Production of three-dimensional electrodes by way of template-assisted electrochemical deposition

Abstract

The present invention relates to a method for producing three-dimensional electrodes or three-dimensional microelectrode arrays by way of template-assisted, in particular electrochemical, deposition, proceeding from two-dimensional substrates.

Description

[0001] Fabrication of three-dimensional electrodes using template-assisted electrochemical deposition

[0002] All documents cited in the present application are incorporated by reference in their entirety into the present disclosure.

[0003] The present invention relates to methods for producing three-dimensional electrodes or three-dimensional microelectrode arrays starting from two-dimensional substrates by means of template-assisted, in particular electrochemical, deposition.

[0004] State of the art:

[0005] Neuronal model systems are used to gain a better understanding of neurological diseases such as Alzheimer's and Parkinson's. In contrast to two-dimensional (2D) systems, three-dimensional (3D) systems (e.g., hydrogel scaffolds, brain organoids, and tissue slices) offer a more complex environment with longer lifetimes and are also more informative, thus better complementing animal models. Although these 3D systems can already closely mimic the brain and other neural tissues, advanced electrophysiological electrodes for long-term stable recordings with single-cell and millisecond resolution are required to better understand the function of such systems.

[0006] While planar 2D electrodes are already widely used, there are few approaches focusing on 3D electrodes. However, as research shifts toward complex 3D cellular systems, the need for truly multilevel 3D electrode systems to better investigate 3D environments is increasing. These systems are 3D arrays of microelectrodes used to measure and monitor neuronal activity in various neuronal tissues, for example, in the brain, retina, or peripheral nerves. Therefore, to further improve signal recording from 3D nervous systems, new innovative methods are needed to investigate three-dimensional neuronal networks at the level of single cells, entire networks, and the 3D space of organoids and tissues.

[0007] Various three-dimensional electrophysiological measurement technologies have already been developed using different manufacturing methods. These mainly include i) 3D inkjet printing methods: A conductive ink (such as silver) is printed dropwise to create the three-dimensional, conductive structures (pillars). Depending on the application, the structures are subsequently passivated and only the tip is opened using etching methods to enable a connection to the cells or tissue (see, for example, Saleh et al., CMU Array: A 3D Nano-Printed, Customizable Ultra-High-Density Microelectrode Array Platform, 2019). ii) Two-photon polymerization printing methods: The two-photon

[0008] Polymerization printing is used to print complex three-dimensional structures, which can optionally be conductive. Various additional micro / nanofabrication steps are then required to produce 3D electrodes. These steps include, for example, selective metal deposition, multiple lithography and etching steps. iii) Kirigami technique: In the kirigami technique, several shafts are separated / cut out of 2D samples and then folded to create a 3D sample. Various methods exist for the folding itself: Using ferromagnetic foils on the back of each shaft, folding is achieved by applying a magnetic field (see, for example, Soscia et al., A flexible 3-dimensional microelectrode array for in vitro brain models, 2020). a. Use of ferromagnetic foils on the back of the shafts to fold the 2D design using a magnetic field. b.The shafts fold without additional assistance. c. Mechanical folding of individual 2D shafts or several at the same time. iv) Stacking of 2D samples: Here, several 2D samples, such as the so-called Michigan arrays, which consist of several gold electrodes arranged one below the other on silicon shafts (several such silicon shafts are usually arranged side by side), are stacked on top of each other to generate a 3D implant. Typically, spacers are placed between the 2D samples to define a precise distance. v) Nanoneedles: The best-known nanoneedle approach is the "Utah array," which consists of silicon needles whose tips are coated with metal or conductive polymer and have a biocompatible sheath. vi) Electrode meshes: Creation of a 2D mesh consisting of holey structures with electrodes at the edges or vertices of the structures.The mesh then deforms and adapts to the 3D biological target (see, for example, Feiner et al, Engineered hybrid cardiac patches with multifunctional electronics for online monitoring and regulation of tissue function, 2016).

[0009] With inkjet printing, the selection of printable materials and geometries is severely limited. For example, the diameter of the printed columns is on the order of 20 pm. The larger the diameter of the columns, the greater the damage to the penetrated tissue and the lower the spatial resolution of the electrode measurement. Furthermore, additional manufacturing steps are required, such as the application of passivation and an etching step. The kirigami and stacking approaches require folding individual shafts, stacking the individual 2D samples, or the use of toxic materials, such as ferromagnetic metals (e.g., nickel), which are hazardous, as well as special equipment to generate the magnetic field.The previously used production of 3D electrodes by 3D printing using two-photon polymerization requires additional, complex microfabrication steps such as selective metal deposition, multiple lithography and etching steps, especially if electrode openings of different, arbitrary heights are desired.

[0010] Silicon nanoneedles are rigid structures on a rigid surface and therefore cause greater tissue damage. Furthermore, this method also requires additional, complex microfabrication steps such as selective metal deposition, multiple lithography, and etching steps, especially when electrodes of varying heights are desired.

[0011] The mesh approach has the major disadvantage of a larger insertion cross-section, which leads to greater tissue damage. Furthermore, it is very difficult to control and define the electrode position in all dimensions.

[0012] Further prior art documents include US 2021 / 0394434 A1 or US 2002 / 0133129 A, as well as Saleh, Mohammad Sadeq, et al., "CMU Array: A 3D nanoprinted, fully customizable high-density microelectrode array platform," Science Advances, 2022, Vol. 8, No. 40, p. eabj4853, and US 2017 / 0231518 A1. Therefore, the search for further methods that allow the reliable production of three-dimensional electrodes or three-dimensional microelectrode arrays is ongoing.

[0013] The object of the present invention was therefore, with regard to the prior art, to find methods for producing three-dimensional electrodes or three-dimensional microelectrode arrays or advantageous three-dimensional electrodes and / or three-dimensional microelectrode arrays which do not have the disadvantages of the prior art and are in particular reliable, as well as corresponding (neuro) implants and arrangements.

[0014] Further tasks for the specialist arise from the following description.

[0015] These and other objects are achieved within the scope of the present invention by the subject matter of the independent claims.

[0016] Preferred embodiments emerge from the dependent claims and the following description.

[0017] Within the scope of the present invention, the term "comprise" also includes "consisting of" as a particularly preferred embodiment; this means that a corresponding list may contain (=comprise) further elements in addition to the explicitly mentioned elements, or it may contain precisely these elements (=consist of) (whereby non-essential elements such as screws, markings, etc. are not taken into account).

[0018] For relative information such as top, bottom, left, right or similar, the reference system used in the present invention is an observer standing upright on the ground in front of the object in question.

[0019] In the context of the present invention, the term "and / or" means that both elements mentioned in the context are included individually as well as the combination of the elements mentioned in the context.

[0020] Within the scope of the present invention, all quantities are to be understood as weight specifications unless otherwise stated. Within the scope of the present invention, the term "microelectrode array" or "MEA" refers to an arrangement of a plurality of electrodes on a flat substrate base. The electrodes themselves are provided with conductor tracks on the base, and contact surfaces may also be present. If the electrodes are based only on the flat base metallization (comprising conductor tracks, electrode surfaces, and optionally contact surfaces) on the substrate base, this is referred to as a 2D MEA within the scope of the present invention. If electrically conductive material is arranged on the electrode surfaces in an orthogonal direction (within 3D-printed hollow cylinders), this is referred to as a 3D MEA or three-dimensionally structured MEA within the scope of the present invention.

[0021] For the purposes of the present invention, the term "ambient temperature" means a temperature of 20°C. Unless otherwise stated, temperatures are in degrees Celsius (°C).

[0022] Unless otherwise stated, the reactions or process steps mentioned are carried out at atmospheric pressure, ie at approximately 1013 kPa.

[0023] Firstly, the present invention relates to a method for producing three-dimensionally structured microelectrode arrays which comprises or consists of the following method steps A), B), C) and D):

[0024] In step A), a 2D microelectrode array comprising a plurality of conductor tracks and a plurality of electrode surfaces is provided. Typically, this also includes a plurality of contact surfaces; this is preferred but not mandatory, because instead of additional contact surfaces, the (ends of) the conductor tracks (=leads to the electrode surfaces) can also serve the same purposes (i.e., in particular, contacting other devices).

