Laser-patterned epoxy-based 3D microelectrode arrays for extracellular recording.

Microelectrode arrays are commonly used to study the electrophysiological behavior of cells. Recently, there has been a growing interest in fabricating three-dimensional microelectrode arrays. Here, we present a novel process for the fast fabrication of epoxy-based 3D microelectrode array platforms with the assistance of laser-patterning technology. To this end, we photopatterned 3D pillars as scaffolds using epoxy-based dry films. Electrodes and conductor traces were fabricated by laser patterning of sputtered platinum films on top of the 3D structures, followed by deposition of parylene-C for insulation. Microelectrodes at the tip of the 3D structures were exposed using a vertical laser ablation process. The final electrodes demonstrated a low impedance of ∼10 kΩ at 1 kHz in electrochemical impedance spectroscopy measurements under physiological conditions. We investigated the maximum compression force of the 3D structures, which could withstand approximately 0.6 N per pillar. The 3D microelectrode arrays were used to record extracellular signals from HL-1 cells in culture as a proof of principle. Our results show regular firing of action potentials recorded at the tip of the 3D structures, demonstrating the possibility of recording cell signals in non-planar environments.

Mechanoactivation of Single Stem Cells in Microgels Using a 3D-Printed Stimulation Device.

In this study, the novel 3D-printed pressure chamber for encapsulated single-cell stimulation (3D-PRESS) platform is introduced for the mechanical stimulation of single stem cells in 3D microgels. The custom-designed 3D-PRESS, allows precise pressure application up to 400 kPa at the single-cell level. Microfluidics is employed to encapsulate single mesenchymal stem cells within ionically cross-linked alginate microgels with cell adhesion RGD peptides. Rigorous testing affirms the leak-proof performance of the 3D-PRESS device up to 400 kPa, which is fully biocompatible. 3D-PRESS is implemented on mesenchymal stem cells for mechanotransduction studies, by specifically targeting intracellular calcium signaling and the nuclear translocation of a mechanically sensitive transcription factor. Applying 200 kPa pressure on individually encapsulated stem cells reveals heightened calcium signaling in 3D microgels compared to conventional 2D culture. Similarly, Yes-associated protein (YAP) translocation into the nucleus occurs at 200 kPa in 3D microgels with cell-binding RGD peptides unveiling the involvement of integrin-mediated mechanotransduction in singly encapsulated stem cells in 3D microgels. Combining live-cell imaging with precise mechanical control, the 3D-PRESS platform emerges as a versatile tool for exploring cellular responses to pressure stimuli, applicable to various cell types, providing novel insights into single-cell mechanobiology.

A multiscale approach to assess thermomechanical performance and force generation in nanorobotic microgels.

We present a multiscale approach to characterize the performance of photothermally powered, nanorobotic 3D microgels. Optically triggered nanoactuators, consisting of a gold nanorod core and thermoresponsive pNIPMAM shell, are used as building blocks to generate the nanorobotic 3D microgels. We use microfluidic encapsulation to physically embed the nanoactuators in an alginate network, to form the microgel droplets. The nanoactuators respond to near-infrared light owing to the synergistic effects of plasmonic and thermoresponsive components, and the nanorobotic 3D microgels generate compressive force under the same light stimulus. We use a multiscale approach to characterize this behavior for both the nanoactuators and the assembled microgels via dynamic light scattering and fluorescence microscopy, respectively. A thermoresponsive fluorescent molecule, Rhodamine B, is integrated into alginate chains to monitor the temperature of the microgels (22-59 °C) during actuation at laser intensities up to 6.4 µW µm-2. Our findings show that nanoactuators and the microgels exhibit reversible deformation above the lower critical solution temperature of the thermoresponsive polymer at 42 °C. 785 nm laser light triggers the generation of 2D radial strain in nanoactuators at a maximum of 44%, which translates to an average 2D radial strain of 2.1% in the nanorobotic microgels at 26.4 vol% nanoactuator loading. We then use a semi-experimental approach to quantify the photothermally generated forces in the microgels. Finite element modeling coupled with experimental measurements shows that nanorobotic microgels generate up to 8.5 nN of force over encapsulated single cells. Overall, our method provides a comprehensive approach to characterizing the mechanical performance of nanorobotic hydrogel networks.

