Title: Three-dimensional electrodes in tissue engineering

Field of the invention

The invention relates to the field of structured electrodes useful for cell and tissue culturing. In particular, the invention relates to the use of structured electrodes to record electrical signals using the electrode's three-dimensional space.

Background of the invention

Studies of biology, drug discovery, diseases, and physiology are often performed in cell culture by studying cells or cell systems. Cell culture in vitro is one of the milestones for understanding biology in health and disease. In vitro cell culture provides an accessible and controlled environment to study cells and perform experiments. Various cell culture techniques and cell culture templates have been developed in the past decades. However, cell culture templates containing three-dimensional structures shaped like fractals to record the electrical activity of cells under culture conditions are unique to understand cell behaviour, human conditions, and treatment.

One of the most popular systems used to investigate cell growth and human conditions are microelectrodes, which measure electrical signals over cell points to understand collective cell interaction between cells in a non-destructive manner. To date, state-of-the-art focus on planarized or pillar-like microelectrodes arrays lacking access to the third dimension to record electrical signals over cell points.

US 2002/182241 describes the preparation of three-dimensional geometries that mimic blood vessels and serve as a template for cell adhesion and growth. In example 1 of US 2002/182241, the preparation of scaffolds from silicon or Pyrex wafers is described, whereby channels are formed by anisotropic etching of the silicon wafers after a layer of silicon dioxide is deposited on the silicon wafer. After etching, the silicon dioxide is removed, and cells are seeded and grown directly on the etched silicon or Pyrex scaffold material. However, US 2002/182241 does not integrate electrical recording elements in three-dimension.

Dituri et al. (2021) describe the use of topographical structures of amorphous silicon dioxide (SiCh) for cell culture. The structures are prepared using a template of silicon material, which is removed in the final steps of the fabrication method. 2

Silicon dioxide is deposited on the template, which is subsequently bonded to a borosilicate glass support via the surface containing the silicon dioxide. The silicon template is then removed to provide a the structures with silicon dioxide at the surface. The cells are subsequently grown on the silicon dioxide at the side that was originally in contact with the silicon template. The structures vary from pyramids to octahedrons to structures named fractals, with increased hierarchy and organized in periodic arrays (square or hexagonal). The pyramids were found to promote complex 2D/3D tissue formation from primary HCC cells.

Diaz Lantrada et al. (2013), reviews fractals in tissue engineering. Described are both fractals prepared from biocompatible materials and fractals prepared from materials that are inadequate for cell culture and tissue repair but that can be coated with a biocompatible layer.

The use of electrodes in cell and tissue culture is used for both electrical stimulation and recording the electrical potential of cells. Metallic or carbon electrodes have been used in vitro (Mobini et al. 2016; Hronik-Tupaj et al. 2011). However, these studies used single Petri dishes and 6-well cell/tissue-culture plates as chambers, limiting the number of cells and samples that can be tested, and the methods are unsuitable for use with cells or tissues grown in 3D, e.g. in a multicellular complex.

Park et al. (2018) describe planar, i.e. 2D, fractal microelectrodes for implantation and subsequent in vivo neurostimulation, instead of cell culturing purposes. For 2D fractal structures as presented in Park et al., although the edges have a ID fractal nature, the 2D structure is still planar. This can be derived from the fact that for a 2D fractal structure, the total surface area converges to a fixed value, whereas the length of the edge becomes infinite. Coating of such 2D fractal structures with a conductive material to function as an electrode is very different from and cannot be directly applied to 3D fractal structures.

There remains a need in the art for systems and methods that allow the use of electrodes in conventional and more complex cellular environments.

Summary of the invention 3

The disclosure provides the following preferred embodiments. In a first aspect, the invention provides a method for producing a device comprising a plurality of three-dimensional structures, the method comprising:

1) providing a monocrystalline substrate, preferably a monocrystalline silicon substrate, comprising a plurality of geometrical cavities,

2) growing or depositing a base three-dimensional structure material, preferably silicon nitride or silicon oxide, on the surface of the monocrystalline substrate including the geometrical cavities,

3) creating apertures in the base three-dimensional structure material, preferably an aperture at the apices of a plurality of geometrical cavities,

4) subtracting a plurality of geometrical features in the monocrystalline substrate through the one or more apertures to create a further plurality of geometrical cavities,

5) growing or depositing one or more materials with different physical and/or chemical characteristics as compared to the base three-dimensional structure material on the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities to form a plurality of structures;

6) optionally removing said material grown or deposited in step 5) from at least the outer surface of the monocrystalline substrate and/or depositing a material that allows for bonding to a support base, preferably silicon, more preferably amorphous silicon, polysilicon or crystalline silicon, to at least the outer surface of the monocrystalline substrate;

7) optionally bonding the plurality of structures to a support base; and

8) removing monocrystalline substrate around at least part of the plurality of geometrical cavities; to thereby provide the device comprising a plurality of three-dimensional structures.

In some embodiments, wherein the one or more materials grown or deposited in step 5) is a conductive material, and the method further comprises:

9) depositing a conductive layer consisting of a material, that is capable of forming an oxide intermediate adhering to the conductive material and to the 4 material deposited in step 5), on the surface of the base three-dimensional structure material to at least partly cover the conductive material;

10) providing a protective layer to at least part of the three-dimensional structures and part of the surface of the monocrystalline substrate comprising said at least part of the three-dimensional structures;

11) removing the part of the conductive layer deposited in step 9) that is not provided with a protective layer in step 10);

12) removing the protective layer provided in step 10);

13) optionally depositing substrate layer, preferably silicon, more preferably amorphous silicon, polysilicon or crystalline silicon,; wherein steps 9) - 13) are performed between steps 6) and 7) of the method of claim 1 and wherein step 7) optionally comprises bonding of the surface of the device that comprises the monocrystalline substrate layer deposited in step 13) to the support base.

In some embodiments, a method of the invention further comprises a step 4a) wherein a base three-dimensional structure material, preferably silicon nitride or silicon oxide, is deposited on at least part of the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities between steps 4) and 5). Preferably, this step comprises oxidation at temperatures below 950 °C. In some embodiments, after bonding to the support base in step 7) and removing monocrystalline substrate around at least part of the plurality of geometrical cavities in step 8) the base three-dimensional material, preferably silicon nitride or silicon oxide, is removed selectively from the apices of the at least part of the three-dimensional structures (i.e. only from the apices), for instance by timed HF wet etching.

In some embodiments, a method of the invention further comprises a step 8a) wherein an active or functional layer is grown or deposited at the surface of at least part of the three-dimensional structures, a step 8b) wherein a conductive material as defined herein is deposited or grown, and a step 8c), wherein active spots in at least the apices of at least part of the three-dimensional structures are created. In some embodiments, the conductive material grown or deposited in step 5), that preferably functions as an electrode, is contacted with the conductive material 5 deposited or grown to the active or functional layer, preferably either on the side of the device that contains the three-dimensional structure (front side, see figure 11) or are side of the device that contains the support base (back side, see figure 10).

In preferred embodiments, the device comprises one or more electrodes, in particular three-dimensional electrodes.

In preferred embodiments, at least part of the three-dimensional structures function as electrodes, in particular three-dimensional electrodes.

In preferred embodiments, at least part of the three-dimensional structures comprise or are electrodes, in particular three-dimensional electrodes.

In a further aspect, the invention provides a device comprising a plurality of three-dimensional structures obtainable with a method according to the invention.

In a further aspect, the invention provides a device comprising a plurality of three-dimensional structures, the device comprising:

- a support base, preferably borosilicate glass;

- a plurality of three-dimensional structures, whereby at least part of the structures comprise a conductive material, preferably at the outer surface;

- wherein the plurality of three-dimensional structures are attached to the support base via a base three-dimensional structure material, preferably silicon oxide or silicon nitride, more preferably silicon dioxide.

In preferred embodiments, the device of the invention comprises one or more electrodes, in particular three-dimensional electrodes. In preferred embodiments, at least part of the three-dimensional structures function as electrodes, in particular three-dimensional electrodes. In preferred embodiments, at least part of the three-dimensional structures comprise or are electrodes, in particular three- dimensional electrodes.

In a further aspect, the invention provides a use of the device according to the invention for cell culturing and/or cell electrical recording.

In a further aspect, the invention provides a use of a device according to the invention for maintaining electrical and/or electrochemical readout in a fouling environment, such as cell culture medium without loss of electrical and/or electrochemical signal.

In a further aspect, the invention provides a method for culturing cells, comprising providing a device according to the invention with cells and culturing 6 the cells. In preferred embodiments, the method comprises producing a structure comprising a plurality of three-dimensional nanostructures with a method according to the invention, providing the surface of the plurality of three- dimensional nanostructures with cells and culturing the cells

Detailed description

As used herein, "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component^) than the ones specifically identified, said additional component^) not altering the unique characteristic of the invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.

The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

As used herein the term “plurality” indicates more than one. In embodiments, a “plurality” refers to at least 5, at least 10, at least 20. A device according to the invention may comprises several hundreds or more three-dimensional structures.

As used herein “conductive” and “conductively attached” refer to electrical conduction. As an example, a “conductive material” as used herein is an electrically conductive material. As another example, two items, e.g. materials or structures, that are “conductively attached” means that electrical current can go from one item to the other item.

