Project Summary. Our overall objective is to develop new bioanalytical methods for exploring brain chemistry dynamics in vivo. Monitoring the concentration dynamics of neurochemicals in vivo is a vital tool for studying brain function, diseases, and treatments. A versatile approach for in vivo monitoring of brain chemistry is to couple sampling methods, such as microdialysis, to analytical measurements. Although this approach is valuable, its utility is limited by low spatial and temporal resolution. Spatial resolution is important because many brain regions are small. Temporal resolution is important because concentrations of neurotransmitters can change rapidly during behavior. We will develop technology that overcomes these limitations of microdialysis. Current microdialysis probes are assembled from fused silica capillaries and dialysis membranes resulting in probes with 250 µm diameter and 2-4 mm sampling length. We microfabricate probes from Si that are 30 µm thick x 55 µm wide to achieve a 30-fold reduction in probe size and 20-fold improvement in sampling spatial resolution based on membrane area. A 3000-fold improvement in spatial resolution will be achieved by microfabricating push-pull probes where sampling only occurs at the probe tip. Enhanced probes with integrated waveguides for optogenetic experiments will be developed. To achieve high temporal resolution, probes will be equipped with internal microfluidics to segment samples into droplets to prevent broadening of sampled concentration pulses as they are transferred to an analytical system. Better than 1 s temporal resolution is possible. To analyze the nanoliter samples that are collected, we will use nanospray ionization mass spectrometry. Preliminary data shows that pumping nanoliter droplet samples into a nanospray source at flow rates of ~20 nL/min will allow detection of low molecular weight neurotransmitters. For measuring neuropeptides and proteins, which require high sensitivity due to their low interstitial concentrations, we will develop immunoassays using photonic ring resonators to detect antibody-antigen binding. The small size of the ring resonators (30 µm diameter) will enable high mass sensitivity. We estimate that detection limits of <1 pM on < 1 µL samples are possible. These techniques will provide in vivo control and measurement of neurochemistry with unprecedented temporal and spatial resolution. We will perform neuroscience studies as a means of testing the methods and demonstrating their utility to the broader community. Applications include determining: 1) concentration dynamics of α-synuclein, a protein involved in pathophysiology of Parkinson's disease; 2) neurochemical changes that precede an epileptic seizure with the goal of determining early warning biomarkers for uncontrolled epilepsy, and 3) neurochemistry of anxiety in a circuit believe to be involved in eating disorders.