Aerosol Jet-Printed High-Aspect Ratio Micro-Needle Electrode Arrays Applied for Human Cerebral Organoids and 3D Neurospheroid Networks.

The human brain is a complex and poorly accessible organ. Thus, new tools are required for studying the neural function in a controllable environment that preserves multicellular interaction and neuronal wiring. In particular, high-throughput methods that alleviate the need for animal experiments are essential for future studies. Recent developments of induced pluripotent stem cell technologies have enabled in vitro modeling of the human brain by creating three-dimensional brain tissue mimic structures. To leverage these new technologies, a systematic and versatile approach for evaluating neuronal activity at larger tissue depths within the regime of tens to hundreds of micrometers is required. Here, we present an aerosol-jet- and inkjet-printing-based method to fabricate microelectrode arrays, equipped with high-aspect ratio µ-needle electrodes that penetrate 3D neural network assemblies. The arrays have been successfully applied for electrophysiological recordings on interconnected neurospheroids formed on an engineered substrate and on cerebral organoids, both derived from human induced pluripotent stem cells.

Inkjet-printed 3D micro-ring-electrode arrays for amperometric nanoparticle detection.

Chip-based impact electrochemistry can provide means to measure nanoparticles in solution by sensing their stochastic collisions on appropriately-polarized microelectrodes. However, a planar microelectrode array design still restricts the particle detection to the chip surface and does not allow detection in 3D environments. In this work, we report a fast fabrication process for 3D microelectrode arrays by combining ink-jet printing with laser-patterning. To this end, we printed 3D pillars from polyacrylate ink as a scaffold. Then, the metal structures are manufactured via sputtering and laser-ablation. Finally, the chip is passivated with a parylene-C layer and the electrode tips are created via laser-ablation in a vertical alignment. As a proof of principle, we employ our 3D micro-ring-electrode arrays for single impact recordings from silver nanoparticles.

Printed Silk Microelectrode Arrays for Electrophysiological Recording and Controlled Drug Delivery.

The use of soft and flexible bioelectronic interfaces can enhance the quality for recording cells' electrical activity by ensuring a continuous and intimate contact with the smooth, curving surfaces found in the physiological environment. This work develops soft microelectrode arrays (MEAs) made of silk fibroin (SF) films for recording interfaces that can also serve as a drug delivery system. Inkjet printing is used as a tool to deposit the substrate, conductive electrode, and insulator, as well as a drug-delivery nanocomposite film. This approach is highly versatile, as shown in the fabrication of carbon microelectrodes, sandwiched between a silk substrate and a silk insulator. The technique permits the development of thin-film devices that can be employed for in vitro extracellular recordings of HL-1 cell action potentials. The tuning of SF by applying an electrical stimulus to produce a permeable layer that can be used in on-demand drug delivery systems is also demonstrated. The multifunctional MEA developed here can pave the way for in vitro drug screening by applying time-resolved and localized chemical stimuli.

4D-Printed Soft and Stretchable Self-Folding Cuff Electrodes for Small-Nerve Interfacing.

Peripheral nerve interfacing (PNI) has a high clinical potential for treating various diseases, such as obesity or diabetes. However, currently existing electrodes present challenges to the interfacing procedure, which limit their clinical application, in particular, when targeting small peripheral nerves (<200 µm). To improve the electrode handling and implantation, a nerve interface that can fold itself to a cuff around a small nerve, triggered by the body moisture during insertion, is fabricated. This folding is achieved by printing a bilayer of a flexible polyurethane printing resin and a highly swelling sodium acrylate hydrogel using photopolymerization. When immersed in an aqueous liquid, the hydrogel swells and folds the electrode softly around the nerve. Furthermore, the electrodes are robust, can be stretched (>20%), and bent to facilitate the implantation due to the use of soft and stretchable printing resins as substrates and a microcracked gold film as conductive layer. The straightforward implantation and extraction of the electrode as well as stimulation and recording capabilities on a small peripheral nerve in vivo are demonstrated. It is believed that such simple and robust to use self-folding electrodes will pave the way for bringing PNI to a broader clinical application.

