Since the advent of biosensing, structuring of electrode surfaces for the improvement of cell‐coupling and electrophysiological properties has been extensively investigated. Most of these methods result in structures with predefined dimensions and regular organization. Nevertheless, natural adhesion surfaces of the cells are hardly uniform. Therefore, this study focusses on fabricating randomly organized nanostructures mimicking the irregular distribution of natural collagen fibers coated on a planar surface. Fiber geometries are replicated by using a spin‐coating process followed by thermal nanoimprint lithography and gold electroplating. Microscopic studies reveal the width of these biomimetic collagen‐like gold nanostructures ranging between 200 nm and 5 µm, with a uniform height of ≈35 nm. In comparison to unstructured gold surfaces, nanostructured surfaces display a decrease in impedance magnitude by 50% for frequencies below 1 kHz and show an increase in critical free surface energy by 35%, the latter translating to an increased surface wettability. Culturing enteric neurons from postnatal mice, a relative hard to handle type of neurons, results in an improved spreading of the neural networks on the nanostructured surfaces.

Nanostructuring is a promising and successful approach to tailor functional layers and to improve the characteristics of biosensors such as signal transmission and tighter cell-surface coupling. One of the major objectives in biosensing and tissue engineering is the development of interfaces that mimic the natural environment of biosystems composed of extracellular matrix biomolecules. Nevertheless, effective techniques to reconstruct the random distribution of these biomolecules are still not well established. For this reason, the presented work demonstrates different methods based on nanoimprint lithography to replicate randomly distributed natural nanostructures with complex geometries into different polymers and metals. The fidelity of the replicated nanostructures has been evaluated by atomic force microscopy and the attributes of the fabrication processes have been discussed. Finally, different replication techniques have been combined for the biomimetic nanostructuring of the dielectric passivation layer as well the metal electrode surface to develop novel whole-surface-nanostructured microelectrode arrays.

Microelectrode surfaces covered with nanostructures derived from components of extracellular matrix, such as collagen fibers, have shown immense beneficial effects in promoting neuronal growth and cellular signaling. Synthetic nanostructures mimicking the features of biological nanostructures with durable conductive materials could promote the cell adhesion on microelectrode surfaces by providing topographical cues and simultaneously improve the charge transfer properties by reducing its global impedance. Therefore, an advanced nanostructuring method mimicking the structural and organizational features of natural collagen fibers onto metallic microelectrode surfaces has been presented here, which is adapted from previous technological achievements of the group and is based on nanoimprint lithography and gold electroplating. Surface characterization methods reveal an increase in surface area between 20% and 68% on the microelectrodes fabricated with two different nanostructure heights. Impedance spectroscopy measurements reveal reduction in impedance magnitude (at 1 kHz) between 22% and 41%, depending upon the nanostructure height and density on the microelectrode, which should subsequently modulate its charge transfer properties for biosensing application. Cell adhesion analysis performed with seal impedance measurements reveals a tighter coupling of enteric neurons on the nanostructured microelectrodes. Finally, extracellular recordings from enteric neurons exhibit a significant improvement in spike detection properties of the nanostructured microelectrodes.

Porous anodic aluminum oxide films (AAO) have been used for a long time as a template in MEMS technology in developing nanostructures such as carbon nanotubes, metal nanodots etc. We in this study have used the same process to nanostructure the electrode surface of a microelectrode array (MEA), since there is literature which proves the significant effect of nanostructured surfaces on cell growth and proliferation. Although AAO templates traditionally result in hexagonally ordered structures; we have used the same process for fabricating randomly ordered structures, since they resemble much more the natural cell interfaces. After fabricating foamy like AAO films with completely randomly placed pores on top of electrode surface of the MEA, we use electroplating to gently fill the random pores with metal. Later the porous films are wet chemically etched leaving standalone random metal nanostructures on top of the electrode surfaces. This process has to be performed for every wafer by complex electrochemical anodization step. We therefore use nanoimprint lithography, to make it more reproducible and cost effective. With just one master substrate with random metal structures as a nanoimprint mask, we fabricate several electrode arrays, reducing the effort for multiple complex oxidations. We analyze these random metal structures with SEM and contact angle measurements. Future experiments based on electrochemical characterization of these electrodes will have to be performed to examine their potential concerning cell recordings. Introduction Since the advent of Microelectrode arrays (MEAs) in electrophysiological measurements, researchers have been continuously trying to improve the quality of cell signal acquisition and stimulation. Apart from the cell culture related improvements a major interest has been laid upon the material and process optimization. One approach used extensively for this optimization improving the electrode surface. Research shows that nanostructuring of the microelectrodes not only decreases the impedance of the MEA, but also enhances the cell adhesion and anchoring [3]. Previous work in our lab included fabrication of organized regular nanostructures in the shape of cones, pillars, mushrooms on top of microelectrode surfaces with predetermined dimensions and spacing. It was observed that the differences in the electrophysiology measurements between the unstructured and nanostructured electrodes were very significant [4]. But the natural cell adhesion surface of the cells is hardly regular and the orientation of the surface profile is very random. Therefore in this work we have focused on fabrication of randomly organized nanostructures on the electrode surface. Materials and Methods Silicon wafers are evaporated with 20 nm of titanium and 200 nm of gold. Afterwards a 500 nm thick passivation layer (SiO2 and Si3N4) is deposited via PECVD. Then with lithography 500 nm DC Sputtered Aluminum layer is patterned on top of the Gold electrode regions. Using the contacts of the MEAs, the Aluminum is anodically oxidized with 0.3M Oxalic acid at 8°C and 40V anodizing potential. Then the pores of the AAO film are filled with metal (Au or Ni) by electroplating. After electroplating, the AAO film is wet etched in 5 wt. % Phosphoric acid to leave standalone randomly organized metal structures on top of the electrode surface regions. This wafer can be used as a master template for nanoimprint lithography to fabricate several wafers without anodizing process. Results We have successful developed AAO films with a foamy texture and randomly organized pores and then filled the pores with metal with electrochemical deposition. The etching of AAO film after deposition was also successful with good adhesion between substrate and the electroplated nanostructures. Conclusion and Discussion The aforementioned process is a modified version of the preexisting work of fabricating nanostructures on MEA electrodes with AAO template. We can eliminate the repeated use of Anodization process which is not only expensive but also time consuming and requires almost impossible reproducible conditions for different wafers. Future work will be based on electrochemical characterization (Cyclic voltammetry and Impedance spectroscopy) of the electrodes and then cell culture experiments to investigate differences between unstructured, regularly structured and randomly structured electrodes.