[0025] A plurality of conductor tracks / electrode surfaces means any desired number of conductor tracks, preferably at least two. Typically, however, this is significantly more, although it is preferred if each electrode surface is assigned a conductor track, i.e., the same number of conductor tracks and electrode surfaces are present. In preferred variants, this is from 2 to 512 (inclusive), preferably 4 to 256 (inclusive), particularly preferably 8 to 128 (inclusive), and in particular 16, 32, or 64.

[0026] Conductor tracks / electrode surfaces. A large number of contact surfaces means a number of at least two. Since these serve for "wiring" or connecting the conductor tracks, there is usually only one large contact surface per conductor track, at each end of each conductor track. However, the contact surfaces can also be grouped together and then there don't need to be as many as there are conductor tracks.

[0027] Step B) involves 3D printing open-topped hollow cylinders orthogonally on the surface of the 2D microelectrode array, with the cylinder bases printed externally around the electrode surfaces.

[0028] This means that the hollow cylinders protrude above the electrode surfaces. The hollow cylinders are manufactured according to the respective requirements, which means that, in particular, their diameter and height are adjusted accordingly. The diameter preferably results from the electrode surfaces over which the hollow cylinders are arranged. Within the scope of the present invention, it is preferred that the cylinder bases are arranged externally around the electrode surfaces so that the entire electrode surfaces lie within the inner diameter of the hollow cylinder bases. This ensures that the entire electrode surface is later available for contact with the electrically conductive material.

[0029] In order to adapt the three-dimensionally structured microelectrode arrays manufactured within the scope of the present invention to the respective desired application, the hollow cylinders can be manufactured in different heights. This is, of course, readily possible during production using 3D printing and can be easily specified within the originally created print file.

[0030] In some particularly preferred variants, the hollow cylinders are uniformly designed, i.e., the wall thicknesses and inner diameters are constant over the entire height. However, since the wall thicknesses can be adapted to requirements, there are also preferred variants in which the wall thickness and inner diameter vary over the height of the cylinders, for example, to increase stability. Preferred inner diameters (=opening diameters) of the hollow cylinders within the scope of the present invention are 0.8 μm to 50 μm, particularly preferably 7 μm to 11 μm, in particular 8 μm or 10 μm.

[0031] Preferred wall thicknesses of the hollow cylinders in the context of the present invention are 0.2 pm to 10 pm, particularly preferably 0.5 pm to 2.5 pm, in particular

[0032] 1 pm or 2 pm.

[0033] Preferred heights of the hollow cylinders in the context of the present invention are 5 pm to 5 mm, particularly preferably 40 pm to 120 pm. In some embodiments, the heights are 20 pm to 110 pm, preferably 35 pm to 95 pm, particularly preferably 50 pm to 80 pm, in particular for in-vivo applications. In some other embodiments, the heights are 30 pm to 140 pm, preferably 45 pm to 125 pm, particularly preferably 60 pm to 110 pm, particularly for in-vitro applications.

[0034] Preferred distances (measured from electrode center to electrode center) between the individual electrodes (hollow cylinders) are

[0035] 2 pm to 200 pm, particularly preferred at 13 pm to 110 pm, especially at 16 pm to 106 pm. It should be noted that the largest distance can be flexibly adjusted to meet specific needs (i.e., it can also be greater than 200 pm), and the smallest distance is the diameter of the electrode plus the wall thickness, in the case where the cylinders are printed directly next to each other.

[0036] The present invention therefore allows the production of electrodes (=the conductive material filled into the hollow cylinders, optionally with an additional coating) with a very high aspect ratio (height of the hollow cylinders:their outer diameter). In preferred embodiments of the present invention, the aspect ratio is therefore 10 or higher, particularly preferably 15 or higher.

[0037] In principle, however, other dimensions are also possible, although it is known to those skilled in the art that the material and the aspect ratio (height:diameter) of the hollow cylinders impose material-technical requirements, in particular with regard to the wall thickness as a function of the height, so that the hollow cylinders remain correspondingly stable (although they are stabilized by the electrically conductive material deposited therein, so that thinner wall thicknesses are possible than with cylinders that remain hollow). It should be expressly stated that within the scope of the present invention, in particular within the scope of the method according to the invention, it is possible to dimension each hollow cylinder (and thus each electrode) individually and independently of all others, in particular with regard to internal diameter and height.

[0038] As a further step Ba), it is optionally possible to detach any remaining carrier substrate from the 2D microelectrode arrays during, before, or after, preferably before or after, the 3D printing of the hollow cylinders. This ultimately depends on whether the carrier substrate is appropriate for the intended application. This is readily apparent to the person skilled in the art within the scope of their general technical knowledge, based on the intended application of the three-dimensionally structured microelectrode arrays produced according to the invention.

[0039] In step C), electrically conductive material is then deposited into the hollow cylinders on the electrode surfaces. This can, in principle, be done using any method familiar to the person skilled in the art, but is usually done using electroplating processes.

[0040] In the context of the present invention, it is preferred if the hollow cylinders open at the top are completely filled with electrically conductive material, i.e., the uppermost surface of the electrically conductive, deposited material is level with the upper end of the respective hollow cylinder. In some variants, it is also possible to deposit a little more electrically conductive material, so that the electrically deposited material then protrudes slightly beyond the previously 3D-printed hollow cylinder.

[0041] As step D), it is optional within the scope of the invention, and preferred in some variants of the present invention, to carry out further coatings of the accessible surfaces of the electrically conductive material deposited in the hollow cylinders. Depending on the deposition, these accessible surfaces are usually only the uppermost surface within the hollow cylinder or at the level of the upper hollow cylinder edge. In the described variants of the present invention, in which slightly more electrically conductive material is deposited than fits into the hollow cylinders, this additional coating naturally also includes the protruding area. This coating of the electrically conductive material is usually carried out with further electrically conductive material, which can be the same as that deposited in the hollow cylinders or a different electrically conductive material.

[0042] Within the scope of the present invention, the additional, identical or different, electrically conductive material is deposited for this optional coating, wherein the exact layer thickness can be adapted to the respective requirements and is preferably in the nanometer to micrometer range.

[0043] It should be noted that, although hollow cylinders are referred to in the present invention, these do not necessarily have to be round cylinders. In principle, it is equally possible to use hollow structures that are n-sided, where n is an integer between 3 and 12. Numbers between 4 and 10 are particularly preferred.

[0044] However, it is preferred within the scope of the present invention if the hollow cylinders are round cylinders, since this has advantages with regard to the application as electrodes, in particular it prevents current peaks from occurring at any protruding corners of the electrically conductive material deposited therein.

[0045] "Round" in this context can also mean oval, although circular is preferred, although technically this cannot always be achieved 100% - the technical limits achievable by means of 3D printing are known to the expert and therefore need not be discussed in detail here.

[0046] In embodiments of the present invention, the method according to the invention further comprises the production of the 2D microelectrode array provided in step A), the production of which comprises or consists of the following method steps: li) provision of a, preferably rigid, substrate, preferably based on or consisting of silicon or quartz, in particular quartz substrate;

[0047] 2i) applying a plurality of conductive tracks and a plurality of electrode surfaces and optionally a plurality of contact surfaces;

[0048] 3i) depositing a passivation layer and then exposing the electrode surfaces and optionally a plurality of contact surfaces; or lii) providing a, preferably flexible, substrate, preferably based on or consisting of parylene-C (PaC) or polyimide, in particular parylene-C, on a carrier substrate, preferably a silicon wafer, 2ii) applying a plurality of conductor tracks and a plurality of

[0049] Electrode surfaces and optionally a variety of contact surfaces;

[0050] 3ii) optionally deposition of a passivation layer and then exposure of the

[0051] Electrode surfaces and optionally a variety of contact surfaces;

[0052] 4ii) alternatively to step 3ii) optionally processing the metal layer applied in 2ii), preferably etching, in particular by reactive ion etching, of selected (mask-defined) areas of the uppermost metal layer (if several are present).

[0053] In preferred embodiments of the present invention, the substrate layer in step lii) has layer thicknesses of 0.5 pm to 15 pm, particularly preferably of

[0054] 3 pm to 8 pm, especially 5 pm.

