer (S) in the z-direction, i.e. the thickness direction of the silicon wafer (S). Each of the trenches therefore has a depth extending in the z-direction.

[0058] At first, a pre-polymer mixture (in this embodiment, a pre-polymer mixture for PDMS) is applied onto the mold (S) e.g. by spin coating, so that the pre-polymer mixture is homogeneously distributed on the main upper surface of the mold (S) and each of the trenches is filled up with the pre-polymer mixture (Fig. 9(b)). Optionally, a coating layer such as parylene may be formed on the surface of the mold (M) prior to the step of applying the pre-polymer mixture. The pre-polymer mixture is then polymerized by e.g. heating process and / or by exposing to light, such as UV light(Fig. 9(c)). After the pre-polymer mixture is polymerized, the polymerized mixture (i.e. the flexible polymer compound) is detached from the mold (S) e.g. by manually peeling off the polymerized mixture from the mold (S) (Fig. 9(d)). The flexible mechanical properties of the polymer compound would be advantageous to facilitate this removing step. The polymerized mixture (i.e. the flexible polymer compound) comprises the base layer (1) and pillar structures (P) protruding from the upper surface of the base layer (1) along the z-direction. The parts of the mixture having been polymerized inside of the trenches correspond to the pillar structures (P). A part of each of the pillar structures (P) is formed into the pillar portion (2-2) of the electrode portion (2), or each of the entire pillar structures (P) corresponds to the pillar portion (2-2).

[0059] The polymerized mixture is then transferred to or onto a support (G), e.g. a glass substrate, such that the lower surface of the base layer (1) is attached to the surface of the support (G) and the pillar structures (P) are oriented to the upper direction, which is opposite to the support (G). A thin layer of organic solvent (E) such as ethanol may be formed between the base layer (1) and the support (G) (Fig. 9(e)). The organic solvent (E) is then dried and the polymerized mixture is directly attached to the support (G) (Fig. 9(f)).

[0060] Subsequently, an electrically conductive material (M) such as Au is applied on the surface of the pillar structures (P) and the upper surface of the base layer (1) between the pillar structures (P) (Fig. 9(g)). Namely, the entire upper surface of the polymerized mixture (which is opposite to the lower surface directly attached to the support) may be covered with the electrically conductive material (M). Prior to applying the electrically conductive material (M), an adhesive material such as Cr may be deposited on the entire upper surface of the polymerized mixture. A part of the electrically conductive material (M) and / or the adhesive layer is then removed from the upper surface of the base layer (1) e.g. by laser- ablation, thereby forming (outlining) leadlines (Fig. 9(h)). Alternatively, the electrically conductive material (M) and / or the adhesive layer may be selectively deposited on the upper surface of the polymerized mixture e.g. by a photolithography process.

[0061] After forming the lead lines, an insulating material (I) is applied on the surface of the electrically conductive material (M), for example by spin-coating (the first spinning process, Fig. 9(i)). The entire surface of the electrically conductive material (M) may be covered by the insulating material (I) (e.g. a pre-polymer mixture such as PDMS) and thereafter a part of the lead lines may be exposed by removing the insulating material (I) e.g. by laser ablation.

[0062] Finally, only a part of the insulating material (I) is removed from the tip portion of the pillar structure (P) by applying a second spinning process. The (intermediate) product, which is the polymerized PDMS covered by the electrically conductive material and the insulating material, is made to spin or rotated by a spin coater around an axis being perpendicular to the upper surface of the base layer (1), before the insulating material (I) is cured and / or hardened. The tip portion of the pillar structure includes the top surface of the pillar structure, which is positioned at the highest position from the upper surface of the base layer (1) in the z-direction. Therefore, centrifugal forcesapplied at the tip portion (and / or the top surface) of the pillar structure are higher than centrifugal forces applied to other parts of the product. The insulating material (I) can be therefore removed only from the tip portion of the pillar structure, while the insulating material (I) remains attached other parts of the microelectrode element. The second spinning process is performed at a rotational speed being higher than a rotational speed for the first spinning process. The second spinning process is performed for a time period being shorter than a time period for the first spinning process. After the second spinning process, the contacting portion covering the top surface of the pillar portion (2-2) is obtained as illustrated in Figs. 6 to 8.

[0063] Alternatively, only the insulating material (I) can be removed from the tip portion of the pillar structure (P), thereby exposing the electrically conductive material covering the top surface of the pillar portion and obtaining the contacting portion covering the top surface of the pillar portion (2- 2) as illustrated in Figs. 6 to 8. In this step of laser- ablation, the base layer (1) may be held vertically, i.e. along the z-direction, and a laser beam maybe radiated in the z-direction to cut off the tip portion or to remove only the insulating material from the tip portion.

