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.

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.

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.

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.

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.

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.

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.