[0055] In preferred embodiments of the present invention, the passivation layers in steps 3i) and 3ii) have layer thicknesses of 0.5 pm to 15 pm, particularly preferably of 2 pm to 7 pm, in particular 2 pm to

[0056] 4 pm on.

[0057] In some preferred variants of the present invention, the conductor tracks and electrode surfaces in steps 2i) and 2ii) respectively consist of several layers of in particular titanium and gold, preferably the layer sequence titanium / gold / titanium and in particular with a total layer thickness of 50 nm to 170 nm, preferably 90 nm to 160 nm, particularly preferably 100 nm to 150 nm, most preferably 110 nm to 140 nm and in particular 120 nm or 130 nm.

[0058] In some preferred variants of the present invention, the passivation layers in steps 3i) and 3ii) are based on or consist of parylene-C or photo-structureable epoxy-based lacquers, such as SU-8.

[0059] The two variants li) to 3i) or alternatively lii) to 4ii) according to the invention are intended in particular for two fundamentally different applications of the electrode arrays, but are not to be used exclusively for each of these, ie are not limited to the respective preferred application.

[0060] The variant of process steps li) to 3i) is preferably used for the production of microelectrode arrays intended for in vitro use. The variant of the present invention with process steps lii) to 4ii), on the other hand, is preferably used for electrode arrays intended for in vivo applications.

[0061] As already mentioned, these two variants are not limited to the respective preferred application for in vitro or in i / i / o applications, but can in principle also be used for the other variant, or for other conceivable applications.

[0062] For the inventive variant of process steps 1i) to 3i), it is preferred if a rigid substrate is provided in step 1i), in particular one based on materials selected from the group consisting of silicon, quartz, ceramics such as Al2O3, SiC, SiSn4, and mixtures thereof, in particular quartz. The term "rigid" in this context means, in particular, elastic moduli of 80 GPa to 120 GPa; for example, quartz with an elastic modulus of 97 GPa and a thickness of 500 μm is preferably used.

[0063] In contrast, in variant lii), a flexible substrate is preferably provided, preferably based on or consisting of materials selected from the group consisting of parylene-C (PaC), polyimide, photo-imageable epoxy-based lacquers, such as SU-8, and mixtures thereof, in particular parylene-C. The term "flexible" in this context means, in particular, elastic moduli of 1.5 GPa to 3.5 GPa; for example, PaC with an elastic modulus of 2.76 GPa at a thickness of 5 pm is preferably used. Furthermore, in preferred variants, these substrates are provided applied to a silicon wafer.

[0064] In steps 2i) and 2ii), a plurality of conductor tracks and a plurality of electrode surfaces plus, in preferred variants, a plurality of contact surfaces are then applied to the provided or prepared substrate, as already explained above in connection with the method according to the invention. In this case, it is also preferred if the number of conductor tracks corresponds to the number of electrode surfaces and the number of contact surfaces is either zero or at most corresponds to the number of conductor tracks.

[0065] In step 4ii), which can be performed alternatively to step 3ii), the uppermost metal layer of the electrode surfaces, or at least a portion thereof, is removed. If a metal layer composed of multiple metals has been constructed, the uppermost metal layer is preferably removed; in the preferred embodiment of a metal layer made of titanium / gold / titanium according to the invention, the titanium layer, or only a portion thereof, in particular only the portion corresponding to the electrode surfaces but not the conductor tracks and, if applicable, contact surfaces, would then be removed. This can preferably be carried out by etching, in particular by reactive ion etching. Depending on the metal substrates used in each case, it will be apparent to the person skilled in the art which specific etching agents they can then select in the respective context.For this purpose, the relevant areas of the metallization are exposed before etching, for example by selective exposure and removal of a photoresist layer; this procedure is known to the person skilled in the art and need not be explained in detail here.

[0066] Furthermore, the process steps li) to 3i) and lii) to 4ii) are in principle known to the person skilled in the art, so that he can determine the precise process steps and process parameters to be carried out on the basis of his general specialist knowledge.

[0067] In some preferred variants of the present invention, the application of a passivation layer during the production of the 2D microelectrode arrays is omitted (i.e. steps 3i) or 3ii) are omitted) and instead an electrically non-conductive material is applied over the entire surface - with the exception of the electrode surfaces (and optionally contact surfaces) - by means of 3D printing, particularly preferably together with the 3D printing of the hollow cylinders, in particular by applying the same material in the same printing process.

[0068] In preferred variants of the present invention, a plate-like structure is printed as a base structure on the surface of the 2D microelectrode array before 3D printing the actual hollow cylinder structure.

[0069] A plate-like structure as a base structure is understood in particular to mean a preferably circular base plate, which is intended to increase the stability of the hollow cylinders, like a kind of foundation. The plate-like structure is printed with a layer thickness of 300 nm to 5 pm, preferably 500 nm to 3 pm; and the diameter of the round plate-like structure corresponds at least to the diameter of the electrodes plus the wall thicknesses plus 0.5 pm to 5 pm, preferably 1 pm to 3 pm, in particular 2 pm.

[0070] In some preferred variants, an electrically non-conductive material suitable for printing using two-photon polymerization is used as the material for 3D printing. Preferred variants are curable (meth)acrylate-based materials and / or mixtures of materials that crosslink via (radical) polymerization of the (meth)acrylate groups.

[0071] Examples of commercially available products are IP-L 780 or IP-Dip from NanoScribe.

[0072] In principle, however, all electrically non-conductive materials that can be 3D printed and that meet or satisfy the (mechanical, biological, etc.) requirements can be used.

[0073] In the context of the present invention, the term "electrically non-conductive" means a conductivity of less than 10' for the material of the hollow cylinders processed by 3D printing. 8 S / cm.

[0074] The electrically conductive material deposited into the hollow cylinders can, in principle, be any electrically conductive, depositable material. In preferred variants of the present invention, the electrically conductive material deposited into the hollow cylinders is selected from the group consisting of gold, platinum, PEDOT:PSS, iridium oxide, chromium, titanium, indium tin oxide, and mixtures or alloys thereof, preferably gold, platinum, PEDOT:PSS, iridium oxide, chromium, and mixtures or alloys thereof, particularly preferably gold.

[0075] The additional, identical or different, electrically conductive material used to coat the upper surfaces of the electrically conductive materials deposited in the hollow cylinders can, in principle, be any electrically conductive, depositable material. In preferred variants of the present invention, it is selected from the group consisting of gold, platinum, PEDOT:PSS, iridium oxide, chromium, titanium, indium tin oxide, and mixtures or alloys thereof, preferably gold, platinum, PEDOT:PSS, iridium oxide, chromium, and mixtures or alloys thereof, particularly preferably PEDOT:PSS.

[0076] A preferred variant according to the invention is when the electrically conductive material deposited into the hollow cylinders is gold and it is (is) coated with PEDOT:PSS as a further electrolytically conductive material.

[0077] In still further preferred variants of the present invention, the 2D microelectrode array has a rigid or flexible substrate layer, wherein the material for the rigid substrate layer is selected from the group consisting of quartz, silicon, ceramics such as Al2O3, SiC, SiSn4 and mixtures thereof, preferably quartz, or the material for the flexible substrate layer is selected from the group consisting of parylene-C, polyimide, photo-structureable epoxy-based lacquers such as SU-8 and mixtures thereof, preferably parylene-C.

[0078] In still further preferred variants of the present invention, the plurality of conductor tracks and the plurality of electrode surfaces and optionally the plurality of contact surfaces consist of a material selected from the group consisting of titanium, gold, platinum, conductive polymers such as PEDOT: PSS, indium tin oxide and mixtures, alloys or layers thereof, particularly preferably a metal layer sequence titanium / gold / titanium or a metal layer sequence titanium / gold.

[0079] Within the scope of the present invention, it is furthermore particularly preferred if—apart from the electrodes, i.e., the material electrically deposited into the hollow cylinders or, if applicable, the coating thereon with further electrically conductive material, and, if applicable, individual contact surfaces (parts)—the three-dimensionally structured electrode arrays are externally non-electrically conductive. However, this is already apparent to the person skilled in the art.

[0080] In still further preferred variants of the present invention, the 3D printing process is a 2-photon polymerization process.