The functionality of the intestine is strongly affected by the microbiome, food, drugs and toxins. This influence can lead to a variety of diseases, which in turn can affect the entire organism¹. For studying the electrophysiological processes of the intestine in health and disease in vitro, a novel microelectrode system is developed and then characterized by electrochemical impedance spectroscopy (EIS). Additionally, EIS will be used to investigate the impedance behavior of different electrode-electrolyte and electrode-cell interfaces. In contrast to conventional planar microelectrode arrays, this novel system utilizes a three-dimensional spiral-like design. This enables to spatially detect the electrophysiological processes of complex tissue-like cell structures (see Figure 1). To ensure flexibility and biocompatibility, a polyimide-based insulating structure with an embedded titanium-gold-titanium metal stack is used. While titanium serves as an adhesion-promoting layer between gold and polyimide, the gold layer itself is used as the electrode material. The sensor is fabricated in a planar configuration on a glass wafer (see schematic representation of the process flow in Figure 2 and the fabricated sensor in Figure 3). First, a 50 nm adhesion-promoting aluminum layer is sputtered on the glass substrate and pre-structured by lithography and wet-etching. An 11 µm thick polyimide layer is then spin-coated and subsequently imidized by a curing process. Then, a metal stack of 20 nm titanium, 200 nm gold and 20 nm titanium is deposited by e-beam evaporation. The conductor tracks of the spiral-shaped electrodes are structured by lithography and wet-etching. An 2 µm thick polyimide layer serves to insulate the electrodes, which is again imidized by curing. The electrode and external contact pad areas are opened by reactive ion etching. For this purpose, a 150 nm sputtered and subsequently structured aluminum layer is used as a hard mask. A combination of SF₆ and O₂ mixture serves as the etching gas. An identical process sequence, which differs only in the mask of the lithography step is used to structure the polyimide frame and thus the contour of the system. Finally, the aluminum sacrificial layer is etched, after which the 3D system can be characterized electrochemically. For all electrode-characterizing electrochemical methods, a potentiostatic three-electrode setup with a 3 M KCl saturated Ag/AgCl reference electrode and a 2 mm gold disc counter electrode in a 0.1 M NaCl electrolyte is used. First, the devices are treated in an oxygen plasma to remove impurities from the openings of the electrodes. Cyclic voltammetry measurements are carried out to receive a stable open circuit potential (OCP), which is then later used as a DC-bias operating point for the EIS measurement. For this, 10 consecutive scans are performed at switching voltages between + 0.1 V and + 0.5 V against Ag/AgCl (3 M KCl) at a scan rate of 0.5 V/s. To obtain reproducible measurement results, the electrolyte is regenerated and the immersion time of the microelectrodes in the electrolyte is kept constant. The EIS measurements are then performed with a sinusoidal AC voltage of 10 mV (amplitude) and a DC operating point of 0 V against OCP in a frequency range from 100 kHz to 1 Hz for three measurement cycles. The electrolyte and the voltage are selected in such a way that faradaic reactions are suppressed and thus the main contribution of the current pertains to charging and discharging of the electric double-layer of the electrode-electrolyte interface. The measurement data is further analyzed in a frequency range where the electric double-layer capacitance is predominant and then fitted to an electrical equivalent circuit using complex non-linear least squares method (see Figure 4). The circuit describes a resistance, which is composed of solution and routing resistance, in series with a parallel circuit consisting of double-layer capacitance and charge-transfer resistance. Due to non-ideal behavior, the double-layer capacitance is modelled by a constant phase element, which is then converted into an average double-layer capacitance using an equation described by Brug et al.². Further EIS measurements will be carried out and modelled using more complex electrical equivalent circuits. For example, more application-based electrolytes such as cell culture medium and hydrogels with and without cells will be used. This serves not to characterize the electrode itself, but to investigate the impedance behavior of the electrode-electrolyte or the electrode-cell interface. Ultimately, electrophysiological recordings of enteric neurons will be spatially evaluated by the 3D microelectrode system. References B. Niesler et al., Nature reviews. Gastroenterology & hepatology, 18(6), 393–410 (2021). G. J. Brug et al., Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 176(1-2), 275–295 (1984). Figure 1