Simultaneous Dopamine and Serotonin Monitoring in Freely Moving Crayfish Using a Wireless Electrochemical Sensing System

Dopamine (DA) and serotonin (5-HT) are neurotransmitters that regulate a wide range of physiological and behavioral processes. Monitoring of both neurotransmitters with real-time analysis offers important insight into the mechanisms that shape animal behavior. However, bioelectronic tools to simultaneously monitor DA and 5-HT interactive dynamics in freely moving animals are underdeveloped. This is mainly due to the limited sensor sensitivity with miniaturized electronics. Here, we present a semi-implantable electrochemical device achieved by integrating a multi-surface-modified carbon fiber microelectrode with a miniaturized potentiostat module to detect DA and 5-HT in vivo with high sensitivity and selectivity. Specifically, carbon fiber microelectrodes were modified through electrochemical treatment and surface coatings to improve sensitivity, selectivity, and antifouling properties. A customized, lightweight potentiostat module was developed for untethered electrochemical measurements. Integrated with the microelectrode, the microsystem is compact (2.8 × 2.3 × 2.1 cm) to minimize its impacts on animal behavior and achieved simultaneous detection of DA and 5-HT with sensitivities of 48.4 and 133.0 nA/μM, respectively, within submicromolar ranges. The system was attached to the crayfish dorsal carapace, allowing electrode implantation into the heart of a crayfish to monitor DA and 5-HT dynamics, followed by drug injections. The semi-implantable biosensor system displayed a significant increase in oxidation peak currents after DA and 5-HT injections. The device successfully demonstrated the application for in vivo simultaneous monitoring of DA and 5-HT in the hemolymph (i.e., blood) of freely behaving crayfish underwater, yielding a valuable experimental tool to expand our understanding of the comodulation of DA and 5-HT.