[0081] In yet further preferred variants of the present invention, the three-dimensionally structured electrode arrays are fixed to circuit boards, for example, printed circuit boards (PCBs), and electrically connected thereto. Particularly preferably, the fixing is carried out using a reversible assembly process.

[0082] A further subject of the present invention are three-dimensionally structured electrode arrays comprising a) 2D microelectrode arrays comprising a plurality of conductor tracks and a plurality of electrode surfaces and optionally a plurality of contact surfaces; b) three-dimensional hollow cylinders filled with electrically conductive material arranged orthogonally on the electrode surfaces.

[0083] In preferred embodiments, the 2D microelectrode array of this subject matter has conductive traces and electrode surfaces on only one surface. In further preferred embodiments of this subject matter, the hollow cylinders are made of a 3D-printed material.

[0084] Thus, preferred embodiments of this subject matter are three-dimensionally structured electrode arrays comprising a) a 2D microelectrode array comprising, on only one surface side, a plurality of conductor tracks and a plurality of electrode surfaces; b) electrically conductive material filled in three-dimensional hollow cylinders made of 3D-printed material, arranged orthogonally on these respective electrode surfaces.

[0085] The material used for 3D printing, and therefore the 3D printed material, is preferably the same as described above.

[0086] In particular, the three-dimensionally structured electrode arrays are / are produced using the method according to the invention as described above and below.

[0087] The MEAs according to the invention or manufactured according to the invention can have different layouts, for example, and thus in preferred variants, 8 x 8 electrodes with a spacing of 200 pm, arranged in a square or 32 electrodes in a diamond arrangement (spacing of the electrodes 30 pm) - however, the present invention is of course not limited to these two specific examples.

[0088] Furthermore, the present invention relates to implants or neuroimplants comprising or consisting of three-dimensionally structured electrode arrays according to the present invention or three-dimensionally structured electrode arrays produced according to the method of the present invention.

[0089] In some preferred embodiments of the present invention, the implants are retinal implants.

[0090] Last but not least, the present invention relates to the use of three-dimensionally structured electrode arrays according to the present invention or those produced by the method according to the invention as or for implants or neuroimplants or for electrochemical and / or electrophysiological measurement in the millivolt range and / or of cell cultures.

[0091] In principle, any electrically conductive material that can be deposited electrolytically or electrolessly can be used as the electrode material within the scope of the present invention. In some variants of the present invention, electrolytically depositable electrically conductive materials are preferred.

[0092] In preferred variants of the present invention, the electrically conductive material which can be deposited galvanically or electrolessly, preferably galvanically, is selected from the group consisting of gold, platinum, PEDOT: PSS, iridium oxide (IrO x ), chromium and mixtures or alloys thereof, with chromium being restricted to applications where biocompatibility is not an issue.

[0093] The electrodes used in the present invention are technically 10 pm or 6 pm openings in the passivation layer (encapsulation). This layer is located above the conductive traces (leads) that conduct current from the electrodes to the edges of the chip, where the so-called bond pads, also referred to as contact areas in the present invention, are located. These bond pads are non-passivated metal contacts that ensure contact between the electrodes and the external electronics.

[0094] The IP-L 780 photopolymer used in the present invention is a photopolymer suitable for the 3D printing process using two-photon polymerization. It was developed by NanoScribe and specifically designed for their 3D printers. Its reactive group is an acrylate group. The cured polymer is a thermoset, and the curing mechanism is radical polymerization. In principle, alternative products are non-conductive materials that can be processed with a resolution of up to 1 pm (either using 3D printing or other microfabrication methods) and that meet the requirements (e.g., mechanical, biological, etc.) appropriate for the application of the particular 3D device used.For 3D printing based on two-photon polymerization, IP-Dip, also developed by NanoScribe for high-resolution 3D printing, is a straightforward alternative that can be used in the present invention. However, it is also possible to use any other photoresin (including suitable solvents and photosensitive agents, etc.) with specific material properties depending on the application of the 3D device. Furthermore, one is, of course, not limited to the NanoScribe 3D printer; any other device from another company or even a home-built device can be used.

[0095] Using a 2-photon polymerization 3D printer, the present invention allows hollow cylinders of varying heights to be printed around the electrode openings of a hard or flexible, planar 2D microelectrode array (MEA) sample. These cylinders serve simultaneously as a template and a passivation layer. To increase the adhesion between the printed cylinders and the sample, a thin (2 to 3 pm) plate is also printed as a base structure.

[0096] An electrochemical deposition process of conductive materials (such as gold, platinum, PEDOTT: PSS, IrOx) through the hollow cylinders then leads to 3D electrodes with different heights, which depend on the heights of the cylinders.

[0097] Therefore, depending on the design and position of the cylinders, which are very flexibly adaptable, a Utah array-like design (nanoneedles with equal or sloping heights) or a 3D Michigan array-like design can be achieved.

[0098] 1) For the first design, cylinders of arbitrary heights are printed onto the 2D MEA sample at various, selected xy locations. The third dimension is then defined by the heights of the cylinders.

[0099] 2) In the second design, groups of three or four cylinders, each with different heights and arbitrarily spaced apart (a few micrometers), are printed together. This creates multiple 3D electrodes with different heights within a small area that depends only on the electrodes' dimensions. (For example, three cylinders with a diameter of 10 pm each result in a total area of ​​30 pm x 10 pm.)

[0100] Furthermore, flexible polymer materials can be selectively chosen for the entire fabrication process, both for the two-dimensional, planar micro-electrode array and for the cylinders.

[0101] The approach of using cylinders as templates for electrode deposition has the great advantage of making the production of 3D electrodes very simple and rapid. No additional complex microfabrication steps are required because the cylinders also serve as passivation. Furthermore, the 3D printing process allows for the realization of arbitrarily different geometries and spacings of the 3D electrodes. Cylinders with different shapes, diameters, aspect ratios, and heights can be easily realized during a single printing process. Therefore, the inventive approach reduces the complexity of fabrication compared to other 3D electrode approaches. Nevertheless, this approach retains a high degree of flexibility, allowing it to be easily adapted to a wide variety of applications.

[0102] Within the scope of the present invention, the heights and diameters, the spacing and shapes of the cylinders, as well as the materials used for implant production and the electrochemical deposition process, can be very flexibly adapted to the application without significantly complicating the fabrication process. The production of 3D electrodes with an aspect ratio of over 15 is readily feasible within the scope of the present invention.

[0103] Advantageously, the present invention enables electrical stimulation and recording of electrophysiological data for various electrogenic tissues such as brain, retina, peripheral nerves, heart, etc., as well as measurement of impedances in a 3D space.

[0104] The 3D devices and designs that can be produced with the present invention can be used for any type of biosensor / neural implant, especially where it is important to measure electrical signals. The design (dimensions, shape, number, etc.) of the cylinders can also be adapted to any need. The individual parts of the products according to the invention, especially implants, are operatively connected to one another in a conventional and known manner.

[0105] In preferred embodiments of the present invention, it does not claim the therapeutic treatment of humans or animals.

[0106] The various embodiments of the present invention, for example - but not exclusively - those of the various dependent claims or individual embodiments described in the figures, can be combined with one another in any desired manner, provided that such combinations do not contradict one another.

[0107] Process steps presented in the examples are to be interpreted as variants of preferred embodiments of the present invention.

[0108] The invention will now be further explained with reference to the following non-limiting examples.

[0109] Example 1 :

[0110] The inventive approach for producing 3D electrodes can be used, for example, to produce a flexible 3D retinal implant.

[0111] Accordingly, a standard 2D implant was first fabricated, using parylene-C (PaC) as the substrate material and for passivation. First, a 5 pm thick PaC layer was deposited on a Si wafer and patterned with a Ti / Au layer for leads, electrodes, and contact areas. Then, a second 5 pm thick PaC layer was applied for encapsulation. In the next etching step, the electrodes and contact areas were opened. Cylinders of various heights and shapes were then printed onto the 2D implant, with the cylinders aligned with the electrode openings. A photoresist capable of printing using the 2-photon polymerization process (IP-L 780) was used as the printed material.

