Real-time tracking of neurotransmitter levels in vivo has been technically challenging due to the low spatiotemporal resolution of current methods. Since the imbalance of cortical excitation/inhibition (E:I) ratios are associated with a variety of neurological disorders, accurate monitoring of excitatory and inhibitory neurotransmitter levels is crucial for investigating the underlying neural mechanisms of these conditions. Specifically, levels of the excitatory neurotransmitter L-glutamate, and the inhibitory neurotransmitter GABA, are assumed to play critical roles in the E:I balance. Therefore, in this work, a flexible electrochemical microsensor is developed for real-time simultaneous detection of L-glutamate and GABA. The flexible polyimide substrate was used for easier handling during implantation and measurement, along with less brain damage. Further, by electrochemically depositing Pt-black nanostructures on the sensor's surface, the active surface area was enhanced for higher sensitivity. This dual neurotransmitter sensor probe was validated under various settings for its performance, including in vitro, ex vivo tests with glutamatergic neuronal cells and in vivo test with anesthetized rats. Additionally, the sensor's performance has been further investigated in terms of longevity and biocompatibility. Overall, our dual L-glutamate:GABA sensor microprobe has its unique features to enable accurate, real-time, and long-term monitoring of the E:I balance in vivo. Thus, this new tool should aid investigations of neural mechanisms of normal brain function and various neurological disorders
