Graphene-Based Photothermal Nerve-on-a-Chip Device for Studying Thermal Sensation

Thermal sensation is an essential function for animals to sense external temperatures, regulate body temperature, and avoid noxious stimuli. The peripheral nerve plays a major role in thermal sensation by receiving temperature, converting it into an electrical signal, and transmitting it to the central nervous system. The discovery of temperature-sensitive ion channels has significantly advanced basic research on the molecular and cellular mechanisms of temperature reception. To examine the cellular responses to local temperature increases, light-sensitive nanomaterials have emerged as promising transducers for high spatiotemporal heat stimulation. However, it is still challenging to investigate how the received temperature is converted into electrical signals and then transmitted in axons due to the difficulty of simultaneously performing heat stimulation and electrical recording on the same axon. In this study, we fabricated a graphene-based nerve-on-a-chip device that enable us to record the electrical signals evoked by thermal stimulations in axon bundles of cultured sensory neurons.<br/> <br/>The device was fabricated by combining a microelectrode array that had both electrodes and heating elements with a polydimethylsiloxane-based culture chamber. The culture chamber had 8 microchannels that guide axon sprouting. Eight recording electrodes were aligned at each microchannel to record extracellular potentials from the guided axons. The heating elements were also positioned between the electrodes to measure the responses to the thermal stimulations upon the axons. To avoid stimulation artifacts caused by resistive heating, the stimulation was performed photothermally using a laser. To fabricate both the electrodes and the heating elements simultaneously, we used a spin-coated layer of graphene dispersion in cyclohexanone/terpineol because of its excellent electrical surface properties and photoabsorbance. The layer was patterned by photolithography and etching with O₂ plasma to form 50 × 50 mm electrodes and 100 × 100 mm heating elements. A laser with a wavelength of 785 nm was focused on the heating elements to deliver local heat to the axons. Temperature measurements using a thermal camera in the air confirmed that the temperature was locally increased and then saturated less than 1 sec after applying the laser. Moreover, the saturated temperature was almost linearly increased by raising the laser power. For example, continuous laser irradiation in the range of 50 to 150 mW increased the temperature in the range of 12°C to 83°C. After confirming the photothermal properties of the heating elements, we seeded primary rat sensory neurons on the fabricated device and tested photothermal stimulation upon the axons. The stimulation was applied in a power range of 10 to 50 mW. At 44 mW, the electrodes aligned with the stimulation site recorded the evoked action potentials. The peak timing of the action potential was delayed in accordance with the distance from the stimulation site and the recording site, demonstrating that the evoked action potential propagated along the axons. Furthermore, calcein-AM staining indicated that there was no apparent laser-induced damage to the axons at 44 mW. In contrast, the axons were ablated, and glial cells were dead on the heating element at a higher laser power of 50 mW. Thus, we have successfully demonstrated that the nerve-on-a-chip device enables us to visualize the generation and propagation of action potential in response to local photothermal stimulation in axons. The device will be helpful in understanding how the sensory axon processes thermal information at the cellular level.