Determination of Stiffness and the Elastic Modulus of 3D-Printed Micropillars with Atomic Force Microscopy-Force Spectroscopy.

Nowadays, many applications in diverse fields are taking advantage of micropillars such as optics, tribology, biology, and biomedical engineering. Among them, one of the most attractive is three-dimensional microelectrode arrays for in vivo and in vitro studies, such as cellular recording, biosensors, and drug delivery. Depending on the application, the micropillar's optimal mechanical response ranges from soft to stiff. For long-term implantable devices, a mechanical mismatch between the micropillars and the biological tissue must be avoided. For drug delivery patches, micropillars must penetrate the skin without breaking or bending. The accurate mechanical characterization of the micropillar is pivotal in the fabrication and optimization of such devices, as it determines whether the device will fail or not. In this work, we demonstrate an experimental method based only on atomic force microscopy-force spectroscopy that allows us to measure the stiffness of a micropillar and the elastic modulus of its constituent material. We test our method with four different types of 3D inkjet-printed micropillars: silver micropillars sintered at 100 and 150 °C and polyacrylate microstructures with and without a metallic coating. The estimated elastic moduli are found to be comparable with the corresponding bulk values. Furthermore, our findings show that neither the sintering temperature nor the presence of a thin metal coating plays a major role in defining the mechanical properties of the micropillar.

Polarity and chirality control of an active fluid by passive nematic defects.

Much like passive materials, active systems can be affected by the presence of imperfections in their microscopic order, called defects, that influence macroscopic properties. This suggests the possibility to steer collective patterns by introducing and controlling defects in an active system. Here we show that a self-assembled, passive nematic is ideally suited to control the pattern formation process of an active fluid. To this end, we force microtubules to glide inside a passive nematic material made from actin filaments. The actin nematic features self-assembled half-integer defects that steer the active microtubules and lead to the formation of macroscopic polar patterns. Moreover, by confining the nematic in circular geometries, chiral loops form. We find that the exact positioning of nematic defects in the passive material deterministically controls the formation and the polarity of the active flow, opening the possibility of efficiently shaping an active material using passive defects.

Filtration-processed biomass nanofiber electrodes for flexible bioelectronics.

An increasing demand for bioelectronics that interface with living systems has driven the development of materials to resolve mismatches between electronic devices and biological tissues. So far, a variety of different polymers have been used as substrates for bioelectronics. Especially, biopolymers have been investigated as next-generation materials for bioelectronics because they possess interesting characteristics such as high biocompatibility, biodegradability, and sustainability. However, their range of applications has been restricted due to the limited compatibility of classical fabrication methods with such biopolymers. Here, we introduce a fabrication process for thin and large-area films of chitosan nanofibers (CSNFs) integrated with conductive materials. To this end, we pattern carbon nanotubes (CNTs), silver nanowires, and poly (3,4-ethylenedioxythiophene):poly (styrenesulfonate) (PEDOT:PSS) by a facile filtration process that uses polyimide masks fabricated via laser ablation. This method yields feedlines of conductive material on nanofiber paper and demonstrates compatibility with conjugated and high-aspect-ratio materials. Furthermore, we fabricate a CNT neural interface electrode by taking advantage of this fabrication process and demonstrate peripheral nerve stimulation to the rapid extensor nerve of a live locust. The presented method might pave the way for future bioelectronic devices based on biopolymer nanofibers.

Prototype Digital Lateral Flow Sensor Using Impact Electrochemistry in a Competitive Binding Assay.