[0112] Four different designs were fabricated. Each design contained 16 electrodes with pitches ranging from 16 pm to 106 pm. The printed cylinders had a height of 40 pm to 80 pm, an opening diameter of 8 pm, and a total diameter of 12 pm. The 2D sample was then detached from the Si substrate and mounted on a circuit board using a flip-top mounting method, where it was electrically connected.

[0113] The 3D electrodes with different heights were then obtained through an electrochemical deposition process of gold. To further improve the chip's electrical properties, a second electrochemical deposition was performed. The gold electrodes were coated with PEDOT:PSS. This reduced the electrode impedance.

[0114] This 3D implant was then used to implant it ex vivo into the retina of rats. This approach made it possible to measure intraretinal activity at different depths.

[0115] Example 2:

[0116] The present invention can also be used to measure and analyze in vitro neuronal cell cultures.

[0117] For this purpose, the cylinders were printed onto a hard silicon or quartz-based 2D MEA chip. First, a Ti / Au layer for leads, electrodes, and contact pads was evaporated onto the wafer and then patterned. A 2 pm to 4 mm thick passivation layer of SU-8 or PaC was then applied for encapsulation. In the next etching step, the electrodes and contact pads were opened. The cylinders were again aligned with the electrode openings and filled using an electrochemical deposition process of gold and PEDOT:PSS. The cylinders had a height of 40 pm to 150 pm, an opening diameter of 8 pm, and a total diameter of 10 pm to 12 pm. The material IP-L 780 was used again.

[0118] Primary rat cortical neurons were then placed onto the 3D sample. These formed a neuronal network that became electrically active after approximately 14 days and exhibited spontaneous electrical activity. Using electrophysiological measurements, neuronal signals in the millivolt range could be measured.

[0119] Examples 1 and 2 are just two examples demonstrating how the present invention can be used to measure various 3D neuronal structures. Accordingly, prototypes were fabricated and used to perform in vitro measurements of neuronal cell cultures as well as ex vivo measurements of intraretinal activity.

[0120] Example 3:

[0121] Two types of microelectrode arrays (MEAs) were fabricated, one for neuronal in vivo applications and one for neuronal in vivo applications. Each MEA according to the invention consists of a 2D part from which the electrodes are "grown" into the third dimension (see their fabrication below).

[0122] The 2D in vitro MEAs were 24 x 24 mm in size and contained a total of 64 electrodes with a pitch of 200 pm arranged in an 8 x 8 grid, or 32 electrodes arranged in a diamond shape with an electrode pitch of 30 pm. They consisted of a rigid quartz-based substrate with a thickness of 500 pm, a thin metal layer of Ti / Au / Ti with a thickness of 10 / 100 / 10 nm, and a 2 pm thick SU-8 encapsulation layer.

[0123] The 2D in vivo MEAs contained 16 electrodes with four different layouts, with electrode spacings of 104 pm, 54 pm, 15 pm, and 10 pm. The idea of ​​the 10 pm electrode spacing layout is to create so-called multisite pillars, i.e., electrodes located close to each other at different heights. In this case, five sets of three electrodes were designed, enabling the fabrication of 3D multisite electrodes at different heights (see the fabrication process below). The 2D in vivo MEAs consisted of a flexible substrate layer based on a 5 pm thick parylene-C (PaC) layer, a thin metal layer of Ti / Au / Ti with a thickness of 20 / 100 / 10 nm, and an encapsulation layer based on a 5 pm thick PaC layer.After fabricating a 2D microelectrode array (2D MEA chip), the template for generating the 3D electrodes was fabricated, followed by the electrodeposition of the metal electrodes to grow them in the third dimension.

[0124] The MEA manufacturing processes described below, except for the deposition of PaC and the electronic deposition by the cylinders, took place in the ISO-certified HNF clean room facility at the Jülich Research Centre and were carried out as follows:

[0125] A) Preparation of 2D-MEA a. 2D in vitro MEA li) Provision of a rigid substrate

[0126] First, the quartz substrates were cleaned with acetone and isopropanol (IPA) and then heated at 150°C for 5 minutes on a direct contact hot plate.

[0127] 2i) Metallization

[0128] To define the metal contact areas, leads (= conductor tracks), and electrodes (or electrode areas), the photoresist LOR 3B (a polydimethylglutarimide-based photoresist from MicroChem Corp, USA) was spin-coated onto the cleaned quartz substrate at 3000 rpm (revolutions per minute) for 45 seconds with a ramp of 500 rpm / s, followed by a hold at 150°C for 5 minutes on a direct-contact hot plate. This first photoresist contributes to clean metal detachment. Afterwards, a second photoresist, nLOF 2020 (negative photoresist based on PGMEA (l-methoxy-2-propanol acetate from MicroChemicals GmbH, Germany), is spin-coated at 3000 rpm for 45 seconds with a ramp of 500 rpm / s and heated at 110°C for 5 minutes. This photoresist was then treated with 20 mJ / cm 2The substrates were exposed to broadband UV using a mask exposure unit, followed by a post-exposure bake step at 110°C for 1 minute on a direct-contact hot plate and a development step in AZ 326 MIF (based on tetramethylammonium hydroxide, MicroChemicals GmbH, Germany) for 40 seconds. The substrates were then evaporated with a metal stack of 10 nm titanium, 100 nm gold, and 10 nm titanium (i.e., a 10 / 100 / 10 Ti / Au / Ti stack) in an electron beam-assisted evaporation system, followed by a stripping process. For nLOF 2020, stripping was performed in acetone for 3 hours, followed by a cleaning step in acetone, isopropanol (IPA), and deionized water. To remove the residues of the second photoresist LOR 3B, the substrates were placed in a bath of AZ 326 MIF for 5 minutes and then cleaned in deionized water.

[0129] 3i) Deposition of passivation layer and definition of passivation openings

[0130] To increase the adhesion of the passivation, the substrates were placed in a piranha solution (H2O2 / H2SO4 2:1) for 5 minutes. To ensure proper passivation between the individual leads, SU-8 2002 (a commercially known photoimageable resist based on bisphenol-A novolak dissolved in an organic solvent and containing up to 10 wt.% mixed triarylsulfonium / hexafluoroanimonate salt) was then spin-coated at 3000 rpm for 45 seconds with a ramp of 500 rpm / s and cured at 90°C for

[0131] Heated for 1 minute. To define the electrode openings and the contact openings in the passivation, SU-8 was exposed to UV light at a wavelength of 365 nm for 27 seconds using a mask exposure unit, followed by a post-exposure bake at 90°C for 1 minute and a development step in MR Dev 600 (solvent-based developer for epoxy-based photoresists) for 100 seconds and IPA for 20 seconds.

[0132] To complete the 2D MEA fabrication, the Ti layer on the Au layer at the electrode openings and the bond pads was removed by reactive ion etching (RIE) using a gas mixture of Ar / Ch (30 / 2 sccm (sccm = standard cubic centimeters per minute)) at 50 / 500 W RF / ICP (inductively coupled plasma / capacitively coupled radio frequency) at 10°C for 15 seconds. b. 2D in vivo MEA

[0133] The fabrication involved the interleaved deposition of two flexible thin-film layers, a metal layer, and an electrode coating. Depending on the electrode coating, seven to eight steps were performed as follows: lii) Provision of a flexible substrate layer

[0134] A first 5 pm thick PaC layer was deposited on a silicon wafer by chemical vapor deposition using a PDS 2010 Labcoater 2 (Specialty Coating Systems Inc., USA), 10 g of PaC dimer and a process vacuum pressure of 3.33 Pa (about 25 mTorr).