[0064] Alternatively, the tip portion of the pillar structure (P), which is covered by the electrically conductive material (M) and the insulating material (I), may be cut off, e.g. by laser ablation, as illustrated by Fig. 9 (j), thereby obtaining the electrode portion (2) in which the electrically conductive layer (2-2) (a remaining part of the electrically conductive material) is exposed on the top surface of the pillar portion (2-1), in a ring-form.Second Embodiment

[0065] Figs. 10, 11, 12 schematically show a perspective view, a top view and a cross-section of a microelectrode element (A) according to the second embodiment, respectively.

[0066] Similar to the first embodiment, the microelectrode element (A) according to the second embodiment comprises an electrically insulative base layer (1) extending in a xy-plane and an electrode portion (2) having a pillar and / or needle structure protruding from the upper surface of the base layer (1). The top surface of the electrode portion (2), i.e. the tip end portion of the electrode portion (2), is for bringing into contact with targeted biological samples. The electrode portion (2) comprises a pillar portion (2-1), which forms a core of the electrode portion (2) and extends through the center part of the electrode part (2) in the z-direction.

[0067] According to the second embodiment, the pillar portion (2-1) comprises and / or is formed by a photoresist, such as an epoxy-based (negative) photoresist, namely, the pillar portion (2-1) is obtained by applying a photolithography process to a photoresist layer. The pillar portion (2-1) obtained by a photoresist layer, having been patterned and polymerized through a lithography process and / or heating treatment would have relatively high buckling forces. Therefore, the microelectrode element (A) according to the second embodiment would be especially suitable fore.g. invasive electroencephalography monitoring, in which the microelectrode element would have to penetrate into target tissues. The pillar portion (2-1) according to the second embodiment has a truncated conical shape and a cross-section of the pillar portion (2-1) in a xy-plane has a circular form or an oval form, which may be further advantageous in its stable mechanical structure, especially against external forces to be applied to the electrode portion (2) along the z-direction. The pillar portion (2-1) may be alternatively formed into a cylindrical form, as shown in the first embodiment.

[0068] According to the second embodiment, the base layer (1) may be a substrate such as a glass plate. Alternatively, the base layer (1) may be formed by the same photoresist such as SU-8 being polymerized by exposing to light and / or heating treatments.

[0069] The electrode portion (2) further comprises an electrically conductive layer (2-2), and according to the second embodiment, the electrically conductive layer (2) covers the entire side surface of the pillar portion (2-1). However, a part of the side surface of the pillar portion (2-1) maybe uncovered by the electrically conductive layer (2-2). As can be seen in the top view of the electrode portion (2) shown in Fig. 11, the electrically conductive layer (2-2) is exposed on the top surface (the tip end portion) of the electrode part (2), so that the electrically conductive layer (2-2) is physically and / or mechanically contactable on the top surface of the electrode portion (2). The electrically conductive layer (2-2) has a ring-form when viewing from the top of the electrode portion (2), which surrounds the pillar portion (2-2) around the axis of the pillar portion (2-2) and is surrounded by the first insulating layer (2-3) around the axis. According to the second embodiment, the electrically conductive layer (2-2) comprises a metal material such as gold (Au) or platinum (Pt). Optionally, an adhesive layer such as chromium (Cr) or titan (Ti) may be formed between the electrically conductive layer (2-2) and the pillar portion (2-1).

[0070] The electrode portion further includes a first insulating layer (2-3) covering the electrically conductive layer (2-2) except the part being exposed on the top surface of the electrode part (2) . The first insulating layer (2-3) is a polymer layer such as paiylene. On the upper surface of the base layer (1), a lead line (3) is formed and connected to the electrically conductive layer (2-2) of the electrode portion (2). In this embodiment, the lead line (3) is formed by the same metal material (e.g. Pt or Au) of the electrically conductive layer (2-2), which would be advantageous in that the electrically conductive layer (2-2) and the lead line (3) can be formed simultaneously. A part of the lead line (3) is covered by a second insulating layer (4), which may be the same insulating material of the first insulating layer (2-3). Another part of the lead line (3), which is opposite to the part being connected to the electrically conductive layer (2-2), is exposed and uncovered by the second insulating layer (4), so that an external device can be further connected to the lead line (3). Similar to the electrode portion (2), an adhesive layer (e.g. Ti or Cr) may be formed between the lead line (3) and the base layer (1).