This work demonstrates a lateral flow assay concept on the basis of stochastic-impact electrochemistry. To this end, we first elucidate requirements to employ silver nanoparticles as redox-active labels. Then, we present a prototype that utilizes nanoimpacts from biotinylated silver nanoparticles as readouts to detect free biotin in solution based on competitive binding. The detection is performed in a membrane-based microfluidic system, where free biotin and biotinylated particles compete for streptavidin immobilized on embedded latex beads. Excess nanoparticles are then registered downstream at an array of detection electrodes. In this way, we establish a proof of concept that serves as a blueprint for future "digital" lateral flow sensors.

On-Chip Electrokinetic Micropumping for Nanoparticle Impact Electrochemistry.

Single-entity electrochemistry is a powerful technique to study the interactions of nanoparticles at the liquid-solid interface. In this work, we exploit Faradaic (background) processes in electrolytes of moderate ionic strength to evoke electrokinetic transport and study its influence on nanoparticle impacts. We implemented an electrode array comprising a macroscopic electrode that surrounds a set of 62 spatially distributed microelectrodes. This configuration allowed us to alter the global electrokinetic transport characteristics by adjusting the potential at the macroscopic electrode, while we concomitantly recorded silver nanoparticle impacts at the microscopic detection electrodes. By focusing on temporal changes of the impact rates, we were able to reveal alterations in the macroscopic particle transport. Our findings indicate a potential-dependent micropumping effect. The highest impact rates were obtained for strongly negative macroelectrode potentials and alkaline solutions, albeit also positive potentials lead to an increase in particle impacts. We explain this finding by reversal of the pumping direction. Variations in the electrolyte composition were shown to play a critical role as the macroelectrode processes can lead to depletion of ions, which influences both the particle oxidation and the reactions that drive the transport. Our study highlights that controlled on-chip micropumping is possible, yet its optimization is not straightforward. Nevertheless, the utilization of electro- and diffusiokinetic transport phenomena might be an appealing strategy to enhance the performance in future impact-based sensing applications.

In vivo closed-loop control of a locust's leg using nerve stimulation.

Activity of an innervated tissue can be modulated based on an acquired biomarker through feedback loops. How to convert this biomarker into a meaningful stimulation pattern is still a topic of intensive research. In this article, we present a simple closed-loop mechanism to control the mean angle of a locust's leg in real time by modulating the frequency of the stimulation on its extensor motor nerve. The nerve is interfaced with a custom-designed cuff electrode and the feedback loop is implemented online with a proportional control algorithm, which runs solely on a microcontroller without the need of an external computer. The results show that the system can be controlled with a single-input, single-output feedback loop. The model described in this article can serve as a primer for young researchers to learn about neural control in biological systems before applying these concepts in advanced systems. We expect that the approach can be advanced to achieve control over more complex movements by increasing the number of recorded biomarkers and selective stimulation units.

Influence of Auditory Cues on the Neuronal Response to Naturalistic Visual Stimuli in a Virtual Reality Setting.

Virtual reality environments offer great opportunities to study the performance of brain-computer interfaces (BCIs) in real-world contexts. As real-world stimuli are typically multimodal, their neuronal integration elicits complex response patterns. To investigate the effect of additional auditory cues on the processing of visual information, we used virtual reality to mimic safety-related events in an industrial environment while we concomitantly recorded electroencephalography (EEG) signals. We simulated a box traveling on a conveyor belt system where two types of stimuli - an exploding and a burning box - interrupt regular operation. The recordings from 16 subjects were divided into two subsets, a visual-only and an audio-visual experiment. In the visual-only experiment, the response patterns for both stimuli elicited a similar pattern - a visual evoked potential (VEP) followed by an event-related potential (ERP) over the occipital-parietal lobe. Moreover, we found the perceived severity of the event to be reflected in the signal amplitude. Interestingly, the additional auditory cues had a twofold effect on the previous findings: The P1 component was significantly suppressed in the case of the exploding box stimulus, whereas the N2c showed an enhancement for the burning box stimulus. This result highlights the impact of multisensory integration on the performance of realistic BCI applications. Indeed, we observed alterations in the offline classification accuracy for a detection task based on a mixed feature extraction (variance, power spectral density, and discrete wavelet transform) and a support vector machine classifier. In the case of the explosion, the accuracy slightly decreased by -1.64% p. in an audio-visual experiment compared to the visual-only. Contrarily, the classification accuracy for the burning box increased by 5.58% p. when additional auditory cues were present. Hence, we conclude, that especially in challenging detection tasks, it is favorable to consider the potential of multisensory integration when BCIs are supposed to operate under (multimodal) real-world conditions.

