Project Summary The real-time measurement of neurotransmitters in vivo in living brain is of utmost importance for understanding brain functions in normal and pathological conditions and to improve diagnosis and treatments of neurological and neuropsychiatric diseases. High surface area carbon (HSAC), or nanocarbon, has been considered the ideal material for electrochemical detection of neurotransmitters, due to its outstanding electrochemical properties and chemical inertness. However, HSAC microelectrode arrays (MEAs) are difficult to fabricate, and the extreme environments needed for the nanocarbon synthesis limit the choice of substrate to rigid materials that can withstand high temperatures. Moreover, chemical doping to improve electrochemical sensing also requires high-temperature post-synthesis processing. Thus, there is an unmet need for fabricating implantable HSAC MEAs on flexible substrates with tunability of morphology and chemistry, for multisite measurements of neurotransmitters at different temporal resolutions (ms to min), within and across brain regions (µm to mm). To fill this gap, this project introduces a new laser-induced nanocarbon (LINC) fabrication technique, capable of patterning customizable types of HSAC on-demand directly on flexible polymers. LINC is a new direct-write process with the unprecedented ability for bottom-up growth of nanocarbons on polymers that act as the carbon source upon laser irradiation. Our inventive approach enables for the first time, a fast, low-cost, batch-fabrication of HSAC MEAs in a highly reproducible way, without the need of high-temperature carbon synthesis, or multistep microfabrication processes. Importantly, LINC allows in situ precise control of the nanocarbon atomic structure, nanoscale morphology, and surface chemistry. Thus, our HSAC MEAs will be tailored for high-sensitivity electrochemical detection of different neurotransmitters using two different electrochemical technique: fast scan cyclic voltammetry (FSCV), for capturing of fast phasic dynamics, and square wave voltammetry (SVW) for detecting tonic levels. Following a meticulous in vitro optimization, we will determine the effectiveness of the proposed HSAC MEA in performing electrochemical sensing of electroactive neurotransmitters for acute in vivo detection of 1) tonic (via SWV) and 2) electrically evoked (via FSCV) dopamine and serotonin release in the rat dorsal striatum and in the hippocampus (CA2 region) of rat brain, respectively or simultaneously. The successful completion of this project will provide 1) a cutting-edge technology with the potential to revolutionize the state- of-the-art of nanocarbon-based MEA fabrication for neurochemical applications, and 2) will provide the scientific community with a platform for unprecedented studies of neurotransmitters and their interactions in normal and pathological brain conditions.