[0071] The base layer (1) has a thickness preferably in a range of 5 pm to 10 mm, more preferably 300 pm to 1 mm. The pillar portion (2-1) has a height in the z-direction preferably in a range of 1 pm to 5 mm, more preferably 500 pm to 1 mm. A cross-section of the pillar portion (2-1) in a xy-plane has a maximum width in the x-direction or in the y-direction preferably in a range of 2 pm to 750 pm, more preferably 10 pm to 500 pm. The electrically conductive layer (2-2) has a thickness (i.e. in a direction perpendicular to the side surface of the pillar portion) preferably in a range of 10 nm to 3 pm, more preferably 30 nm to too nm. The first insulating layer (2-3) has a thickness (i.e. in the direction perpendicular to the side surface of the pillar portion) preferably in a range of 50 nm to 3 pm, more preferably 1 pm to 3 pm. The lead line (3) has a thickness (i.e. in the z-direction) preferably in a range of 10 nm to 3 pm, more preferably 30 nm to too nm. The lead line (3) extends from the pillar portion in the y-direction to a part being exposed and has a width in the x direction. The width of the lead line (3) is preferably 10 pm to 1 mm, preferably 20 to 50 pm. The second insulating layer (4) has a thickness (i.e. in the z-direction) preferably in a range of 50 nm to 3 pm, more preferably 1 pm to 3 pm.

[0072] A plurality of the microelectrode element (A) according the second embodiment may form a microelectrode array (B) similar to the first embodiment as illustrated in Figs. 4 and 5. The microelectrode elements (A) are connected by joint portions being integrally formed with the base layer (1) and the second insulating layer (2) of the microelectrode elements. The plurality of the microelectrode elements (B) and the joint portions between the microelectrode elements (B) are therefore manufactured simultaneously. Similar to the variation of the first embodiment shown in Figs. 6 to 8, the electrode portion (2) may further comprise a contacting portion, which covers the upper surface of the pillar portion (2-1). The contact portion may be integrally formed with the electrically conductive layer (2-2) and consists of the same material of the electrically conductive layer (2-2). Alternatively, a separate contact portion, such as a metal cap, maybe attached to the top surface of the electrode portion (2) and connected to the electrically conductive layer (2-2).

[0073] < Manufacturing of the Microelectrode Element>Figs. 13 (a) to (h) schematically illustrate steps of the manufacturing process of the microelectrode element (A) according to the second embodiment. Fig. 13 shows only a single microelectrode element. However, a microelectrode array (B) may be manufactured by forming a plurality of the microelectrode elements simultaneously by applying the same steps.

[0074] At first, a photoresistive polymer layer (2’) (i.e. a photoresist layer) is attached to the base layer (1) (Fig. 13(a)). For example, the photoresistive polymer layer (2‘) may be formed by attaching a photoresist film, e.g. SU-film, to the upper surface of the base layer (1). Optionally, the stack of the photoresistive polymer layer (2’) and the base layer (1) may be heat-treated before the photolithography process. The photoresistive polymer layer (2’) is then exposed to a pattern of light, e.g. UV light (Fig. 13(b)), and then optionally subsequently heat-treated. The photoresistive polymer layer (2’) having been exposed to the patterned light is then developed in developer. For example, when the photo resistive polymer layer (2’) is a negative photoresist, a first part having been notexposed to the light would be removed by the step of developing. A second part having been exposed to the light would remain on the surface of the base layer (t) after the step of developing. The second part remained on the base layer (t) after the step of developing has a pillar structure (P), which will be formed into the pillar portion (2-2) or correspond to the pillar portion (2-2) (Fig. 13(c)).

[0075] Subsequently, the pillar structure (P) and the upper surface of the base layer (1) are (entirely) covered by an electrically conductive material (M) (Fig. 13(d)). Optionally, prior to applying the electrically conductive material (M), an adhesive material such as Ti or Cr maybe deposited on the pillar structure (P) and the upper surface of the base layer (1). A part of the electrically conductive material (M) and / or the adhesive material is removed by e.g. laser ablation from the upper surface of the base layer (1), thereby a lead line is formed (outlined) on the upper surface of the base layer (1) (Fig. 13(e)). Alternatively, the electrically conductive material (M) and / or the adhesive layer may be selectively deposited on the upper surface of the base layer, e.g. by a photolithography process.

[0076] After forming the lead line, an insulating material (I) is applied on the surface of the electrically conductive material (M) (Fig. 13(f)) . The entire surface of the electrically conductive material (M) may be covered by the insulating material (I) and thereafter a part of the lead lines may be exposed by removing the insulating material (I) e.g. by laser ablation.

[0077] Finally, a tip portion of the pillar structure (P), which is covered by the electrically conductive material (M) and the insulating material (I), is cut off, e.g. by laser ablation (Fig. 13 (g)), thereby obtaining the electrode portion (2) in which the electrically conductive layer (2-2) (a remaining part of the electrically conductive material) is exposed on the top surface of the pillar portion (2-1) (Fig. 13(h)).