Single-Impact Electrochemistry in Paper-Based Microfluidics.

Microfluidic paper-based analytical devices (µPADs) have experienced an unprecedented story of success. In particular, as of today, most people have likely come into contact with one of their two most famous examples─the pregnancy or the SARS-CoV-2 antigen test. However, their sensing performance is constrained by the optical readout of nanoparticle agglomeration, which typically allows only qualitative measurements. In contrast, single-impact electrochemistry offers the possibility to quantify species concentrations beyond the pM range by resolving collisions of individual species on a microelectrode. Within this work, we investigate the integration of stochastic sensing into a µPAD design by combining a wax-patterned microchannel with a microelectrode array to detect silver nanoparticles (AgNPs) by their oxidative dissolution. In doing so, we demonstrate the possibility to resolve individual nanoparticle collisions in a reference-on-chip configuration. To simulate a lateral flow architecture, we flush previously dried AgNPs along a microchannel toward the electrode array, where we are able to record nanoparticle impacts. Consequently, single-impact electrochemistry poses a promising candidate to extend the limits of lateral flow-based sensors beyond current applications toward a fast and reliable detection of very dilute species on site.

Temperature profile characterization with fluorescence lifetime imaging microscopy in a thermophoretic chip.

This study introduces a thermophoretic lab-on-a-chip device to measure the Soret coefficient. We use resistive heating of a microwire on the chip to induce a temperature gradient, which is measured by fluorescence lifetime imaging microscopy (FLIM). To verify the functionality of the device, we used dyed polystyrene particles with a diameter of 25 nm. A confocal microscope is utilized to monitor the concentration profile of colloidal particles in the temperature field. Based on the measured temperature and concentration differences, we calculate the corresponding Soret coefficient. The same particles have been recently investigated with thermal diffusion forced Rayleigh scattering (TDFRS) and we find that the obtained Soret coefficients agree with literature results. This chip offers a simple way to study the thermophoretic behavior of biological systems in multicomponent buffer solutions quantitatively, which are difficult to study with optical methods solely relying on the refractive index contrast.

Impedance scaling for gold and platinum microelectrodes.

Objective.Electrical measurement of the activity of individual neurons is a primary goal for many invasive neural electrodes. Making these 'single unit' measurements requires that we fabricate electrodes small enough so that only a few neurons contribute to the signal, but not so small that the impedance of the electrode creates overwhelming noise or signal attenuation. Thus, neuroelectrode design often must strike a balance between electrode size and electrode impedance, where the impedance is often assumed to scale linearly with electrode area.Approach and main results. Here we study how impedance scales with neural electrode area and find that the 1 kHz impedance of Pt electrodes (but not Au electrodes) transitions from scaling with area (r-2) to scaling with perimeter (r-1) when the electrode radius falls below 10µm. This effect can be explained by the transition from planar to spherical diffusion behavior previously reported for electrochemical microelectrodes.Significance.These results provide important intuition for designing small, single unit recording electrodes. Specifically, for materials where the impedance is dominated by a pseudo-capacitance that is associated with a diffusion limited process, the total impedance will scale with perimeter rather than area when the electrode size becomes comparable with the diffusion layer thickness. For Pt electrodes this transition occurs around 10µm radius electrodes. At even lower frequencies (1 Hz) impedance approaches a constant. This transition tor-1scaling implies that electrodes with a pseudo-capacitance can be made smaller than one might expect before thermal noise or voltage division limits the ability to acquire high-quality single-unit recordings.