The present inventors have developed a device comprising a plurality of structures for use in cell culturing and that is provided with a coating that can serve as an electrode. These three-dimensional electrodes can be used to culture 7 cells in 2D or 3D for disease modelling and drug development. This is the case of epithelial and endothelial cells that grow in 2D and form a barrier in the body. The strength and integrity of these barriers can be assessed by measuring electrical resistance across the cell layer in-vitro using three-dimensional electrodes, such as fractals, instead of the commonly used planarized counterpart. In the case of the 3D cell environment, the three-dimensional electrodes can allow 3D cell culture (spheroids) and organoid to understand cell interaction by measuring electrical signals from the three-dimensional cell environment. The procedure by which the device is fabricated and optionally coated allows for tunability and a great diversity in configurations of the electrode(s). I.e. different structures can be internally connected to allow electrical conductivity between the structures thereby creating different subsets of structures, whereby the structures within each subset are connected but not electrically connected with the structure of other subsets. In addition, it is possible to attach the electrodes of a single structure or a subset of structures with external electrodes.

In a first aspect, the invention provides a method for producing a device comprising a plurality of three-dimensional structures, the method comprising:

1) providing a monocrystalline substrate, preferably a monocrystalline silicon substrate, comprising a plurality of geometrical cavities,

2) growing or depositing a base three-dimensional structure material, preferably silicon nitride or silicon oxide, on the surface of the monocrystalline substrate including the geometrical cavities,

3) creating apertures in the base three-dimensional structure material, preferably an aperture at the apices of a plurality of geometrical cavities,

4) subtracting a plurality of geometrical features in the monocrystalline substrate through the one or more apertures to create a further plurality of geometrical cavities,

5) growing or depositing one or more materials with different physical and/or chemical characteristics as compared to the base three-dimensional structure material on the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities to form a plurality of structures; 8

6) optionally removing said material grown or deposited in step 5) from at least the outer surface of the monocrystalline substrate and/or depositing a material that allows for bonding to a support base, preferably silicon, more preferably amorphous silicon, polysilicon or crystalline silicon, to at least the outer surface of the monocrystalline substrate;

7) optionally bonding the plurality of structures to a support base; and

8) removing monocrystalline substrate around at least part of the plurality of geometrical cavities; to thereby provide the device comprising a plurality of three-dimensional structures.

Figure 5 schematically shows an example of a method of the invention.

The term “device” as used herein refers to a three-dimensional device that can be prepared with a method of the invention. The device comprises a plurality of three-dimensional structures which can be used to culture cells on. I.e. the structures comprises a surface to which cells can attach.

Steps of a method of the invention as indicated herein are numbered. The steps of the method are performed in the numbered order indicated, unless stated otherwise.

The device of the invention is prepared using a monocrystalline substrate. A single -crystal or monocrystalline solid is a material in which the crystal lattice of the entire sample is continuous and unbroken to the edges of the sample, with no grain boundaries. Monocrystalline substrates are composed of a single crystal throughout, while polycrystalline is composed of an aggregate of very small crystals in random orientations. Examples of monocrystalline are monocrystalline silicon, sapphire, Quartz, Ge (germanium), or GaN (gallium nitride). In preferred embodiments, the monocrystalline substrate is monocrystalline silicon. Monocrystalline silicon, is also called single-crystal silicon, in short, mono c-Si or mono-Si. It consists of silicon in which the crystal lattice of the entire solid is continuous, unbroken to its edges, and free of any grain boundaries. Silicon is tetrahedrally coordinated by oxygen in the low-pressure SiO2 polymorphs; quartz, tridymite, cristobalite, and in its high-pressure polymorph coesite. Silicon is coordinated by six oxygens in the high- pressure SiO2 polymorph stishovite. 9

The monocrystalline substrate that is provided comprises a plurality of geometrical cavities. Such monocrystalline substrate comprising a plurality of geometrical cavities can be prepared using methods known in the art, e.g. as described in Berenschot et al. (2013) and Dituri et al. 2021. These references describe processes whereby geometrical features are subtracted from a monocrystalline substrate by etching or by drilling. Preferably subtraction of material from the monocrystalline substrate is performed by using etching. For example the geometrical cavity is etched in the substrate by means of anisotropic etching. Anisotropic etching is a subtractive microfabrication technique that aims to remove material in specific directions to obtain a geometrical shape. Preferably, the wet etching technique can be used as anisotropic etching. Wet techniques exploit the crystalline properties of a structure to etch in directions governed by crystallographic orientation. For instance, potassium hydroxide (KOH) is used for anisotropic etching of the monocrystalline substrate.

The geometrical feature and resulting geometrical cavity can have various shapes, such as a pyramid, an octahedron, a tetrahedron, a cube, a cuboid, or a cone. Preferably the geometrical feature and resulting geometrical cavity has one or more apices. In preferred embodiments, the geometrical features and cavities have the shape of an octahedron or part thereof. Parts of an octahedron include pyramidal features and pyramidal cavities. Figure 1 schematically shows an octahedron structure being subtracted partially or entirely in a monocrystalline substrate. In some embodiments, the monocrystalline substrate comprises a plurality of geometrical cavities of a single generation. I.e. the plurality of geometrical cavities are present in the monocrystalline substrate in a single layer. For instance, all geometrical cavities are cavities such as demonstrated in figure 1.

In step 2) of a method of the invention, a base three-dimensional structure material is grown or deposited on the surface of the monocrystalline substrate including the geometrical cavities. In some embodiments, this step is performed by treating the monocrystalline substrate to form the base three-dimensional structure material. In preferred embodiments, the base three-dimensional structure material is silicon oxide or silicon nitride, more preferably silicon dioxide. 10

The material grown or deposited in step 2) of a method of the invention is compatible with cell culture and/or cell growth for cells culture applications. In preferred embodiments the provided monocrystalline substrate is a silicon containing material. In preferred embodiments the provided monocrystalline substrate is silicon, more preferably silicon oxide or nitride, e.g. Si, SiCh, SiaN4, SiRN. In preferred embodiments, the growth or deposition of a base three- dimensional structure material in step 2) is performed by thermal oxidation resulting in a layer of silicon oxide, preferably amorphous silicon dioxide.

In some embodiments, the base three-dimensional structure material is thermally grown oxide, a layer of silicon nitride or a combination thereof. A layer of silicon nitride is for instance applied by low-pressure chemical vapor deposited (LPCVD), followed by corner lithography, and local oxidation of silicon. Thermally grown oxide is for instance achieved by thermal oxidation. The monocrystalline substrate including the geometrical cavities are for instance exposed to thermal oxidation at a temperature between 950-1500 °C, e.g. at 1100 °C. At this temperature, the surfaces of the subtracted structure will oxidize. If the monocrystalline substrate is monocrystalline silicon, an amorphous silicon dioxide layer is conformally grown. The thickness of the layer depends on the temperature and the duration of the thermal oxidation step. In preferred embodiments, the oxide layer is at least 25 nm thick, a preferable thickness is about 160 nm. In some embodiments, the oxide layer is between 25 and 160 nm thick, an amorphous silicon dioxide layer is conformally grown, except at the concave corners. Hence, in this step, preferably a conformal layer around convex corners is obtained. In intersections of multiple planes, e.g., three or four planes, oxide sharpening occurs. This aspect yields the possibility to solely remove silicon oxide from apices by means of timed isotropic etching, while the oxide layer remains in ribbons and on planes (see figure 4)). In some embodiments, a process like, plasma oxidation of silicon, anodic oxidation of silicon, or nitridation (by means of thermal conversion of silicon into nitride) can be performed to grow or deposit the base three-dimensional structure material.

In step 3) of a method of the invention, apertures are created in the base three-dimensional structure material, preferably an aperture at the apices of a 11 plurality of geometrical cavities, i.e. preferably at every apex in the geometrical cavities. This aperture allows subtraction of an additional layer of cavities to create multilevel three-dimensional structures. Various techniques known in the art can be used to make an aperture, for example, corner lithography or timed isotropic etching. In some embodiments, the apertures are created by means of timed isotropic etching. In this technique, the aperture is created by solely removing the base three-dimensional structure material from the apices. This can be done by timed wet etching using hydrogen fluoride, e.g., 1% hydrogen fluoride. Alternatively, for the fabrication of apertures, other methods might apply, for example, low-temperature oxidation and selective etching.

In step 4) of a method of the invention, a plurality of geometrical features is subtracted in the monocrystalline substrate through the one or more apertures to create a further plurality of geometrical cavities. The geometrical features can be the same as indicated herein above, for instance a pyramid, an octahedron, a tetrahedron, a cube, a cuboid, or a cone. In preferred embodiments, the geometrical feature is an octahedron or part thereof. The subtracting is performed through the one or more apertures formed at the one or more apices. In preferred embodiments the subtracting in step 4) is performed by means of anisotropic etching. Figure 1 (Gl) schematically shows this round of subtracting, creating octahedral cavities at each apex of the previous cavity. For example, the next round of geometrical cavities can be created by selectively etching at each apex the underlying silicon with anisotropic etching in TMAH (tetramethylammonium hydroxide). This etching step will form cavities at all apices simultaneously.