[0078] Alternative to the step as illustrated in Fig. 13(h), only a part of the insulating material (I) can be removed only from the tip portion of the pillar structure (P), thereby obtaining the contacting portion covering the top surface of the pillar portion (2-2) similar to the first embodiment shown in Figs. 6 to 8. In this step of laser-ablation, the base layer (1) may be held vertically, i.e. along the z- direction, and a laser beam may be radiated in the z-direction to cut off the tip portion or to remove only the insulating material from the tip portion.

[0079] Alternative to the laser-ablation step, only a part of the insulating material (I) is removed from the tip portion of the pillar structure (P) by applying a spinning process. The (intermediate) product, which is the photoresist layer covered by the electrically conductive material and the insulating material, is made to spin or rotated by a spin coater around an axis being perpendicular to the upper surface of the base layer (1), before the insulating material (I) (for example a pre-polymer mixture such as PDMS) is cured and / or hardened. The tip portion of the pillar structure includes the top surface of the pillar structure, which is positioned at the highest position from the upper surface of the base layer (1) in the z-direction, and therefore centrifugal forces applied at the tip portion (and / or the top surface) of the pillar structure are higher than that of other parts of the product. Theinsulating material (I) can be therefore removed only from the tip portion of the pillar structure, while the insulating material (I) remains attached other parts of the microelectrode element. The second spinning process is performed at a rotational speed being higher than a rotational speed for the first spinning process. The second spinning process is performed for a time period being shorter than a time period for the first spinning process. After the second spinning process, the contacting portion covering the top surface of the pillar portion (2-2) is obtained, similar to the first embodiment as illustrated in Figs. 6 to 8.Examples

[0080] Certain aspects and embodiments of the present disclosure will be illustrated by way of examples. Such examples of the present disclosure are representative only and should not be taken to limit the scope of the present disclosure to only such representative examples.

[0081] First ExampleMicroelectrode array samples, which are examples of the microelectrode array including microelectrode elements according to the first embodiment, were manufactured and its electrochemical characteristics were analyzed:

[0082] < Manufacturing of a Mold>Prior to the manufacturing of the microelectrode array sample, three different molds were prepared: For each of the molds, an upper surface of a silicon wafer (an example of silicon wafer (S)) was etched by applying reactive ion etching, and a plurality of trenches is formed. The silicon wafer used is 4 inch sized. The mold accordingly has a main surface extending in a xy-direction and the plurality of trenches extending from the main surface in the depth direction of the silicon wafer. In this example, each of the trenches was formed into a cylindrical form with a bottom, so that a cylindrical formed pillar structure can be obtained by using the mold. The three molds are different from each other in the depth of the trenches: The first mold for “Sample (a)” has trenches, each of which with a depth 200 pm and a diameter 30 pm. The second mold for “Sample (b) ” has trenches, each of which with a depth 200 pm and a diameter 40 pm. The third mold for “Sample (c)” has trenches, each of which with a depth 200 pm and a diameter 10 pm.

[0083] < Manufacturing of Samples>The main surface and surfaces of the trenches were coated with parylene (an example of the coating layer) by chemical vapor deposition into a thickness of 5 pm. Subsequently, 5 ml PDMS prepolymer solution (an example of the pre-polymer mixture) was spin coated (the fist spinning process, 400 rpm for 30 sec) onto the main surface, thereby the PDMS pre-polymer solution was distributed on the main surface of the mold and fill up each of the trenches. The mold with the PDMS prepolymer solution was then cured at 70 °C for 3 hours. The polymerized / cured PDMS (an example of the polymerized mixture) was then manually peeled off from the silicon mold, and the polymerized PDMS including a base layer in a thickness of 400 pm and a plurality of pillar structures wassuccessfully obtained. Subsequently, 200 ill ethanol was applied on a glass wafer (an example of the support (G)) and the polymerized PDMS was then transferred on the ethanol so that the lower surface the base layer was adhered to the glass wafer. The polymerized PDMS and the glass wafer were then left overnight to dry the ethanol.

[0084] Subsequently, 5 nm Cr layer (an example of the adhesive layer) and too nm Au layer (an example of the electrically conductive material / layer) were sputtered onto the entire surface of the upper surface of the PDMS base layer and the PDMS pillar structure. A laser-patterning system (MD- U1000C, Keyence Japan, Japan) was then used to pattern lead lines by selectively ablating the Au and Cr layers on the PDMS base layer to form lead lines. The entire surfaces of the remained Au layer on the PDMS base layer and the PDMS pillar structures were coated with PDMS by spincoating at 6000 rpm for 40 to 50 sec (the first spinning process), thereby the insulating layer being an example of the first and second insulating layers is obtained. Subsequently, only a part of the PDMS pre-polymer solution was removed from the tip portion of the pillar structures by a spinning process at 9000 rpm for 2 sec (the second spinning process), thereby the Au layer is exposed on the tip portion of each of the pillar structures.

