| A microfluidic separations-based sensor was developed to incorporate push-pull perfusion sampling, on-line derivatization, flow-gated injection, electrophoretic separation and fluorescence detection for neurotransmitter monitoring. The device allows in vivo measurements of neurochemical changes with both high temporal and spatial resolution, which is important for uncovering the role of neurotransmission in behavior, pharmacological response, and disease.; First, peptide neurotransmitter candidate activity screening is described to illustrate the previous state-of-the-art in high speed neurotransmitter monitoring. The microdialysis, capillary electrophoresis laser-induced fluorescence (CE-LIF) instrument employed allows for separation of 5 amino acid neurotransmitters (glutamate, aspartate, gamma-aminobutyric acid (GABA), glycine, and taurine) as well as several metabolites in under 10 s. Three of six novel proenkephalin peptides tested elicited a response when perfused into the striatum. While a powerful analytical technique, spatial resolution is limited by the 1-4 mm length of the dialysis probes preventing measurements in all but the largest brain regions of rats. Furthermore, the method requires dedicated personnel to build and operate the instrumentation thus preventing widespread use in the neuroscience community.; A microfluidic device was sought to enable facile operation and portability through miniaturization. Devices were created in poly(dimethylsiloxane) (PDMS) because its tunable elasticity allows the formation of valves and pumps through multilayer soft-lithography. A microfluidic push-pull perfusion sampling device was created and shown to allow on-line sampling with improved spatial resolution over microdialysis. After demonstration of feasibility, other analytical operations were incorporated onto the device including flow-gated injection onto an embedded fused silica capillary, separations in PDMS microchannels, and embedded optical fibers and waveguides for fluorescence excitation and collection of emission.; The final prototype incorporates sampling, derivatization, injection, separation, and detection and is capable of detecting dynamic changes in eight neurotransmitter concentrations simultaneously and serially for up to 4 hours. Separation efficiencies of 500,000 theoretical plates and detection limits between 30 and 60 nM were demonstrated with the device. The capability to deliver pharmacological agents and quantify subsequent changes in the neurotransmitter profile was also demonstrated. Future directions of this technology are also proposed. |