Inkjet-Printed and Electroplated 3D Electrodes for Recording Extracellular Signals in Cell Culture.

Recent investigations into cardiac or nervous tissues call for systems that are able to electrically record in 3D as opposed to 2D. Typically, challenging microfabrication steps are required to produce 3D microelectrode arrays capable of recording at the desired position within the tissue of interest. As an alternative, additive manufacturing is becoming a versatile platform for rapidly prototyping novel sensors with flexible geometric design. In this work, 3D MEAs for cell-culture applications were fabricated using a piezoelectric inkjet printer. The aspect ratio and height of the printed 3D electrodes were user-defined by adjusting the number of deposited droplets of silver nanoparticle ink along with a continuous printing method and an appropriate drop-to-drop delay. The Ag 3D MEAs were later electroplated with Au and Pt in order to reduce leakage of potentially cytotoxic silver ions into the cellular medium. The functionality of the array was confirmed using impedance spectroscopy, cyclic voltammetry, and recordings of extracellular potentials from cardiomyocyte-like HL-1 cells.

Upscaling of pneumatic membrane valves for the integration of 3D cell cultures on chip.

Microfluidic large-scale integration (mLSI) technology enables the automation of two-dimensional (2D) cell culture processes in a highly parallel manner. Despite the wide range of biological applications of mLSI chips, manufacturing limitations of the central functional element, the pneumatic membrane valve (PMV), make the technology inaccessible for integrating tissue cultures and organoids with dimensions larger than tens of microns. In this study, we developed microtechnology processes to upscale PMVs for mLSI chips by combining 3D printing and soft lithography. Therefore, we developed a robust soft lithography protocol for the production of polydimethylsiloxane chips with PMVs from 3D-printed acrylate and wax molds. While scaled-up PMVs manufactured from acrylate-printed molds exhibited channel profiles with staircases, owing to the inherent 3D stereolithography printing process, PMVs manufactured from reflowed wax molds exhibited a semi-half-rounded channel profile. PMVs with different channel profiles showed closing pressures between 130 and 22.5 kPa, respectively. We demonstrated the functionality of the scaled-up PMVs by forming and maintaining 3D cell cultures from mouse fibroblasts (NIH3T3), human induced pluripotent stem cells (hiPSCs), and human adipose-derived adult stem cells (hASCs), with a narrow size distribution between 124 and 136 µm. Further, parallel and serial design of PMVs on an mLSI chip is used to first form and culture 3D cell cultures before fusing them within a defined flow process. Unit cell designs with upscaled PMVs enabled parallel formation, culturing, trapping, retrieval, and fusion of 3D cell cultures. Thus, the presented additive manufacturing strategy for mLSI chips will foster new developments for highly parallel 3D cell culture screening applications.

Lab-on-a-chip based mechanical actuators and sensors for single-cell and organoid culture studies.

All living cells constantly experience and respond to mechanical stresses. The molecular networks that activate in cells in response to mechanical stimuli are yet not well-understood. Our limited knowledge stems partially from the lack of available tools that are capable of exerting controlled mechanical stress to individual cells and at the same time observing their responses at subcellular to molecular resolution. Several tools such as rheology setups, micropipetes, and magnetic tweezers have been used in the past. While allowing to quantify short-time viscoelastic responses, these setups are not suitable for long-term observations of cells and most of them have low throughput. In this Perspective, we discuss lab-on-a-chip platforms that have the potential to overcome these limitations. Our focus is on devices that apply shear, compressive, tensile, and confinement derived stresses to single cells and organoid cultures. We compare different design strategies for these devices and highlight their advantages, drawbacks, and future potential. While the majority of these devices are used for fundamental research, some of them have potential applications in medical diagnostics and these applications are also discussed.