[0085] Scanning electron microscopy (SEM, JSM-6060 LV, JOEL, Japan) was used to image different pillar structures; (i) Pillar structures, each of which has a diameter of 10 pm, before the first spinning process (“Sample (a)”, Figs. 14 (e), (f)), (ii) Pillar structures, each of which has a diameter of 30 pm, directly after the first spinning process (“Sample (c)”, Figs. 14(c), (d)), (iii) Pillar structures, each of which has a diameter of 40 pm, directly after the second spinning process (“Sample (b)”, Figs. 14(a), (b)). The height of the pillar structure of each of “Sample (a)”, “Sample (b) ” and “Sample (c)” is 200 pm. As can be seen from Figs. 14(e), (f), the pillar structures were successfully covered by the Au layer after the step of applying the Au. Figs. 14(c), (d) show that the top surface of the pillar structure is completely covered by the PDMS layer, whereas Figs. 14(a), (b) show the Au layer on the top surface of the pillar structure was successfully removed and exposed by the second spinning process, while the side surface of the pillar structure and the upper surface of the base layer were still covered by PDMS.

[0086] We could further confirm the exposure of the Au surface at the tip portion of the pillar structure achieved by the second spinning process, by electrochemical characterizations: Fig. 15 shows average absolute impedance (solid lines) and phase (dashed lines) recorded with the Sample (a), (b) and (c). As can be especially seen from the dashed lines, the high impedance of the Sample (c) indicates that the Au layer of the Sample (c) at the tip portion of the electrode portion was completely passivated by PDMS. The impedance was decreased approximately by a factor of three for the Sample (b), which is comparable to that of the Sample (a), which is a clear indication that the top surface of the pillar structure is uncovered by parylene and the Au surface is exposed.

[0087] Second ExampleMicroelectrode array samples, which are examples of the microelectrode array including microelectrode elements according to the second embodiment, were fabricated and their characteristics were analyzed:

[0088] < Manufacturing of Samples>A glass wafer (a diameter of 76.2 mm and thickness of 0.5 mm wafer) being an example of the base layer (1) were cleaned by subsequent ultrasonication in acetone, isopropanol, and deionized water for 10 minutes each. The procedure was repeated twice, and afterward, the samples were blow- dried with compressed air. A dehydration bake of the wafer was then conducted at 7O°C for 15 minutes.

[0089] A SU-8 film (SUEX series dry films, micro resist technology GmbH, Germany) were applied on the surface of the glass wafer, which is an example of the photoresistive polymer layer (2’), by rolling onto the substrates at 7O°C using a manual laminating roller. A soft bake at 95°C for 30 minutes was then carried out to adhere the films well onto the substrate. Photolithography was implemented using a maskless alignment system (LIMLA, Heidelberg Instruments, Germany). A defocus of +10 with exposure doses of 18 J / cm2was used (the wavelength of the light source was 365 nm). After exposure, the samples were subjected to a post-exposure bake by heating for 1 minute at 6s°C and then ramping up the temperature to 95°C in 3 minutes. The samples were then transferred to a beaker containing 10 ml of developer (mrDev-600, micro resist technology GmbH, Germany), and development was performed with periodic shaking of the beaker for 20 minutes. Once the pillar structures (being examples of the pillar structure (P)) were visible, the samples were rinsed with isopropanol.

[0090] After SU-8 pillar structures were formed on the glass wafer, metal layers (10 nm Ti followed by 150 nm Pt), being examples of the adhesive layer and the electrically conductive material (M), were sputtered (BAL-TEC MED 020, LabMakelaar Benelux BV, Zevenhuizen, the Netherlands) onto the entire surfaces of the pillar structures and the glass wafer. A laser-patterning system (MD-U1000C, Keyence Japan, Japan) was used to pattern lead lines by selectively ablating the metal layer. Subsequently, 5 pm of paiylene-C being an example of the insulating material (I) (PPS Parylene 3000, Plasma Parylene System GmbH, Germany) was deposited to passivate the Pt layer. A ring electrode were exposed via vertically aligned laser ablation of the pillar structures (average laser power: 2.8 W, scan speed: 800 mm / s, pulse frequency: 40 KHz, repetition: 30,000), thereby obtaining a microelectrode array comprising multiple microelectrode elements.

[0091] <SEM Images >Scanning electron microscopy (SEM, JSM-6060 LV, JOEL, Japan) was used to image the pillars structures and the microelectrode array. Fig. 16 (a) and (b) show SEM images of the SU-8 pillar structures, each of which has a top surface of ~ 40 pm diameter. Fig. 16 (c) and (d) show SEM images of the microelectrode array and the electrode element (i.e. with the electrically conductive layer and the first and second insulating layers). Each of the pillar portion shown in Figs. 16(c) and (d) has atop surface ~ 80 pm. As clearly seen in Fig. 16(c), a ring-formed gold layer is exposed on the top surface of the pillar portion.