Steps 2)-4) can be repeated to create multilevel, or multi-generation, geometrical cavities with a higher level of complexity. Each repeat of these steps results in an extra layer of octahedral structures (also called extra generations of structures herein), as exemplified between sequence Figure 2C-2G. Each following layer will comprise smaller geometrical cavities. Preferably, each following layer will comprise smaller octahedrons at each apex of the previous layer. Preferably, repetition of the sequence of anisotropic etching of the monocrystalline substrate, thermal oxidation, and isotropic etching of the base three-dimensional structure 12 material is performed to create multilevel, or multi- generation, geometrical cavities with a higher level of complexity. Each following layer of the device will comprise smaller geometrical cavities, preferably created at each of the apices of the geometrical cavities.

As an example, Figure 2a) and 2b) show the top view scanning electron micrographs (SEM) of two different layouts of the initiator, configured in a square or hexagonal lattice. Figure 2c) shows a tilted view of a single initiator feature, as sketched in the most right image of Figure 1. Exemplary structures of a geometrical shape of octahedrons are shown. Figure 2C shows a simple three- dimensional structure that can be created with 1 round of subtraction. Figure 2D shows a three-dimensional structure that can be created with 2 rounds of subtraction. Figure 2E shows a three-dimensional structure that can be created with 3 rounds of subtraction. Figure 2F shows a three-dimensional structure that can be created with 4 rounds of subtraction. And figure 2G shows a three- dimensional structure that can be created with 5 rounds of subtraction.

In step 5) of a method of the invention, one or more materials with different physical and chemical characteristics as compared to the base three-dimensional structure material are grown or deposited on the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities to form a plurality of structures. This allows for yielding material interfaces, including conductivity, refractive index, fermi level and combinations thereof. Further examples of such materials are materials with different fermi levels or energy band gaps for electrical, (electro)optical and electrochemical applications and/or creation of junctions. In preferred embodiments, the one or more materials are grown or deposited over essentially the entire surface of the of the monocrystalline substrate including the base three- dimensional structure material and the geometrical cavities.

In preferred embodiments, a conductive material is deposited or grown on the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities. The conductive material can be any conductive material known in the art. Non-limiting examples include metals, silicides, such as platinum silicide (PtSi), oxides of 13 transition metals and rare-earth elements, semiconductor oxides and conductive polymers. In preferred embodiment, the conductive material is a metal or a silicide. In preferred embodiment, the metal is platinum, gold, silver or a combination thereof, or PtSi.

The material grown or deposited in step 5) of a method of the invention can be grown or deposited using any method known in the art. Examples of suitable methods are atomic layer deposition (ALD), physical vapour deposition (PVD), sputtering and evaporation. In preferred embodiments, the material is deposited by ALD.

The material grown or deposited in step 5) of a method of the invention is compatible with cell culture and/or cell growth for cells culture applications, at least part of the cells are provided to structures at the surface of this layer. In some embodiments, after producing the device, the outside of the layer of this material forms the functional layer of the device and will be the outer surface. The cells will use this outer surface to attach and/or grow on. I.e. the outer layer of this material, which is in contact with the monocrystalline substrate before removal thereof, will in such embodiments become the surface of the three-dimensional structures formed on which cell can be cultured. Reference is made to exemplary Figure 3, which shows an example of fractal structures with Pt suitable for cell culture.

As indicated herein below, it is possible to deposit one or more further layers between steps 4) and 5), and e.g. selectively remove this from the apices. In such embodiments, the cells will grow on the one or more further layers and the material grown or deposited in step 5) at the apices.

It is generally known in the art that cells are able to grow on the materials listed above, in particular on conducive materials, including metals, such as platinum, gold and/or silver. After removal of the monocrystalline substrate, this layer will form the three-dimensional structures. Therefore, this layer should have a thickness sufficient to create a self-contained device. While not wishing to be bound by theory, the material, the thickness of the material and the form of the device together contribute to the strength of the device. The device should be sturdy enough to carry cells that potentially grow on the structures. In preferred embodiments, the formed layer is at least 10 nm thick, such as 20 nm, 25 nm or 50 nm thick. If the material grown or deposited in step 5) is a conductive material, in 14 particular a metal such as platinum, silver, gold or combinations thereof, the formed layer is preferably at least 15 nm thick, such as about 20 nm thick, to allow for free standing, self-contained structures.

In preferred embodiments, the material grown or deposited in step 5) is able to function as an electrode or functions as an electrode.

In preferred embodiments, the device comprises one or more electrodes, in particular three-dimensional electrodes.

In preferred embodiments, at least part of the three-dimensional structures comprise or are electrodes, in particular three-dimensional electrodes.

An example of step 5) of a method of the invention is schematically shown in figures 5A and 6A.

In step 6) of a method of the invention, the material grown or deposited in step 5), preferably the conductive material, is optionally removed from at least the outer surface of the monocrystalline substrate and/or optionally a material that allows for bonding to a support base is deposited to at least the outer surface of the monocrystalline substrate. This allows for the bonding of this outer surface of the substrate to a support base, if the material grown or deposited in step 5) is not compatible with binding to a support base. This is for instance the case if the material grown or deposited in step 5) is a conductive material, such as metals, such as platinum, gold and/or silver.

In preferred embodiments, the material grown or deposited in step 5), preferably the conductive material, is optionally removed from at least the outer surface of the monocrystalline substrate. The result of this step is that the material, preferably conductive material is only present in the geometrical cavities, but not at the outer surface of the monocrystalline substrate that will be bonded to the support base. Removal of this material can be achieved by any method known in the art. In preferred embodiments, the material is removed by ion beam etching, in particular angled ion beam etching. The use of angled ion beam etching allows for removal of the material from the outer surface of the monocrystalline substrate, while maintaining the material inside the three-dimensional geometric cavities. Because the ion beam is applied under an angle, it is not able to reach the material 15 deposited inside the geometric cavities. In preferred embodiments the angle is between 5 and 54 degrees, such as about 20 degrees. It is further preferred that the angled ion beam etching is performed under concentric rotation of the structures, e.g. at a rotational speed of 5 rpm.

An example of such step 6) of a method of the invention is schematically shown in figures 5B and 6B.

Alternatively, in step 6) material that allows for bonding to a support base is optionally deposited to at least the outer surface of the monocrystalline substrate. As indicated herein above, said material serves to allow bonding to a support base, if the material grown or deposited in step 5) is not compatible with binding thereto. In preferred embodiments, said material is a silicon containing material. In preferred embodiments, said non-conductive material is silicon. In preferred embodiments said material is amorphous silicon, polysilicon or crystalline silicon. Deposition of this material can be achieved using any method known in the art. In preferred embodiments, the material is deposited by directional deposition, such as sputtering.

In step 7) of a method of the invention, the plurality of structures are optionally bonded to a support base. In particular, the outer surface that comprises the base three-dimensional structure material deposited in step 2) is bonded to a support base. Suitable examples of a support base are supports comprising or consisting of ceramics (such as silicon nitride, alumina, zirconia), glass (such as borosilicate glass, and soda-lime glass), or polymeric surfaces (such as polystyrene, permanox, polydimethylsiloxane). In preferred embodiments, the outer surface that comprises the base three-dimensional structure material is bonded to borosilicate glass. In preferred embodiments, the support base comprises or consists of borosilicate glass and the outer surface of the device that comprises the base three- dimensional structure material is bonded to the borosilicate glass.

Bonding in this step can be achieved by various techniques known in the art. In some embodiments, bonding to the support base is by anodic bonding. In preferred embodiments, bonding to the support base is by direct bonding, anodic bonding or thermal compression. In further preferred embodiment, bonding to the 16 support base is by anodic bonding. For example, anodic bonding with a Mempax glass wafer at 400 °C.

An example of such step 7) of a method of the invention is schematically shown in figure 5C.

Figure 7 shows two example device of the invention wherein the three- dimensional structures are not bonded to a support base, i.e. wherein step 7 is not performed.

In step 8) of a method of the invention, monocrystalline substrate is removed around at least part of the plurality of geometrical cavities. The monocrystalline substrate can be removed by any known technique, for instance by a wet-etching step. For example, removal of the monocrystalline substrate, preferably silicon, is done with prolonged exposure to tetramethylammonium hydroxide. The outside of the three-dimensional structure which consists of the material grown or deposited in step 5) of a method of the invention is now accessible, for example, for cells to attach.

In preferred embodiments, the monocrystalline substrate is partially removed, in particular partly etched away, in step 8). This result in monocrystalline substrate remaining and at least partially covering part of the plurality of three-dimensional structures. Partial removal of the monocrystalline substrate allows the creation of multiple compartments with one or more three- dimensional structures exposed. These compartments can be in the form of wells, by leaving rings of bulk-monocrystalline substrate unetched. The silicon rings will separate the wells and allow the wells to contain fluid. These wells are suitable to culture cells. Furthermore, structures of the left bulk-monocrystalline substrate can protect the fractal structures. Hence, in some embodiments, the monocrystalline substrate around part of the plurality of geometrical cavities is maintained to create a barrier between geometrical cavities. In some embodiments, some of the three-dimensional structures are covered by monocrystalline substrate while other three-dimensional structures are being exposed, i.e. free of monocrystalline substrate, for example for cells to attach.

In preferred embodiments, the monocrystalline substrate is removed by etching. 17

An example of such step 8) of a method of the invention is schematically shown in figure 5D.