[0135] 2ii) Metallization

[0136] In a second step, metallization was performed using the stripping process. The metal layer for contact areas, leads, and electrodes was patterned after the negative photoresist AZ LNR-003 (containing, among other ingredients, hexakis(methoxymethyl)melamine and l-methoxy-2-propanol acetate; MicroChemicals GmbH, Germany) was spin-coated at 4000 rpm for 45 seconds with a ramp of 500 rpm / s, followed by holding at 120°C for

[0137] 2 minutes on a direct contact hot plate. The photoresist was then heated at 320 mJ / cm 2The wafer is exposed to UV light at a wavelength of 375 nm using a maskless exposure system (MLA 150, Heidelberg Instruments, Germany) with a Defoc setting of 2 and a CDB (critical dimension bias) of 800. This is followed by a post-exposure bake step at 100°C for 1.5 minutes on a direct contact hot plate, a development step in AZ 326 MIF (MicroChemicals GmbH, Germany) for 1.5 minutes, and a cleaning step in deionized water. Subsequently, the wafer is evaporated with 20 nm of titanium, 100 nm of gold, and 10 nm of titanium (corresponding to a metal stack of 20 / 100 / 10 nm Ti / Au / Ti) using an electron beam-assisted evaporation system (Balzer PLS 570, Pfeiffer, Germany). A tungsten crucible and deposition rates of 0.1 nm / s for titanium and 0.5 nm / s for gold were used. Subsequently, a stripping process was carried out in an acetone bath for 2.5 hours to wash off the sacrificial material and photoresist.After detachment, the wafer is rinsed in isopropanol for 2 minutes and dried with a nitrogen gun. In some cases, the UV exposure step was also performed with standard UV photolithography using a broadband mask exposure system (Süss MA8 / BA8, Germany) with a dose of 100 mJ / cm. 2 carried out.

[0138] 3ii) optional deposition of a passivation layer and exposure of the electrode surfaces

[0139] A flexible PaC passivation layer with a thickness of 5 pm was deposited as described in step 1.

[0140] The flexible polymer was then removed from the contours of the support substrate, the contact pads, and the electrode openings. First, an etch mask was patterned using a thick positive photoresist at least 1.5 times the thickness of the polymer to be etched. An etch mask was patterned on top of the last PaC layer by spin-coating the photoresist AZ 12XT (photoresist based on l-methoxy-2-propanol acetate) (MicroChemicals GmbH, Germany) at 1000 rpm for 180 seconds with a ramp of 200 rpm / s, holding at 110°C for 4 minutes using a hot plate, and applying a dose of 350 mJ / cm 2with UV light of 375 nm wavelength, a Defoc setting of 2, and a CDB of -800 using a maskless exposure unit. The wafer was then heated using a hot plate at 90°C for 1 minute, followed by a 2-minute development step using AZ 326 MIF. After patterning the etch mask, an RIE step was performed with a Ch / CF^2 gas mixture (36 / 4 sccm) at RF / ICP powers of 50 / 500 W to etch PaC. 4ii) alternative to step 3ii) optional reprocessing of the metal layer applied in 2ii).

[0141] In some cases, steps 3 and 4 were not performed, and instead a second RIE step was performed to etch the top 10 nm thick Ti layer using a C / Ar gas mixture (20 / 20 sccm) and an RF power of 150 W.

[0142] After RIE, the etch mask was removed with AZ 100 remover (ethanolamine-containing solvent) (MicroChemicals GmbH, Germany) in a two-bath system. The first bath used a low-power ultrasonic bath for 5 minutes, followed by a second bath with fresh AZ 100 remover without ultrasonication. The wafer was then rinsed in three baths with isopropanol.

[0143] B) 3D printing of top-open hollow cylinders orthogonally on the surface of the 2D microelectrode array (electrodeposition template for 3D electrodes). The electrodeposition templates were based on structures 3D-printed using two-photon polymerization. The 3D structures were designed using CAD software, exported as STL files, and converted into printing instructions using Describe (software from NanoScribe GmbH). The 3D structures for the templates were then printed (Photonic Professional GT2 from NanoScribe GmbH, Germany). To create the 3D electrodes, hollow cylinders were first printed on top of a 2D MEA chip, which was fabricated as previously described. The hollow cylinders were aligned with the MEA layout and printed around the electrode openings. Two lenses, a Zeiss 25XNA0.8 and a Zeiss 63XNA1.4, were used to print the cylinders, depending on the electrode layout.When using the 25X objective, a scan speed of 50,000 pm / s, a laser power of 100%, and a power scaling of 1.2 resulted in the best print result with sufficient resolution and stability. The layer pitch was set to 700 nm and the voxel pitch to 400 nm. For the 63X objective, a scan speed of 8,000 pm / s, a laser power of 100%, and a power scaling of 1.0 were used. The layer pitch was 300 nm and the voxel pitch was 200 nm.

[0144] In principle, the heights and diameters of the cylinders were (and are) limited only by the lowest resolution of the 3D manufacturing process. Nevertheless, the aspect ratio (height divided by the outer diameter) and the wall thickness of the cylinders were (and are) the most important parameters limiting the stability of the printed cylinders.

[0145] Therefore, various cylinder dimensions were tested to obtain stable cylinders with high aspect ratios. The highest aspect ratio that still resulted in a stable print result under the specified conditions (printer, photo resin, etc.) was achieved by printing cylinders with a height of 500 pm, an outer diameter of 12 pm, and a wall thickness of 2 pm, resulting in an aspect ratio of just under 42.

[0146] The 3D printing approach, in which cylinders are printed around the electrodes of a 2D component to create 3D electrodes, was (and is) not limited to a specific 2D component or layout design. The design and all structural dimensions in the x, y, and z axes could (and can) be adapted to the 2D component and its specific application. Cylinders with varying and randomly distributed heights could (and can) be easily printed in a single print. The limiting factor was (and is) the decreasing stability of the cylinders with increasing aspect ratio.

[0147] After printing, a development step was performed to wash away any unpolymerized photopolymer residue. The samples were placed in a bath of Mr-Dev 600 developer (a solvent-based developer for epoxy-based photoresists) for 10 minutes, followed by another 10-minute bath of fresh Mr-Dev 600 to ensure complete development of the high-aspect-ratio cylinders. Finally, the sample was rinsed in fresh IPA for another 5 minutes and then air-dried.

[0148] Ba). Assembly (only for in vivo MEA)

[0149] After the 3D templates were printed onto the flexible 2D component, the component was detached from the silicon wafer using water droplets and tweezers. The component was then mounted on a printed circuit board (PCB). First, the PCB was preheated to 180°C on a direct-contact hot plate, and the low-temperature solder alloy Sn42 / Bi58 (AMTECH, USA) was applied to the contact pads of the PCB, allowing the formation of liquid bumps on each contact pad. By lowering the temperature to 160°C, the flexible MEAs were aligned and placed onto the liquid solder paste bumps, which solidified after the new chip was quickly removed from the hot plate and cooled to room temperature. The freshly soldered area is sealed, for example, with a polydimethylsiloxane (PDMS) coating in a mixing ratio of 1:10 (for example Sylgard 184, Dow Corning, USA) and cured in an oven at 120°C for 30 minutes.Other epoxy-based polymers could (and can) also be used as sealing coatings.

[0150] C) Deposition of electrically conductive material into the hollow cylinders on the electrode surfaces

[0151] To obtain electrodes with different heights, a template-assisted gold (Au) electrodeposition step was used. Au was electrodeposited through the printed hollow cylinders. The cylinders served both as a template to guide the electrodeposition (growth of Au) and to passivate the 3D electrodes along the third dimension, making this method a very simple, flexible, and fast 3D MEA fabrication method.

[0152] A liquid gold bath containing an aqueous solution of 50 mM AuCU was used for electrodeposition. The electrodeposition of gold was carried out in two steps. In the first step, a constant potential (chronoamperometry) of -1.3 V was applied until the current reached -100 nA. In a second step, a homogeneous cap was created on top of the cylinder by gently and controlled overgrowth of the gold. For this purpose, a constant current of -100 nA was applied for 20 seconds. The deposition process was carried out using a multi-channel potentiostat from CH Instruments and a 3-electrode cell setup with an Ag / AgCl reference electrode and a Pt counter electrode.

[0153] D) (optional) Coating the top surfaces of the electrical materials deposited in the hollow cylinders with additional electrically conductive material. To improve the electrochemical properties of the Au-based electrodes, an optional PEDOT:PSS coating was sometimes electrodeposited. The electrodeposition of PEDOT:PSS was performed using cyclic voltammetry (CV), which led to the electropolymerization of PEDOT:PSS from an EDOT:PSS solution. A CH Instruments multi-channel potentiostat with a 3-electrode cell setup was used. Five to 15 CV cycles were performed along the potential boundaries of 0 V and 1 V at a scan rate of 0.1 V / s. Instead of the gold explicitly mentioned in this example, any conductive material that can be electrodeposited can be used to fabricate the 3D electrode, as already mentioned.Other preferred materials according to the invention are platinum, PEDOT:PSS, iridium oxide (IrO. x ) or chromium for applications where biocompatibility is not an issue.