[0092] < Buckling Force Measurements>Buckling force measurements were conducted to assess the maximum compression force that the electrode portion (i.e. with the pillar portions) can withstand. The tensile test instrument can measure the buckling force in the compression mode. We observed that the buckling force of the electrode portion of the microelectrode array sample could withstand approximately 0.6 N / pillar.

[0093] < Electrochemical Characterization >Electrochemical impedance spectroscopy (EIS; 200 mV offset vs. Ag / AgCl, 10 mV amplitude, 1 Hz to 10 kHz scan range) was carried out using a VSP-300 potentiostat (Biologic Science Instruments, France) in a three-electrode configuration with an Ag / AgCl reference electrode (Dri- Ref, Flexref from World Precision Instrument, USA) and a coiled platinum wire as the counter electrode. The characterization experiments were conducted in phosphate-buffered saline (PBS). Before EIS, the microelectrode array sample were activated in 150 mM H2SO4 using cyclic voltammetry (scan rate 500 mV / s, -0.2 to 1.5 V vs Ag / AgCl, 20 cycles).

[0094] Fig. 17 (a) shows average absolute impedance and phase recorded from eight electrodes of the microelectrode array according to Sample 2. The impedance decreases with increasing frequency but does not follow a purely capacitive (t / f) relation. Specifically, at 1 kHz, the impedance magnitude is around 10 l<U. The relatively low impedance significantly differs from the expectation for a small, exposed electrode area of ~28 pm2. A possible reason for this deviation could be ascribed to the laser ablation process. While the paiylene typically ensures good insulation quality, after laser ablation, the ring electrode area may no longer be tightly surrounded by paiylene. Thus, the inner electrode shafts could be exposed to electrolyte via a nano- or microscale gap at the pillar tip. This would result in a larger effective electrode / electrolyte interface compared to a standard ring electrode. The decreased impedance would be advantageous in extracellular recordings with an improved signal- to-noise ratio.

[0095] < Extracellular Recording in HL-1 Cells >HL-t cells were cultured on the microelectrode array sample: The microelectrode array sample were plasma-treated and pre-coated with fibronectin (5 pg mL-1, Sigma-Aldrich, USA) and gelatin (0.2 mg mL-t, Sigma-Aldrich, USA) for ~1 h at 37 °C for cell adhesion. The detection was performed with a 64-channel home-built amperometric amplifier system (10 kHz sampling rate per channel, 3.4 kHz bandwidth) in a two-electrode setup using a Ag / AgCl electrode as a combined reference and counter electrode 24. All the experiments were performed in a grounded Faraday cage. The cell activity was stopped by adding SDS (toopL from 1 M stock solution pipetted into 1 m medium on the chip, Sigma-Aldrich, USA)).

[0096] As shown in Figs. 17 (b) to (d), we recorded extracellular signals from HL-1 cells. The cell growth was observed for five days, and the medium was changed daily until confluency was reached. As shown in Fig. 17(b), spontaneous action potentials recorded from 6 channels, and at 10 s, the cell contractions were stopped by adding 1% sodium dodecyl sulfate (SDS). Before application of SDS we observed a stable but phase-shifted beating frequency at -0.7 Hz across all channels. The recorded peak-to-peak current amplitudes ranged from around 60 to 200 pA. The difference in amplitudes recorded at different channels can be explained by variations in the junction resistance at the interface of individual cells and microelectrodes. Signal traces from a single channel are exemplary shown in Fig. 17(c), and average spike shape of all recorded spikes is shown in Fig. 17(d). The average noise was calculated by evaluating the root mean square (RMS) of the trace. For the given channel, the RMS value was found to be 24.2 ± 1.3 pA with a peak-to-peak amplitude of 363.5 ± 21.7 pA (approximate SNR of 15). This proof-of-concept experiment demonstrated the applicability of the microelectrode array sample for extracellular signal recording from cardiomyocyte cells.

[0097] < Reference numbers >A: microelectrode element, 1: base layer, 2: electrode portion, 2-1: pillar portion, 2-2: electrically conductive layer, 2-3: first insulating layer, 3: lead line, 4: second insulating layer, B: microelectrode array, S: mold, P: pillar structure, M: electrically conductive material, I: insulating material, 2’: photoresistive polymer layer.

Claims

Claims1. A microelectrode element (A) for being implanted into tissues such as a nerve or for being brought into contact with a single cell or cells, in order to apply and / or record an electrical signal to and / or from the tissues or the single cell or the cells, wherein the microelectrode element (A) comprises a base layer (t) extending in a xy-plane being perpendicular to a z-direction, the base layer (t) being electrically insulative, and an electrode portion (2) comprising a pillar portion (2-1) protruding from an upper surface of the base layer (1) along the z-direction and comprising a polymer compound, an electrically conductive layer (2-2) covering at least a side surface of the pillar portion (2-1), and a first insulating layer (2-3) at least partly covering the electrically conductive layer (2-2), wherein a part of the electrically conductive layer (2-2) is contactable.