In some embodiments a method of the invention, in particular if the one or more materials grown or deposited in step 5) is a conductive material, further comprises:

9) depositing a conductive layer consisting of a material, that is capable of forming an oxide intermediate adhering to the conductive material and to the material deposited in step 5), on the surface of the base three-dimensional structure material to at least partly cover the conductive material;

10) providing a protective layer to at least part of the three-dimensional structures and part of the surface of the monocrystalline substrate comprising said at least part of the three-dimensional structures;

11) removing the part of the conductive layer deposited in step 9) that is not provided with a protective layer in step 10);

12) removing the protective layer provided in step 10);

13) optionally depositing substrate layer, preferably silicon, more preferably amorphous silicon, polysilicon or crystalline silicon; wherein steps 9) - 13) are performed between steps 6) and 7) of the method of claim 1 and wherein step 7) optionally comprises bonding of the surface of the device that comprises the monocrystalline substrate layer deposited in step 13) to the support base.

Figure 6 schematically shows an example of such method of the invention.

In step 9) of a method of the invention, a conductive layer consisting of a material, that is capable of forming an oxide intermediate adhering to the conductive material and to the material grown or deposited in step 5), is deposited on the surface of the base three-dimensional structure material to at least partly cover the conductive material. The purpose of this step is to create a layer that adheres to both the base three-dimensional structure material, preferably silicon oxide or silicon nitride, already present on the surface, and the substrate layer deposited in step 13) after bonding to the support base. As explained herein below, 18 this substrate layer deposited in step 13) is preferably a silicon containing material, preferably silicon, which is converted into silicon oxide if the bonding to the support base is performed by anodic bonding. I.e. the layer deposited in step 9) functions both as a conductive material and as a bonding agent.

In preferred embodiment, the material of the conductive layer is any material which forms an oxide intermediate bond with SiCh. In preferred embodiments, the material is a chromium (Cr), titanium (Ti), aluminum (Al), molybdenum (Mo), tantalum (Ta), niobium (Nb), vanadium (V) and/or hafnium (Hf) containing material. In preferred embodiments, the material is a Cr and/or Ti containing material, more preferably a Cr containing material. The material further preferably comprises a metal, preferably platinum, gold, silver or a combination thereof, more preferably platinum. Suitable examples of suitable materials are Cr/Pt/Cr (e.g. 5nm/20nm/5nm), Ti/Pt/Ti (e.g. 5nm/20nm/5nm). However, a skilled person is well capable of selecting further appropriate materials.

In preferred embodiments, the formed layer is at least 1 nm thick, more preferably at least 10 nm, more preferably at least 20 nm, more preferably at least 30 nm thick. In some embodiments, the formed layer is preferably between 10 and 100 nm thick.

Deposition of this layer can be achieved using any method known in the art. In preferred embodiments, the material is deposited by directional deposition, such as sputtering.

An example of such step 9) of a method of the invention is schematically shown in figure 6C.

In step 10) of a method of the invention, a protective layer is provided to at least part of the three-dimensional structures and part of the surface of the monocrystalline substrate comprising said at least part of the three-dimensional structures. This protective layer protects the conductive layer deposited in step 9) from being removed in stem 11). Hence, the protective layer allows to selectively remove part of the conductive layer deposited in step 9). I.e. in this step the configuration of the device obtained with a method of the invention in terms of which structures are conductively connected and which structures are not conductively connected and/or which structures can be conductively connected to 19 one or more electrodes located outside the device itself, is determined. For instance the structure can be connected to one or more electrodes located outside the device itself through one or more openings in the support base

The protective layer can be of any material that is resistant to the method by which part of the conductive layer is removed in step 11). In particular, the protective layer is a material that is resistant to ion beam etching. In preferred embodiment the protective layer comprises or is a photoresist layer. In preferred embodiments, the photoresist layer is provided by lithography, preferably contact UV lithography.

An example of such step 10) of a method of the invention is schematically shown in figure 6D.

In step 11) of a method of the invention, the part of the conductive layer deposited in step 9) that is not provided with a protective layer in step 10) is removed. In this step conductive connection between structures is abolished.

Removal of part of the conductive layer can be achieved by any method known in the art. In preferred embodiments, the removal is performed by ion beam etching, in particular angled ion beam etching. The use of angled ion beam etching allows for removal of the material from the outer surface of the monocrystalline substrate, while no affecting any material deposited inside the three-dimensional geometric cavities. Because the ion beam is applied under an angle, it is not able to reach the material deposited inside the geometric cavities. In preferred embodiments the angle is between 5 and 54 degrees, such as about 45 degrees. It is further preferred that the angled ion beam etching is performed under concentric rotation of the structures, e.g. at a rotational speed of 5 rpm.

An example of such step 11) of a method of the invention is schematically shown in figure 6E.

In step 12) of a method of the invention, the protective layer provided in step 10 is removed. The method of removal depends on the protective layer that has been provided. In preferred embodiments, the protective layer comprises or is a photoresist layer and the protective layer is removed by stripping of the 20 photoresist. This can be performed by methods known in the art to remove photoresist.

An example of such step 12) of a method of the invention is schematically shown in figure 6F.

In step 13) of a method of the invention, a substrate layer is optionally deposited to the surface of the device obtained in step 12). This allows for the bonding of the outer surface of the structures to a support base, for instance if the material deposited in step 9) is not compatible with binding to a support base. In preferred embodiments, essentially the entire surface of the device obtained in step 12) is provided with the substrate layer. In preferred embodiments, said layer can be of any silicon containing material. In preferred embodiments, said non- conductive material is silicon. In preferred embodiments said material is amorphous silicon, polysilicon or crystalline silicon. Deposition of this material can be achieved using any method known in the art. In preferred embodiments, the material is deposited by directional deposition, such as sputtering.

An example of such step 13) of a method of the invention is schematically shown in figure 6G.

In a method of the invention in which steps 9-13 are performed, step 7), if performed, comprises bonding of the surface of the structures that comprise the substrate layer deposited in step 13) to the support base.

An example of such step 7) of a method of the invention is schematically shown in figure 6H. Anodic bonding performed in step 7) of a method of the invention converts silicon containing material, preferably silicon, deposited in step 13 into silicon oxide.

Figure 61 shows a representation of the exemplary device with the different materials indicated.

An example of a step 8) of a method of the invention in which steps 9-13 are performed is schematically shown in figure 6J. 21

An advantage of the use of a silicon, deposited in step 13), in the devices of the invention is that with anodic bonding of the silicon-containing surface, the silicon is converted to silicon oxide, which is similar to the material that is preferably deposited in step 2). If the silicon layer deposited is thin enough, e.g. between 5 and 20 nm of amorphous silicon, it will be completely converted to silicon oxides, as exemplified in figure 61. In this figure, the black layer of silicon is converted to silicon oxide where the anodic bonding has taken place.

In some embodiments of a method of the invention, the device, in particular the three-dimensional structures consists of the layers and materials that are deposited and/or grown (and not removed) in steps 1) - 13) of a method of the invention.

In some embodiments of a method of the invention, one or more additional layers or materials may be deposited or grown on the structures.

In some embodiments, in particular wherein in step 5) a conductive material as defined herein is grown or deposited, a base three-dimensional structure material, preferably silicon nitride or silicon oxide, is deposited on at least part of the surface of the monocrystalline substrate including the base three-dimensional structure material and at least part of the plurality of geometrical cavities between steps 4) and 5). Preferably, this step (also referred to herein as step 4a) comprises oxidation at temperatures below 950 °C. This results in sharpened oxides, which means that the resulting layer is thinner at the apices. In some embodiments, after bonding to the support base in step 7) and removing monocrystalline substrate around at least part of the plurality of geometrical cavities in step 8) the base three-dimensional material, preferably silicon nitride or silicon oxide, is removed selectively from the apices of the at least part of the three-dimensional structures (i.e. only from the apices), for instance by timed HF wet etching. Figure 8 schematically shows such process. Such process gives the possibility to exclusively open the apices in a post processing step, so every apex will be active. As another example, such process gives the possibility to protect the conductive or other type layer inside the structures until it is opened in a post processing step. The post processing could be such that only at least one or a selection of structures is 22 stripped. This allows to selectively remove or add material to the structure and allows for selective measurements.

In some embodiments, wherein in step 5) a conductive material as defined herein is grown or deposited, following step 8) an active or functional layer is grown or deposited at the surface of at least part of the three-dimensional structures (also referred to herein as step 8a), followed by deposition or growing of a conductive material as defined herein (also referred to herein as step 8b), followed by creating active spots in at least the apices of at least part of the three- dimensional structures (also referred to herein as step 8c), for instance by contacting the conductive material grown or deposited in step 5), that preferably functions as an electrode, to the conductive material deposited or grown to the active or functional layer. Active spots can for instance be created only in the apices or of the entire structure or part thereof. Figure 9 (active spots in the apices) and figures 10 and 11 (active structures) schematically shows such processes. The conductive material grown or deposited in step 5), that preferably functions as an electrode, can be contacted with the conductive material deposited or grown to the active or functional layer either on the side of the device that contains the three- dimensional structure (front side, see figure 11) or are side of the device that contains the support base (back side, see figure 10).

The invention also provides a device comprising a plurality of three- dimensional structures obtainable with a method.