[0154] Figures:

[0155] The embodiments of the present invention explained in more detail below with reference to the figures represent various preferred embodiments. Many of the features or embodiments shown in individual figures below can be combined with features and embodiments shown in other figures or the rest of the description, particularly where the features are described accordingly. Furthermore, the figures are not to be interpreted in a limiting manner and are not true to scale. Furthermore, the figures do not contain all features that conventional devices / systems have, but are reduced to the features essential to the present invention and its understanding. For example, screws, hoses, brackets, etc. are not shown or not shown in detail.

[0156] The same reference symbols / numbers mean the same or equivalent device parts.

[0157] Figure 1 schematically shows the method according to the invention with its three essential process steps. Conductor tracks and contact surfaces are not shown in Figure 1 for clarity. The top row shows a schematic view from the top right of a section of a 2D microelectrode array (2DE).

[0158] The first of the three images in the top row shows two electrode areas EF on the substrate S; thus, the image shows step A), the provision of a 2D microelectrode array 2DE. As mentioned, this is merely a schematic, simplified section, and a "real" 2D microelectrode array 2DE would normally include more electrodes (areas) EF. Below is the same step A) in a side view. Based on the illustration, it can be seen that a passivation layer P is arranged on the substrate S, which is interrupted at the locations where the electrode areas EF are located so that they are accessible in the next step.Although it is not visible here, there can be a free area (i.e., an "open" substrate surface) between the edge of the electrode surfaces EF and the edges of the passivation layer P, for example, a ring around a circular electrode surface EF, which can then serve as the target surface for 3D printing in the next step. However, printing can also be done directly onto the electrode surface EF if desired (or even onto the passivation layer P). In the bottom row, on the left, the same step A) is shown in a top view of the section of the 2D microelectrode array 2DE. Here, two electrode surfaces EF and the substrate S / or the passivation layer P can be seen.

[0159] The second of the three images in the top row shows step B), i.e., the 3D printing. Here, you can see that two hollow cylinders HZ of different heights have been printed; in this case, the printing was carried out such that the hollow cylinders HZ are arranged around the electrode surfaces EF and not on them. The image below shows, for this step B), that the hollow cylinders HZ abut the inner edges of the recesses in the passivation layer P with their outer edges. The lower view then shows that the 3D-printed hollow cylinders HZ, in this example, are arranged concentrically around the electrode surfaces EF; however, this is not required, and other arrangements (polygon and circular surface or circle around polygonal surface, etc.) are conceivable and possible within the scope of the present invention.

[0160] The right image in the top row illustrates step C) where the hollow cylinders HZ are filled with electrically conductive material eM up to the top edge of the hollow cylinders HZ. The images below show this from the other two perspectives.

[0161] Figure 2 schematically shows the inventive method for producing a three-dimensional MEA starting from a 2D MEA, with the three essential steps already shown in Fig. 1 and an additional step D). Furthermore, Fig. 2 shows the method in a slightly different representation. Basically, Figure 2 shows a lateral section through a section of a microelectrode array. In contrast to Fig. 1, this time four electrode surfaces EF are shown. Step A, i.e., the preparation of the 2D microelectrode array 2DE, is shown on the far left in Fig. 2. Here, the substrate S and the passivation layer P thereon can be seen. The passivation layer P has four recesses for the electrode surfaces EF.

[0162] The second image in Fig. 2 shows step B), i.e. the 3D printing of the hollow cylinders HZ, which in this sectional view only show the right and left outer edges. As can be seen from Fig. 1, the hollow cylinders HZ can be designed with different heights and widths - depending on the desired specifications. However, within the scope of the present invention, all hollow cylinders HZ can just as easily be the same height, or all different heights, or some can be the same and the rest different heights. The respective diameters of the hollow cylinders HZ can also be determined independently of the others within the scope of the present invention, so that all can have the same diameter, all different diameters, or some can be the same and others different diameters (i.e. have different widths). Different widths are shown here, but it is often preferable to have the same widths, i.e. diameters.Here, as in Fig. 1, it can be seen that the hollow cylinders HZ are flush with the passivation layer P; this is preferred in some variants according to the invention.

[0163] The third image of Fig. 2 then shows how the hollow cylinders HZ are filled with electrically conductive material eM, in particular gold, slightly beyond the upper edge of the hollow cylinders HZ. This illustration also clearly shows how the conductive material eM located in the hollow cylinders HZ directly merges into the conductive material of the electrode surfaces EF previously applied to the substrate.

[0164] Finally, the last image of Fig. 2 illustrates again how the exposed surfaces of the conductive material eM deposited in the hollow cylinders HZ (i.e. essentially their surfaces) are coated with another, here different, conductive material, in particular PEDOT:PSS, which then results in the optional (but generally preferred) electrically conductive coating eB, which can further improve the electrochemical properties of the electrodes; in particular, in some variants of the present invention, a combination of gold electrode (i.e. gold as electrically conductive material eM) and PEDOT:PSS (as electrically conductive coating eB, i.e. EDOT:PSS is polymerized to PEDOT:PSS and used as a further electrically conductive material) is preferred.Figure 3, in its images a) to e), shows a highly schematic representation of a possible manufacturing sequence for an in vitro 2D MEA in a cross-sectional view of a section of a 2D-MEA. Image a) illustrates a substrate S coated with a photoresist PR / PRe. By placing an exposure mask Ma over a portion of the photoresist (either directly on top or between the photoresist and the exposure source), this portion is only partially accessible during subsequent exposure (indicated by the wavy arrows), namely the portion PRe not covered by the mask Ma, which is exposed to radiation. The portion covered by the mask Ma is not exposed to radiation. If a photoresist is used that is subjected to radiation in such a way that it can be subsequently washed off, it is possible to remove the mask Ma after irradiation and then wash off the exposed portion of the photoresist PRe.The unexposed part would then remain, which can be exploited in the next step. In the next step b) of Fig. 3 it is shown that the exposed part of the photoresist PRe has been removed, but the unexposed part of the photoresist PR still remains. Metal is deposited on the substrate S and the remaining photoresist PR, in this illustrated example in two layers, for example, which is also preferred in some variants of the present invention, a layer of gold and / or platinum and on top of that a layer of titanium. As can be seen, the metal coating in the right-hand part of this image does not come into contact with the substrate S, but only with the layer of photoresist PR. This is then removed (lifted off / lift-off process), as illustrated by the arrow, exposing a part of the substrate S on which there is then neither a metal nor a photoresist layer. In image c) of Fig.Figure 3 then illustrates how a passivation layer P is deposited (shown here consisting of photoresist, applied, for example, by spin coating), both on the metal-coated area and the exposed area. An exposure mask Ma is then placed (either directly on top or between the photoresist and the exposure source), leaving a portion PRe of the photoresist exposed. A subsequent exposure (indicated by the wavy arrows) then allows the exposed portion to be washed out. Image d) of Figure 3 then shows a section of the resulting MEA in which the exposed photoresist PRe has been washed out and the mask Ma has been removed. Finally, image e) of Figure 3 indicates, by the star shapes Ä, that a portion of the metal layer—in the described / illustrated case, the titanium layer—is removed, for example, by etching, in particular reactive ion etching (RIE).The result is, for example, a 2D microelectrode array (2DE).

[0165] It is important to note, however, that Fig. 3 describes only one possible manufacturing route for an in vitro 2D MEA, not the only one. Other photoresists can also be used, for example, those that polymerize upon irradiation, and the unexposed portion can then be washed away. These variations are well known to those skilled in the art. In particular, it is also possible to use etching processes, for example, using reactive gases, instead of the exposure method described here and the corresponding exposure masks and photoresist, and then to use the corresponding etching masks and etching resists.

[0166] Figure 4 shows a highly schematic illustration of a possible manufacturing sequence for an in vivo 2D MEA in a cross-sectional view for a section of a 2DE.