2. The microelectrode element (A) according to claim 1, wherein the polymer compound of the pillar portion (2-1) is obtained by a photoresistive polymer, wherein preferably the photoresistive polymer comprises an epoxy-based polymer and / or negative photoresist, or wherein the polymer compound of the pillar portion (2-1) comprises a flexible polymer compound, wherein the flexible polymer compound is an elastomer and / or polydimethylsiloxane and / or hydrogel and / or natural rubber and / or styrene butadiene rubber and / or chloroblen rubber and / or acrylonitrile rubber and / or butyl rubber and / or ethylene propylene rubber and / or urethane rubber and / or silicone rubber and / or fluorine rubber.

3. The microelectrode element (A) according to claim 1 or 2, wherein the electrically conductive layer (2-2) covers a top surface of the pillar portion (2-1), orwherein a top surface of the electrode portion (2) comprises a part of the electrically conductive layer (2-2) being ring-formed when viewing from the top of the electrode portion (2).

4. The microelectrode element (A) according to any one of claims 1 to 3 comprising a lead line (3) being electrically conductive and being stacked on a part of the upper surface of the base layer (1) in the z-direction and being connected to the electrically conductive layer (2-2), a second insulating layer (4) being stacked at least on a part of the lead line (3) in the z-direction.

5. The microelectrode element (A) according to any one of claims 1 to 4 further comprises an adhesive layer between the electrically conductive layer (2-2) and the side surface of the pillar portion (2-1), and / or an adhesive layer between the lead line (3) and the upper surface of the base layer (1).

6. The microelectrode element (A) according to any one of claims 1 to 5, wherein a cross-sectional area of the pillar portion (2-1) in a xy-plane being perpendicular to the z-direction is constant at least 90 % of the extension of the pillar portion (2- 1) in the z-direction.

7. The microelectrode element (A) according to any one of claims 1 to 6, wherein the pillar portion (2-1) has a cylindrical form, and a cross-section of the pillar portion in a xy-plane being perpendicular to the z-direction has a circular form or an oval form, or wherein the pillar portion (2-1) has a prism form, and a cross-section of the pillar portion (2-1) in a xy-plane being perpendicular to the z-direction has a polygonal form.

8. The microelectrode element (A) according to any one of claims 1 to 5, wherein a cross-sectional area of the pillar portion (2-1) in a xy-plane being perpendicular to the z-direction becomes smaller in the z-direction from the upper surface of the base layer (1) to the top surface of the pillar portion (2-1).

9. The microelectrode element (A) according to any one of claims 1 to 5 and 8,wherein the pillar portion (2-1) has a truncated conical shape and a cross-section of the pillar portion (2-1) in a xy-plane being perpendicular to the z-direction has a circular form or an oval form, or wherein the pillar portion (2-1) has a truncated pyramid shape and a cross-section of the pillar portion (2-1) in a xy-plane being perpendicular to the z-direction has a polygonal form.

10. The microelectrode element (A) according to any one of claims 1 to 9, wherein the pillar portion (2-1) and the base layer (1) are integrally formed and comprise the same polymer compound.

11. The microelectrode element (A) according to any one of claims 1 to 10, wherein the base layer (1) is obtained by a photoresistive polymer, wherein preferably the photoresistive polymer comprises an epoxy-based polymer and / or negative photoresist, or wherein the base layer (1) comprises a flexible polymer compound, wherein the flexible polymer compound is an elastomer and / or polydimethylsiloxane and / or hydrogel and / or natural rubber and / or styrene butadiene rubber and / or chloroblen rubber and / or aciylonitrile rubber and / or butyl rubber and / or ethylene propylene rubber and / or urethane rubber and / or silicone rubber and / or fluorine rubber.

12. The microelectrode element (A) according to any one of claims 1 to 11 further comprising a contacting portion being electrically conductive, wherein the contacting portion is attached to a top surface of the pillar portion (2-1) and being connected to the electrically conductive layer (2-2).

13. A microelectrode array (B) comprising a plurality of microelectrode elements (A) according to any one of claims 1 to 12.