The invention further provides a device comprising a plurality of three- dimensional structures, the device comprising:

- a support base, preferably borosilicate glass;

- a plurality of three-dimensional structures, whereby at least part of the structures comprise a conductive material, preferably at the outer surface;

- wherein the plurality of three-dimensional structures are attached to the support base via a base three-dimensional structure material, preferably silicon oxide or silicon nitride, more preferably silicon dioxide.

Suitable examples of a support base are supports comprising or consisting of ceramics (such as silicon nitride, alumina, zirconia), glass (such as borosilicate glass, and soda-lime glass), or polymeric surfaces (such as polystyrene, permanox, 23 polydimethylsiloxane). In preferred embodiments, the support base comprises borosilicate glass, such that the three-dimensional structures are attached to the borosilicate glass via an, preferably silicon-containing, layer. In preferred embodiments, the support base consists of borosilicate glass.

The conductive material can be any conductive material known in the art. In preferred embodiments, the conductive material comprises or consists of one or more metals, a silicide, such as platinum silicide (PtSi), oxides of transition metals and rare-earth elements, semiconductor oxides and conductive polymers. A silicide such as PtSi is formed when a metal, e.g. platinum, after deposition is in contact with silicon and at elevated temperature the silicon is in part converted to PtSi. In preferred embodiments, the conductive material is a metal. In further preferred embodiments, the metal is platinum, gold, silver or a combination thereof.

In preferred embodiments, the conductive material further is or comprises a chromium (Cr), titanium (Ti), aluminum (Al), molybdenum (Mo), tantalum (Ta), niobium (Nb), vanadium (V) and/or hafnium (Hf) containing material. In preferred embodiments, the material is a Cr and/or Ti containing material, more preferably a Cr containing material. The material further preferably comprises a metal, preferably platinum, gold, silver or a combination thereof, more preferably platinum. Suitable examples of suitable materials are Cr/Pt/Cr (e.g. 5nm/20nm/5nm), Ti/Pt/Ti (e.g. 5nm/20nm/5nm). However, a skilled person is well capable of selecting further appropriate materials.

In further preferred embodiments, the conductive material is a combination of 1) a material comprising or consisting of one or more metals, a silicide, such as platinum silicide (PtSi), oxides of transition metals and rare-earth elements, semiconductor oxides and conductive polymers, preferably a metal or silicide, more preferably platinum, gold, silver or a combination thereof or PtSi, more preferably platinum or PtSi, and 2) a material comprising or consisting of a Cr, Ti, Al, Mo, Ta, Nb, V and/or Hf containing material, preferably Cr or Ti containing material, the material further comprising a metal, preferably platinum, gold, silver or a combination thereof, more preferably platinum.

In preferred embodiments, the base three-dimensional structure material is a silicon containing material. In further preferred embodiments the non-conductive layer is silicon, more preferably silicon oxide or nitride, e.g. Si, SiCh, SisN4, SiRN. 24

In preferred embodiments the plurality of geometrical cavities consists of geometrical cavities of different generations. In preferred embodiments, this is achieved by repeating steps 2)-4) of a method of the invention one or more times before continuing with step 5). Reference is made to Figures 2 and 3 showing examples of multiple generation structures. Figure 2C shows a simple three- dimensional structure that can be created with 1 round of subtraction (Generation -G- 0). Figure 2D shows a three-dimensional structure that can be created with 2 rounds of subtraction (Gl). Figure 2E shows a three-dimensional structure that can be created with 3 rounds of subtraction (G2). Figure 2F shows a three-dimensional structure that can be created with 4 rounds of subtraction (G3). And figure 2G shows a three-dimensional structure that can be created with 5 rounds of subtraction (G4).

In preferred embodiments, the device comprises one or more electrodes, in particular three-dimensional electrodes. In preferred embodiments, at least part of the three-dimensional structures function as electrodes, in particular three- dimensional electrodes. In preferred embodiments, at least part of the three- dimensional structures comprise or are electrodes, in particular three-dimensional electrodes.

In some embodiments, the base structure material embeds a further conductive layer that is conductively attached to at least two three-dimensional structures of the plurality of structures comprise a conductive material.

In preferred embodiments of a device of the invention, the three-dimensional structures that comprise a conductive material are conductively attached to the one or more electrodes located outside the device via the further conductive layer.

In some embodiments, the three-dimensional structures consist at least partially of the conductive material. In such embodiment, the conductive material preferably serves as the surface for cell growth.

In some embodiments, the three-dimensional structures consist of conductive material. In such embodiment, the conductive material preferably serves as the surface for cell growth. In such embodiment, the conductive material preferably serves as an electrode to record electrical potentials from the cell environment. 25

In some embodiments, the device, in particular the three-dimensional structures consists of the layers and materials that are deposited and/or grown (and not removed) in steps 1) - 13) of a method of the invention.

In some embodiments, at least part of the three-dimensional structures comprising a conductive material as defined herein, comprise one or more additional layers or materials.

In some embodiments, at least part of the three-dimensional structures further comprise a base three-dimensional structure material as an additional layer or material, preferably silicon nitride or silicon oxide, which is at least absent at the apices of the three-dimensional structures. In preferred embodiments, the base three-dimensional structure material, preferably silicon nitride or silicon oxide, is only absent at the apices of the three-dimensional structures. In such embodiment, both the base three-dimensional structure material and the conductive material preferably serve as the surface for cell growth. In preferred embodiments, the conductive material is present at the apices of the structures, preferably at all apices of the structure. This allows electrical recording of the cells and cell environment.

In some embodiments, at least part of the three-dimensional structures further comprise an active or functional layer and a further layer of conductive material as defined herein as additional layers or materials.

In some embodiments, at least part of the three-dimensional structures that comprise a conductive material are conductively attached to one or more electrodes located outside the device. For instance, the structure can be connected to one or more electrodes located outside the device itself through one or more openings in the support base.

The three-dimensional structures of a device of the invention or prepared with a method of the invention have the material deposited and/or grown before step 6) of a method of the invention is performed, in particular deposited and/or grown in steps l)-5) of a method of the invention, as the outer surface on which cells can be cultured. In particular, the outer surface of the material deposited in these steps are the surface on which cells can be cultured. 26

The plurality of three-dimensional structures of the invention or prepared with a method of the invention are produced by micro- and nanofabrication. Each three-dimensional structure preferably, independently, has a size between 10 nm and 500 pm. In preferred embodiments, the three-dimensional structures have a size between 100 nm and 500 pm, more preferably between 100 nm and 250 pm.

In preferred embodiments, the structures have a maximum height of between 0.1 and 50 pm. The size of all structures in a structure of the invention can be the same or different. In preferred embodiments, the structures are oriented perpendicular to the support base and have a dimension in the range of 1 nm to 100 pm, preferably 50 nm to 50 pm. In preferred embodiments, the volume and area of the three-dimensional structures is defined by the size of the first generation geometrical cavity. Preferably the areal dimensions, also called the footprint, of the first generation geometrical shape are between 1 and 2500 pm ² .

It will be understood that the three-dimensional structure typically decrease in size with every next generation.

In preferred embodiments, the three-dimensional structure of the invention or prepared with a method of the invention is a fractal structure. Fractal structures exhibit similar patterns at different scales called self-similarity. As used herein, the term "fractal" means and includes a pattern (i.e., shape or geometry) that can be repeatedly divided into smaller parts or repeatedly multiplied into more significant parts that are the same or similar to the original pattern (i.e., shape or geometry). Examples are shown in figures 2 and 3.

The distance between the three-dimensional structures or fractals can vary. The distance between the centers of any of two adjacent three-dimensional structures can also be called a pitch. Preferably the pitch between the three- dimensional structures is 5 - 100 pm, preferably 10-50 pm, more preferably 10-25 pm, most preferably 12 - 20 pm. The pitch between the three-dimensional structures depends on the placing, the orientation, and the size of the three- dimensional structures. For example, in preferred embodiments, the pitch between the three-dimensional structures placed in a hexagonal orientation is 12 pm, and the pitch between three-dimensional structures placed in a square orientation is 20 pm. 27

In preferred embodiments the device of the invention or prepared with a method of the invention comprises a surface defining a regular pattern of protrusions; the protrusions are built up from octahedral structures; and the octahedral structures are becoming narrower to the outside of the three- dimensional structure.

In some embodiments, the three-dimensional structure has any of the following topographies (see figure 2):

- a pyramid (GO),

- a pyramid with on the apex an octahedral (Gl),

- a pyramid with on the apex an octahedral and on each apex of the octahedral a second level of octahedral structures (G2),

- a pyramid with on the apex an octahedral and on each apex of the octahedral a second level of octahedral structures and on each apex of the second level a third level of octahedral structures (G3), or

- a pyramid with on the apex an octahedral and on each apex of the octahedral a second level of octahedral structures and on each apex of the second level a third level of octahedral structures and on each apex of the third level a fourth level of octahedral structures (G4),

- a pyramid with on the apex an octahedral and on each apex of the octahedral a second level of octahedral structures and on each apex of the second level a third level of octahedral structures and on each apex of the third level a fourth level of octahedral structures (G4), on each apex of the n-lth level a nth level of octahedral structures (Gn) n being 5-10.

In preferred embodiments, the plurality of three-dimensional structures is evenly distributed on the support.

In some embodiments the plurality of three-dimensional structures comprise are placed on the surface of the support base in a lattice configuration, preferably a square or hexagonal lattice configuration.