[0167] The first image (corresponding to step lii)) shows how a substrate layer S, for example based on parylene-C or polyimide, in particular parylene-C, is arranged over a carrier layer T (for example applied via chemical vapor deposition); in some variants, a metal layer can be arranged between these two layers as a sacrificial layer O, which is indicated here, for example based on three metal layers of chromium / gold / chromium.

[0168] The second image in Fig. 4 (corresponding to step 2ii)) shows how a structured metal layer M (seen in three parts in this section, left and right later serve as conductor tracks and the middle as electrode surface) is applied (for example made of titanium and / or gold, in particular titanium / gold, by means of vapor deposition or similar processes), which forms conductor tracks, electrode surfaces and contact surfaces.

[0169] The third and fourth images of Fig. 4 (corresponding to the optional step 3ii)) first show how a passivation layer P is applied, for example, via chemical vapor deposition. The second part then shows how the passivation layer is partially opened, for example, via masking and etching, for example, via reactive ion etching (if the passivation layer consists of parylene-C and / or polyimide, for example, via O2 / CF4), whereby the structured metal layer becomes partially accessible again (i.e., in particular at / above the electrode surfaces EF (only one visible in the middle in this section); the conductor tracks L (one each visible on the left and right in this section) remain covered by the passivation layer P). Figure 5 shows two abstracted photographs of in vitro 2D microelectrode arrays, fabricated as described in Example 3A)b.

[0170] Figure 5A) shows a 2D microelectrode array (2DE) in a layout with 64 electrodes spaced 200 pm apart, arranged in an 8 x 8 square matrix. The respective conductive traces L (leads to the electrodes) and the electrode areas EF arranged at the ends of the respective conductive traces are clearly visible. Also visible here are two enlarged electrodes serving as reference electrodes R, in particular as internal reference electrodes for electrical measurements (these have a diameter of approximately 150 pm here, but this diameter can also vary in other embodiments).

[0171] Figure 5B) shows a 2D microelectrode array (2DE) in a layout with 32 electrodes with a minimum electrode spacing of approximately 30 pm (measured from electrode center to electrode center; the minimum spacing is given in each case, as the spacing always varies slightly depending on the layout), arranged in a diamond-shaped matrix. Here, too, the respective conductor tracks L (leads to the electrodes) and the electrode surfaces EF, which are arranged at the ends of the respective conductor tracks, can be seen.

[0172] Figure 6 shows four abstracted photographs of in vivo 2D microelectrode arrays, manufactured as described in Example 3A)b. In this figure, the respective conductor tracks L (leads to the electrodes) and the electrode areas EF, which are arranged at the ends of the respective conductor tracks, are also clearly visible. Also visible here is an enlarged electrode, which serves as a reference electrode R, in particular as an internal reference electrode for electrical measurements. The four individual images in Fig. 6 show different 2D microelectrode array layouts: Image A shows a layout with an electrode spacing of approximately 16 pm, Image B with an electrode spacing of approximately 112 pm, Image C with an electrode spacing of approximately 62 pm, and Image D with an electrode spacing of approximately 21 pm (each measured from electrode center to electrode center; the smallest spacing is given in each case, since the spacings always vary slightly depending on the layout).

[0173] It should be explicitly pointed out that the above descriptions of the figures represent particularly preferred embodiments of the present invention and are to be regarded as descriptions of such even without the figures, in particular this applies to the photographs 5A, 5B and 6.

[0174] List of reference symbols:

[0175] 2DE 2D microelectrode array

[0176] 3DE three-dimensional structured microelectrode array

[0177] T carrier substrate

[0178] S substrate layer

[0179] M structured base metal layer (conductor tracks and electrode surfaces and

[0180] contact surfaces)

[0181] O sacrificial layer

[0182] P passivation layer

[0183] R Reference electrode eM Electrically conductive material (of the hollow cylinder filling) eB Electrically conductive coating (further electrically conductive material)

[0184] EF electrode area

[0185] L Conductor track

[0186] HZ hollow cylinder

[0187] Ma (etching or exposure) mask

[0188] Ä Etching solution

[0189] PR photoresist

[0190] PRe photoresist exposed

Claims

Claims 1. A method for producing three-dimensionally structured microelectrode arrays comprising or consisting of the following process steps: A) providing a 2D microelectrode array comprising a plurality of conductive paths and a plurality of electrode surfaces; B) 3D printing of top-open hollow cylinders orthogonally on the surface of the 2D microelectrode array, with the cylinder bases printed externally around the electrode surfaces; Ba) optionally removing any carrier substrate that may be present; C) Depositing electrically conductive material into the hollow cylinders onto the electrode surfaces; D) optionally coating the accessible surfaces of the electrically conductive material deposited in the hollow cylinders with further electrically conductive material.

2. The method according to claim 1, characterized in that the production of the 2D microelectrode array comprises or consists of the following method steps: li) providing a substrate; 2i) applying a plurality of conductive tracks and a plurality of electrode surfaces and optionally a plurality of contact surfaces; 3i) depositing a passivation layer and then exposing the electrode surfaces and optionally a plurality of contact surfaces; or lii) providing a substrate on a carrier substrate, 2ii) applying a plurality of conductive tracks and a plurality of electrode surfaces and optionally a plurality of contact surfaces; 3ii) optionally depositing a passivation layer and then exposing the electrode surfaces and optionally a plurality of contact surfaces; 4ii) alternatively to step 3ii) optionally reprocessing the metal layer applied in 2ii).

3. Method according to one of the preceding claims, characterized in that before the 3D printing of the actual hollow cylinder structure, a plate-like structure is printed as a base structure on the surface of the 2D microelectrode array.

4. Method according to one of the preceding claims, characterized in that the material used for 3D printing is electrically non-conductive and is preferably selected from the group consisting of polymers, ceramics, glass and mixtures thereof.

5. Method according to one of the preceding claims, characterized in that the electrically conductive material deposited in the hollow cylinders is selected from the group consisting of gold, platinum, PEDOT:PSS, iridium oxide, chromium and mixtures or alloys thereof, preferably gold, platinum, PEDOT:PSS, iridium oxide, chromium and mixtures or alloys thereof, particularly preferably gold.

6. Method according to one of the preceding claims, characterized in that the further, identical or different, electrically conductive material used for coating the upper sides of the electrically conductive materials deposited in the hollow cylinders is selected from the group consisting of gold, platinum, PEDOT:PSS, iridium oxide, chromium and mixtures or alloys thereof, preferably gold, platinum, PEDOT:PSS, iridium oxide, chromium and mixtures or alloys thereof, particularly preferably PEDOT:PSS.

7. Method according to one of the preceding claims, characterized in that the 2D microelectrode array has a rigid or flexible substrate layer, wherein the material for the rigid substrate layer is selected from the group consisting of silicon or quartz, in particular quartz, or the material for the flexible substrate layer is selected from the group consisting of parylene-C or polyimide, in particular parylene-C.

8. Method according to one of the preceding claims, characterized in that the plurality of conductor tracks and the plurality of electrode surfaces are made of a material selected from the group consisting of titanium, gold, platinum, conductive polymers such as PEDOT: PSS, indium tin oxide and mixtures, alloys or layers thereof, particularly preferably a metal layer sequence titanium / gold / titanium or a metal layer sequence titanium / gold.

9. Method according to one of the preceding claims, characterized in that the 3D printing method is a 2-photon polymerization method.

10. Three-dimensionally structured electrode arrays comprising a) a 2D microelectrode array comprising a plurality of conductor tracks and a plurality of electrode surfaces, wherein the 2D microelectrode array preferably has conductor tracks and electrode surfaces on only one surface side; b) three-dimensional hollow cylinders arranged orthogonally on the electrode surfaces and filled with electrically conductive material, wherein the hollow cylinders consist of a 3D-printed material.

11. Three-dimensionally structured electrode arrays produced by a method according to one of claims 1 to 9.

12. Implants or neuroimplants comprising three-dimensionally structured electrode arrays according to claim 10 or according to claim 11 or consisting of these.

13. Use of three-dimensionally structured electrode arrays according to claim 10 or according to claim 11 or manufactured according to one of claims 1 to 9 as or for implants or neuroimplants or for electrochemical and / or electrophysiological measurement in the millivolt range and / or of cell cultures.

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