14. A method for manufacturing a microelectrode element (A) according to any one of claims 1 to 12 and / or a microelectrode array according to claim 13 comprising a step of applying a pre-polymer mixture onto and / or into a mold (S) and polymerizing the mixture, a step of detaching the polymerized mixture from the mold (S), the polymerized mixture comprising the base layer (1) and a pillar structure (P) protruding from the upper surface of the base layer (1) along the z-direction,a step of applying an electrically conductive material (M) on a surface of the pillar structure (P), thereby forming the electrically conductive layer (2-2), a step of applying an insulating material (I) on a surface of the electrically conductive material (M) being on the surface of the pillar structure (P), thereby forming the first insulating layer (2-3) on the electrically conductive layer, a step of removing at least a part of the insulating material (I) from a tip portion of the pillar structure (P), so that a part of the electrically conductive layer (2-2) is contactable.

15. A method for manufacturing a microelectrode element (A) according to any one of claims 1 to 12 and / or a microelectrode array according to claim 13 comprising a step of forming a photoresistive polymer layer (2’) on the base layer (1), a step of exposing the photoresistive polymer layer (2’) to a pattern of light, subsequent to the step of exposing, a step of developing the photoresistive polymer layer (2’) and removing only a first part of the photoresistive polymer layer (2’), thereby forming a pillar structure (P) protruding from the upper surface of the base layer (1) along the z-direction, the pillar structure (P) being a second part of the photoresistive polymer layer (2’), the second part being different from the first part, a step of applying an electrically conductive material (M) on a surface of the pillar structure (P), thereby forming the electrically conductive layer (2-2), a step of applying an insulating material (I) on a surface of the electrically conductive material (M) being on the surface of the pillar structure (P), thereby forming the first insulating layer (2-3) on the electrically conductive layer (2-2), a step of removing at least a part of the insulating material (I) from a tip portion of the pillar structure (P), so that a part of the electrically conductive layer (2-2) is contactable.

16. The method according to claim 14 or 15, wherein the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) includes a step of cutting off the tip portion of the pillar structure (P) together with a part of the electrically conductive material (M) covering the tip portion and a part of the insulating material (I) on the tip portion, ora step of removing a part of the insulating material (I) only from the tip portion of the pillar structure (P), while the electrically conductive material (M) remains attached on the tip portion.

17. The method according to claim 15 or 16 comprising a step of depositing the adhesive layer directly on the surface of the pillar structure (P), prior to the step of applying the electrically conductive material (M).

18. The method according to any one of claims 14 to 17 comprising a step of applying the electrically conductive material (M) on the upper surface of the base layer (1), wherein preferably the method further comprises a step of depositing the adhesive layer directly on the upper surface of the base layer (1), prior to the step of applying the electrically conductive material (M), wherein the method further comprises a step of removing a part of the electrically conductive material (M) and / or the adhesive layer from a part of the upper surface of the base layer (1), thereby forming the lead line (3) on the upper surface of the base layer (1), and a step of applying the insulating material (I) on a surface of the electrically conductive material (M) being on the upper surface of the base layer (1), thereby forming the second insulating layer (4).

19. The method according to claim 18, wherein the step of applying the electrically conductive material (M) on the upper surface of the base layer (1) and the step of applying an electrically conductive material (M) on the surface of the pillar structure (P) are simultaneously performed, and / or the step of applying the insulating material (I) on the surface of the electrically conductive material (M) being on the upper surface of the base layer (1) and the step of applying the insulating material (I) on the surface of the electrically conductive material (M) being on the surface of the pillar structure (P) are simultaneously performed.

20. The method according to any one of claims 14 to 19, wherein the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) is performed by laser-ablation, orwherein the step of removing at least a part of the insulating material (I) from the tip portion of the pillar structure (P) is performed by a spinning process, thereby removing the insulating material (I) only from the tip portion by centrifugal forces.

21. The method according to claim 19 or 20, wherein the step of removing a part of the electrically conductive material (M) and / or the adhesive layer from a part of the upper surface of the base layer (1) is performed by laser-ablation.

22. The method according to any one of claims 14, 16 to 21 further comprising a step of forming a coating layer on a surface of the mold (S) prior to the step of applying the pre-polymer mixture, wherein the coating layer is preferably paiylene.

23. The method according to any one of claims 14, 16 to 22 further comprising a step of preparing the mold (S) by applying reactive ion etching on an upper surface of a silicon wafer (S), thereby forming a trench extending from the upper surface of the silicon wafer (S) in a thickness direction of the silicon wafer (S), the trench being for forming the pillar structure (P) and the upper surface of the silicon wafer (S) being for forming the base layer (1).

24. The method according to any one of claims 14, 16 to 23 further comprising, a step of transferring the polymerized mixture to a support (G), such that a lower surface of the base layer (1) is attached to a surface of the support (G), subsequent to the step of detaching the polymerized mixture from the mold (S) and prior to the step of applying the electrically conductive material (M) on the surface of the pillar structure (P), wherein preferably the step of transferring comprises a step of applying organic solvent between the support and the polymerized mixture and subsequently diying the organic solvent (E).

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