As detailed herein above, a method of the invention allows for timability and a great diversity in configurations of the three-dimensional structures, in particular of structure composed of a conductive material and/or structures that function as electrode(s). I.e. different structures can be internally connected to 28 allow electrical conductivity between the structures thereby creating different subsets of structures, whereby the structures within each subset are connected but not electrically connected with the structure of other subsets.

It is further possible to create different types of electrodes within a single device or within three-dimensional structures, in particular within fractals. For instance, in part of the three-dimensional structures electrodes are formed by performing steps l)-5) as described herein, and in another part of the three- dimensional structures electrodes are formed by performing steps 1)-13) as described herein. Both of these types of electrodes can also be combined with further types of electrodes as described herein, e.g. whereby in part of the three- dimensional structures a method of the invention comprises step 4a) as described herein and/or whereby in part of the three-dimensional structures a method of the invention comprises steps 8a)-8c) as described herein. As yet another example, a device can be produced whereby in part of the three-dimensional structures a method of the invention comprises step 4a) as described herein and whereby in part of the three-dimensional structures a method of the invention comprises steps 8a)-8c) as described herein. It is further also possible to combine within a single device three-dimensional structures that function as electrodes with three- dimensional structures that do not function as electrodes, for instance that are formed of silicon dioxide or that are formed of a material grown or deposited in step 5) of a method of the invention other than a conductive material. Figure 3g shows an example of a device in which different types of electrodes are combined.

In addition, it is possible to attach the electrodes of one or more single structures or one or more subset of structures with external electrodes. Figure 12 schematically shows examples of different configurations. As will be understood by a person skilled in the art, the possibilities is conductively connecting structures or subsets of structures are fully timeable and as such innumerable.

Three-dimensional structures comprised within a device of the invention and comprising or consisting of a conductive material as defined herein are able to function as an electrode. Reference is made to Figure 3, which shows an example of fractal structures with Pt suitable for cell culture. Hence, in preferred embodiments, at least part of the three-dimensional structures comprise in a device 29 of the invention or produced with a method of the invention comprise an electrode. In other preferred embodiments, at least part of the three-dimensional structures comprise in a device of the invention or produced with a method of the invention is an electrode.

The conductive material in the three-dimensional portions can facilitate an electric current. An electric current may influence the cells in culture. For example, an electric current may influence cell morphology and/or cell spreading a cell culture.

In some embodiments, the conductive material serves as one or more electrodes that are used for external stimulation of the cells or tissues in culture. For example, external stimulation of cells and/or tissues in cell culture can be used to induce a synthesized rhythm in the waves.

In other embodiments, the conductive material serves as one or more electrodes that are used for measuring and/or recording electrical pulses or signals. The electrical recording includes the ability to record in folding environments, such as cell culture medium containing serum (demonstrated in the examples and shown in Figure 13), without affecting the electrical signal. This includes the capability of measuring electrical capacitance, e.g., impedance (as shown in Figure 14).

Also, combinations of electrical stimulation and measuring and/or recording of electrical pulses is possible. Examples of cells for which such applications are useful are muscle cells, especially cardiac muscle cells, and neurons. Therefore, a device of the invention can improve muscle cell culture technologies and/or cardiac cell culture technologies. Furthermore, neurons and the synapses of neurons can be stimulated by an electric field or by a varying magnetic field. Therefore, a device of the invention can be used to culture neurons and/or neuronal tissues and simulate these cells during cell culture.

In preferred embodiments, at least part of the three-dimensional structures are conductively connected. This allows for electrical stimulation and/or recording electrical potential of cells grown or cultured in different structures.

In preferred embodiments, at least part of the plurality of structures is used as an electrode to acquire or emit a signal upon potential or current application in 30 full-cell or half-cell configurations. Such electrodes function similar to conventional electrode.

In preferred embodiments, at least part of the plurality of three-dimensional structures is coated. Preferably the structures are used as electrodes for electroplating for the deposition of metals and/or halogenated metals. Preferably the coating is a silver, silver chloride, or semiconductor, preferably titanium nitride or iridium oxide, coating.

In some embodiments, the conductive layer grown or deposited in step 5) and/or the conductive layer deposited in step 9) is connected to a conductive material, in particular to a conductive material located outside of the device, to form one or more electrodes. For instance the structure can be connected to one or more electrodes located outside the device itself through one or more openings in the support base.

The device comprising a plurality of three-dimensional structures of the invention can be used for various cell culture purposes, for example, 2D or 3D cell culture, inducing stem cell differentiation, and/or culturing multicellular organoids. Cells can grow on conductive material as defined herein, particularly on metal surfaces. Furthermore, cells can grow on base three-dimensional structure material, particularly silicon oxide or silicon nitride, e.g. on the surface of three- dimensional structures and fractal structures (see e.g. Dituri et al. 2021).

The three-dimensional cell culture template, as described herein, can be used to culture various cell types, alone or in co-culture and can be used with various types of cell culture media. In some embodiments, the cultured cells are eukaryotic cells, preferably mammahan cells. In preferred embodiments, the cultured cells are human primary or immortalized cells. Cells can be grown in adherent cultures or in suspension. In some embodiments, the cells are attached to the three- dimensional structure of the cell culture template.

Also provided is a use of the device according to the invention for cell culturing.

Also provided is a use of the device according to the invention for cell electrical recording. 31

Also provided is a use of device according to the invention for maintaining electrical or electrochemical readout in a fouling environment, such as cell culture medium, without loss of electrical or electrochemical signal.

Also provided is a method for culturing cells, comprising providing a device according to the invention with cells and culturing the cells. In some embodiments, the method comprises producing a device comprising a plurality of three- dimensional structures with a method according to invention, providing the surface of the plurality of three-dimensional structures with cells and culturing the cells.

In preferred embodiments, the device or three-dimensional structures are sterilized before providing and culturing cells. For example, the structures can be sterilized by chemical means, high temperature treatment, irradiation, such as autoclave and UV light. In preferred embodiments, the three-dimensional structures or the entire device are sterilized by using UV, chemical means and/or high temperature treament.

In some embodiments, cells are provided to the surface of the plurality of three-dimensional nanostructures under growth permitting conditions.

In some embodiments, cells are provided to the conductive material, and optional to base three-dimensional structure material, preferably silicon oxide or silicon nitride, more preferably silicon dioxide, if present at the surface of the three-dimensional structures.

Culturing of cells and tissues requires the supply of medium and nutrients. Hence, in some embodiment, the three-dimensional structures are provided with cell culture medium. The culture environment should be stable in terms of pH, oxygen supply, and temperature. Cell culture media often comprise balanced salt solutions, amino acids, vitamins, fatty acids and lipids to support the growth of the cells and/or tissues. The precise media formulations have often been derived by optimizing the concentrations of every constituent. Different cell types may need of different media compositions, which can be assessed by a person skilled in the art.

Furthermore, culturing of cells often requires the addition of serum. The serum is a complex mix of proteins, peptides, growth factors, and growth inhibitors. The most commonly used serum is fetal calf serum, which is used for a wide range 32 of cell types. In addition, the medium may be supplemented with growth factors and cytokines.

In preferred embodiments, the cells are in the form of tissue or organoid.

In some embodiments, the cells are primary cells, preferably primary tumour cells. Primary cells are cells that are isolated directly from tissues. For example, these primary cells can be epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic, and mesenchymal stem cells. The cultures can be heterogeneous. The device can also be used to co-culture different cell types. In some embodiments, the primary cells cultured in the three- dimensional structures are epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic and/or mesenchymal stem cells. In some embodiments, the cultures are heterogeneous, comprising various cell types. Furthermore, primary cells can be derived from healthy or diseased tissue, for example, tumors. Primary cells derived from tumors are called primary tumor cells. These cells can be tumor cells but also cells that are present in the microenvironment of the tumor and support the tumor cells. For example, cancer- associated fibroblasts. In some embodiments, the cultured cells are cancer- associated fibroblasts. In some embodiments, the cells are cancer-associated fibroblasts (CAFs) activated by the material, shape, and/or the pattern of the three- dimensional structures. Cancer-associated fibroblasts are non-tumor cells that are present in the tumor microenvironment. The tumor-microenvironment is a multicellular tumor-supportive system and comprises cells from mesenchymal, endothelial and hematopoietic origin. The cells interact closely with the tumor cells and contribute to tumorigenesis. The tumor microenvironment is also a target for the development of anti-cancer drugs. Culturing cells from the tumor microenvironment, for example, tumor-associated fibroblasts is therefore of value for studies to tumor- targeting drugs.

In a preferred embodiment of the method for culturing cells or tissues as described herein, the cells are stem cells, preferably mesenchymal stem cells, adult stem cells, adipose adult stem cells and/or induced pluripotent stem cells. In some embodiments, the cells are progenitor cells. In preferred embodiments, the stem cells are not derived from embryo’s or embryonic tissue. Preferably, the stem cells are not embryonic stem cells. 33

In some embodiments, the cell culture template, as described herein, can be used to grow or create functional 3D structures. In some embodiments, cells in the method for culturing as described herein form complex cellular assemblies, preferably a multicellular organoid. An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions. These organoids are multicellular and show realistic micro- anatomy. They are derived from one or a few cells from a tissue, stem cell, or introduced pluripotent stem cell. The cells in these organoids are organized and can be polarized, having an apical and a basal side. The three-dimensional structures of the described cell culture template can attribute to the formation of organoid structures and support these structures to grow.

