PROJECT SUMMARY How does chemical signaling in the brain control function? Answering this question requires fast sensors to measure at the synapse. Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes (CFMEs) has enabled in vivo detection of neuromodulators. However, most sensors are too big to measure at the synapse and there are challenges to distinguish neurochemicals and monitor multiple neuromodulators simultaneously. Thus, new sensor technology is needed to target the synapse and measure multiple neuromodulators in real- time. In the previous period, our lab developed new approaches to electrode development, including testing new carbon nanomaterials and 3D printing nanolithography. However, these methods have not been customized to meet the experimental requirements for emerging applications in neurochemical research. The goal of this project is to develop customized carbon electrodes and tune their properties for applications at the synapse, including (1) nanoelectrodes for monoamine detection in the Drosophila neuromuscular junction (NMJ) synapse, (2) trapping electrodes for highly sensitive and selective measurements of neuropeptides in Drosophila NMJ, and (3) a microelectromechanical systems (MEMS) platform for multianalyte detection of dopamine and glutamate simultaneously in vivo and octopamine and glutamate simultaneously in the Drosophila NMJ. This work is significant because it will transform microelectrode design to facilitate complex measurements of neurochemistry that will lead to a better understanding of neurochemical signaling at the synapse. In Aim 1, we will create practical nanoelectrodes for measurements in smaller organisms by coating etched metal wires with carbon nanospikes and 3D printing long nanofibers through shrinkage-induced pulling. These small, less than 200-500 nm nanoelectrodes will be used to measure octopamine in the Drosophila NMJ synapse. In Aim 2, we will design electrodes with trapping effects to improve sensitivity and selectivity. These carbon nanotube (CNT) yarn electrodes and 3D printed electrodes with arrays of carbon pillars will be used to measure neuropeptides in the Drosophila NMJ. In Aim 3, we will develop a Si-based platform for biosensors and direct electrochemistry, enabling multianalyte measurements. The Si-cantilever microneedle will be implantable in vivo and in Drosophila NMJ for simultaneous measurements of neurotransmitters. The proposed research is innovative because it uses new technology to radically change electrode fabrication and enable novel electrode designs. This work will demonstrate proof of principle that these electrodes are capable of measuring many neuromodulators in a model synapse Drosophila NMJ as well as in vivo. With a focus on easy, batch fabrication, these electrodes will be made available to the neuroscience community, to facilitate studies of real-time neuromodulation and how it malfunctions during disease.