Features maybe described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The invention will be explained in more detail in the following, non-limiting examples.

34

Brief description of the drawings

Figure 1: Initiator: Etching of the monocrystalline substrate to subtract at least one, or part of one geometrical feature with anisotropic etching to produce a geometrical cavity. The displayed geometrical cavities are an octahedral cavity or a part of an octahedral cavity. This cavity renders as the initiation for a three- dimensional structure, thereby preferably forming one or more apices. In the middle planes, the octahedral cavity in the monocrystalline substrate has broad access to the outside of the substrate. In the right plane, the octahedral cavity in the monocrystalline substrate has the widest point of the octahedral shape as opening and access to the outside of the substrate. Generation 1 (Gl): Schematic display of the second round of anisotropic etching, creating octahedral cavities at each apex of the previous cavity in the monocrystalline substrate.

Figure 2: Scanning electron micrographs of the amorphous silicon dioxide fractals. A) square orientation with a 20 pm pitch; B) hexagonal orientation with a 12 pm pitch; the structure of C) generation (G)0; D) Gl; E) G2; F) G3; G) G4. The size bar in A) and B) indicates 20 pm; for the images in C)-G) it is 2 pm.

Figure 3: Representation of a fractal (a) and (g); and scanning electron micrographs (b-f) of Pt material (bright) supported on amorphous silicon dioxide (gray) shaped as fractals.

Figure 4: Selective opening of the thermally grown amorphous silicon dioxide at the apex of the pyramidal pit after HF etching. Note that stress-induced oxidation retardation is more pronounced in concave corners when more than two planes intersect.

Figure 5: Exemplary process of a method of the invention. TMAH = tetramethylammonium hydroxide

Figure 6: Exemplary process of a method of the invention. IBE = ion beam etching; TMAH = tetramethylammonium hydroxide.

Figure 7: Exemplary devices of the invention wherein the three-dimensional structures are not bonded to a support base.

Figure 8: Exemplary process of the invention.

Figure 9: Exemplary process of the invention.

Figure 10: Exemplary process of the invention. 35

Figure 11: Exemplary process of the invention.

Figure 12: Examples of different configurations of three-dimensional structures in a device of the invention.

Figure 13: CV scans of (A) Pt octahedra in 0.1 M H2SO4, (B) Pt octahedra, thin film and foil in the electrolyte with 0 mg ml ¹ BSA concentration (C) 10 mg ml" ¹ BSA concentration and (D) 40 mg ml ¹ BSA concentration which is the human blood equivalent amount [D01:10.1016/j.bpj.2009.09.056]. CV scans provided here were measured with 100 mV S" ¹ scanning rate.

Figure 14: A. i _p as a function of square root of the scanning rate for Pt octahedra collected at different BSA concentrations and B. EIS results acquired from Pt octahedra, thin film and foil at 40 mg ml ¹ BSA concentration. Note that the sweep rate dependency of the Pt fractals was not affected from the change in concentration of BSA meaning that Pt fractals can be utilized as electrochemical readout elements in a fouling environment without loss of electrochemical signal.

Examples

Example 1 - Fabrication of Pt octahedra based electrodes

A phosphorus-doped n-type single-crystalline silicon (c-Si) (100) substrate 100 mm in diameter and 380 pm thick was cleaned with ozone steam combined with a pre-and post-cleaning silicon dioxide strip in 1% aqueous hydrofluoric acid. Substrates were immediately transferred to a high-temperature furnace where dry thermal oxidation (O2 environment) at 1100 °C for 95 minutes was used to grow a 160 ± 1 nm thick t-SiCh film, verified by ellipsometry measurements. A 1.7 pm thick positive type photoresist layer was spun on top of the t-SiCh at 4000 rpm, preexposure baked at 95 °C, and transferred to a mask aligner where it was exposed under a mask for 4.3s at 12 mW/cm ² . The mask consisted of a hexagonal array of circular openings with a 6 pm diameter and 13 pm pitch. After exposure, the photoresist was developed in two sequential cycles of the 30s in separate beakers, after which the substrate was quick to dump rinsed (QDR) and spin-dried at 2500 rpm. A short UV-Ozone cycle enhances the wettability of the patterned surface, after which the substrate was submerged in buffered hydrofluoric acid (BHF) for 03:30 (mm:ss) patterning the t-SiCh film. The photoresist was stripped in 99% 36 aqueous nitric acid (HNOa) in two sequential cycles of 5 minutes in separate beakers. Before etching the Oth generation pyramidal pits in the c-Si using 25% aqueous potassium hydroxide (KOH), a 1 -minute etching cycle in 1% HF was performed. Stripping the photoresist in HNOa chemically grows a thin SiOa layer at the position of the patterned oxide (exposed c-Si), which otherwise impairs the etching in KOH. The KOH etching was conducted at 75 °C for 09:30 (mm:ss). A 37% aqueous hydrochloric acid, hydrogen peroxide, DI water solution was mixed in a 1:1:5 ratio and heated to 70 °C to remove ionic contaminants from the substrate (RCA-2). The Oth generation structure was completed by stripping the t-SiOa in 50% aqueous HF for 45s.

Fabrication of the 1st generation fractal structures was achieved using a Oth generation substrate and an Ozone/steam cleaning. Substrates were again immediately transferred to a high-temperature furnace where dry thermal oxidation at 1100 °C for 95 minutes was used to grow a 162 ± 1 nm thick t-SiCh film, verified by ellipsometry measurements on a monitor substrate within processed in the same batch. Etch-rate characterization was performed, measuring an etch rate of 4.3 nm/min after 10 minutes of submersion in 1 % HF, yielding a film thickness of 119 ± 1 nm. Another 11 minutes of etching in 1% HF yields the target thickness of 75 nm facilitating the local opening of the SiCh nozzle existing because of the retarded oxide growth at the apices. The 1st generation octahedral features were etched using an aqueous 25% tetramethylammonium hydroxide (TMAH) solution kept at 70 °C by submerging the substrate for 10 minutes. Nozzle openings were verified by SEM inspection and checking that all features were open. Afterward, the substrates were submerged for another 153 minutes in 25% TMAH to finish etching the 1st generation octahedral features. Lastly, the t-SiCh was stripped in 50% HF for 30s to acquire the c-Si fractal cavities. For 2nd and 3rd generation fractal structures, the method for obtaining 1st generation fractal structures was repeated, using a substrate containing previous generation features, but decreasing the total time the substrate was submerged in 25% TMAH to obtain a fractal-like decrease of the octahedral size.

The 4th generation fractal structures contain octahedral features that are too small to grow the 160 nm thick t-SiCh, as the obtained nozzle opening manufactured in the 3rd generation structure would be too big for fabricating the 37

4th generation octahedral features. Therefore, a slightly different approach is taken. The 3rd generation fractal structure containing substrate was Ozone/steam cleaned. It was directly transferred to a furnace where t-SiO ₂ was grown in an O2 environment at 750 °C for 180 minutes, yielding a 6.3 ± 0.1 nm thick film verified by ellipsometry measurements on a batch-processed monitor substrate. Subsequently, the substrate was transferred into a low-pressure chemical vapor deposition (LPCVD) system for stoichiometric silicon nitride ( Si ₃ N ₄ deposition. Timed isotropic etching of the Si ₃ N ₄ in aqueous 85% phosphoric acid (H3PO4) at 180 °C leaves Si ₃ N ₄ plugs at the apices of the 3rd generation octahedral features. The substrate is then again exposed to a high-temperature furnace where dry thermal oxidation of silicon is conducted at 1050° for 45 minutes, giving an 82.0 ± 1.5 nm thick t-SiO2 film on a c-Si (111) dummy substrate and a 72.0 ± 2.5 nm thick t-SiO2 film on a c-Si (100) substrate. Then, a 30s submersion in 1% HF is used to remove the part of the Si ₃ N ₄ that is converted into t-SiO2. The remaining Si ₃ N ₄ plugs are removed in 85% H3PO4 at 140 °C to expose the locally thin t-SiO2 at the apices of the 3rd generation octahedral features. The 3rd generation nozzle is opened by a timed 1% HF etch for 02:30 (mm:ss). Finally, the 4th generation octahedral features are etched by wet anisotropic etching of c-Si in 25% TMAH at 70 °C for 8 minutes. After etching, a negative silicon replica of the 4th generation octahedra is produced.

Once the negative silicon replicas containing 1st, 2nd, 3rd, or 4th generations are produced, atomic layer deposition (ALD) of Pt is used to create Pt electrodes within the fractal structure. The procedure to produce the most complex fractal generation (4th) is explained for simplicity. The deposition of Pt was performed using an ALD reactor. The pumping system of the ALD reactor consisted of a turbopump connected to a rotary pump, keeping the pressure of the reactor chamber close to I0 ^-6 mbar. A shutter valve allowed to isolate the reactor from the pumping system. The walls of the chamber were heated to 90 °C, while the temperature of the substrate holder was maintained to 300 °C. The reactor was equipped with an inductively coupled plasma source (I CP), used for the plasma cleaning of the substrate which was performed before each deposition. The plasma cleaning procedure carried out flowing O2 gas at the pressure of 7.5xl0 ² mbar and then ignited the plasma for 15 minutes at the power of 100 W. 38

Trimethyl(methylcyclopentadienyl)platinum(rV) (MeCpPtMea) was used as precursor. The MeCpPtMea was placed in a stainless-steel cylindrical bubbler heated to 40 °C. Argon gas was used to carry the MeCpPtMea vapor from the bubbler to the reactor through a line heated at 60 °C. O2 gas at the pressure of 1.0 mb ar was used as a reactant.

For the conformal deposition of platinum into the fractal structure, MeCpPtMea pulses were performed while keeping the shutter valve between the reactor and the pumping system closed. The cycle started by flowing Ar in order to stabilize the gas flow for 7 s. Then MeCpPtMea was dosed for 1 s by diverting the Ar flow from the line to the precursor’s cylinder. Then Ar flow was diverted back for 6 s for the purge of the line. The precursor dosing was repeated three times before performing the reactant dosing. Subsequently, the O2 dosing was performed for 10 s followed by 10 s of pumping down. The O2 reactant step was repeated two times.

Example 2 - Fabrication of back contact layered electrodes

Electrical contacts were obtained by a fabrication process that focuses on partial electrical isolation of areas containing the fractal structures. The idea was that removing the ALD deposited Pt layer disconnects all fractals, whereas the redeposition of an electrically conductive layer and patterning of this layer only connects patches of neighbouring fractals. The schematic representation of this fabrication process is shown in Figure 6). The fabrication is initiated by rotatingangled- and timed ion beam etching (rat-IBE) using argon ions impeding under a 20-degree angle relative to the flat substrate surface (Figure 6A). By applying rat- IBE, partial removal of the ALD deposited Pt layer is possible, constricted solely to the flat substrate and part of the pyramidal cavity, leaving the Pt inside the fractal structure intact. This effect is due to a self-shadowing effect because incident argon ions predominantly move in straight line paths (Figure 6B). Concentric rotation of substrate ensures full removal of the Pt over part of the GO pyramidal cavity, leaving electrically isolated fractal structures instead of an interconnected array. To electrically reconnect patches of fractals, a tri-layer stack consisting of 5 nm chromium (Cr) adhesion layer, 20 nm Pt, and another 5 nm Cr adhesion layer is sputter-deposited over the substrate, Figure 6C), and patterned by applying, 39 exposing, and developing a positive tone photoresist (Figure 6D) and sequential rat-IBE under a 45-degree incidence angle relative to the flat substrate surface, Figure 6E). This partially removes the tri-layer stack revealing the t-SiO ₂ and electrically disconnecting some of the fractal structures once more, defining specific electrically connected areas. To facilitate anodic bonding of the fractal containing single crystalline silicon substrate, first, the resist is removed (Figure 6F), and a 10 nm thick amorphous silicon layer (a-Si) is sputter-deposited over the patterned tri-layer stack, this means that the a-Si has interfaces both with the thermally grown SiO2 as shown in Figure 6G). A borosilicate substrate is bonded to the silicon substrate using anodic bonding at 400 °C and 1000V, as shown in Figure 6H. A few remarks are in place regarding this approach. The choice of the materials for forming the tri-layer stack was based on that the materials should be compatible with anodic bonding at the stated temperature, the material is anodically bondable to glass, and should stand the etchant used to remove the silicon wafer in order to obtain the final freestanding patterned and electrically connected fractal structures. More importantly, the 10 nm a-Si is fully converted into SiO2 during the anodic bonding process forming the amorphous reaction layer that bonds the two substrates together both at the borosilicate glass- SiO2- Si(100) interface, and the (Cr+Pt+Cr), SiO2, Si (100) interface.

Example 3 - Electrochemical measurements with Pt octahedra based electrodes

Fabricated Pt octahedra-based electrodes were utilized as a working electrode for the electrochemical measurements in comparison to the flat counterparts (thin films). Three different Pt electrodes were used to evaluate how 3D modifications influence the electrochemical signal detection in the presence of bovine serum albumin (BSA). Those electrodes were i) Pt foil, ii) Pt thin film and iii) Pt octahedra.

All the electrochemical measurements were carried out in a three-electrode system. A Pt coil is used as counter electrode together with the Ag/AgCl reference electrode. The first electrolyte, 0.1M H2SO4, was used to activate Pt surface before the fouling experiments, and the electrochemical response was recorded. The second electrolyte containing 2 mM K2Fe(CN) ₆ in 0.1 M KC1 which was buffered 40 with 0.1 M KH2PO4 to keep the pH at 7 was used as the general medium for the rest of the experiments. BSA was used as biofouling agent, and added as 4, 10 and 40 mg ml ¹ for different measurements. Pt working electrodes were tested with potentio-static/dynamic electrochemical techniques with a potentiostat/galvanostat/zero resistance ammeter. Cyclic voltammetry (CV) scans were conducted with scanning rates varying in between 10 to 200 mV s ¹ .

Electrochemical impedance spectroscopy (EIS) tests were carried out in between 10 mHZ to 10 kHZ with a 10 mV amplitude (Vrm ₈ = 7 mV). Electrochemical cell was kept at open circuit potential (Voc) in between different measurements to prevent double layer’s contribution to the results.

Initial CV scan of the Pt octahedra measured in 0.1 M H2SO ₄ is provided in Fig. 13A below. H adsorption/desorption and oxide formation/reduction peaks were evident for the electrode (see Fig 8A). EIS measurements were fitted with an equivalent circuit and Rct values obtained were found to be 160 Q cm ² compatible with the findings in the literature (https://doi.org/10.1016/jjjhydene.2019.03.076).

In the light of these findings, it is certain that the electrochemical signal collected from the structures was only due to the presence of the Pt on the Pt fractal electrode. After the Pt activity verification, the ability of a redox molecule to exchange electrons with the Pt surface in an environment with and without fouling agents (BSA) was tested. Fouling experiments were carried out by means of CV scans in different BSA concentrations, namely 0, 4, 10 and 40 mg ml ¹ in a solution of potassium ferricyanide (Fe(CN)6 ^3- , 2 mM). Expected redox reaction within the system is given in Eq. 1 below.

FeIII(CN)6 ³ " -> FeII(CN)6 ⁴ " Equation 1

A diffusion controlled reversible redox reaction was observed with the experiments done without BSA (0 mg ml ¹ ) on all three electrode structures (see Fig. 13B). Analysis of the CV scans are tabulated and given in Table 1. 41

Table 1. Voltage and current related finding collected from CV at different BSA concentrations. Note that for Pt foil, no data could be collected when a BSA concentration of 10 mg ml ¹ is maintained in the electrolyte environment.

CV scans with elevated BSA concentrations are provided in Fig. 13C and 13D for all the electrodes to visualize the effect of BSA over redox reaction occurring at different electrode architectures. A considerable decrease in peak current was observed for the Pt-based electrodes here, indicating that BSA acted as a fouling agent and prevented efficient electron exchange for the FefCN)^ ion. Increasing BSA concentration led to lower redox reaction efficiency as expected. For Pt foil, a decrease of 80% in the peak current density was observed for 10 mg ml ¹ BSA concentration, which translated into to 0.2 mA cm ² for 40 mg ml ¹ BSA concentrations later. Pt thin film followed a similar trend with 50% reduction only at current density with 10 mg ml ¹ BSA and only reached a value of 0.51 mA cm ² at 40 mg ml ¹ BSA. Decrease observed on the Pt foil and thin film electrodes can be explained with flat surface structure being blocked by the large proteins and resulting in lowered redox reaction efficiency. Pt octahedra on the other hand seemed to be less vulnerable to the fouling agents’ presence, such that even at 40 mg/ml BSA concentrations a peak current density of 2.3 mA cm ^-2 was still measurable. This large difference, quadruple peak current density, proves that 3D engineered Pt fractal structure can efficiently create reaction sites and cannot be blocked by large molecules. Considering 40 mg ml ¹ BSA as the equivalent amount 42 for the human body environment, Pt octahedra were shown to be efficient structures for bio-compatible redox detection.

CV scans with varying scan rates were used to check the effects of BSA concentration on the nature of electron transfer process. Peak current (ip) values of the electrodes are plotted against scanning rate, n (mV a ¹ ) and given in Fig. 14A. In the experiments with no BSA (0 mg ml ¹ ), all three electrodes here showed a reversible electron transfer process involving freely diffusing redox species since a linear relationship in between ip and n ^1/2 . Therefore, no deviations for the nature of electron transfer were observed. Corresponding diffusion coefficients calculated by steady state peak currents. Results indicated that analyte has the highest diffusivity on Pt octahedra with a 1.04X10" ⁶ cm ² a ¹ , followed by Pt thin film and Pt foil with fluxes of 9.05X10" ⁸ and 6.19X10" ⁸ cm ² a ¹ . When BSA is added, diffusivity of the analyte is changed, and a non-zero intercept for was found showing that experimental conditions do not meet linear diffusion criteria (https://doi.org/10.1002/elan.201700695). This is due to the presence of fouling agents within the electrolyte, which deviates diffusion behavior from ideality. Additionally, no detectable change was observed for EIS spectra on high frequency range for Pt samples (see Fig. 14B); however, responses at low frequencies were all deteriorated. Observation of finite length diffusion behavior in Warburg resistance here is an indication of limited mass transfer due to the folding agents present in the electrode-electrolyte interface.

References

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