In vivo neurochemical monitoring with high temporal and spatial resolution using segmented flow microfluidics.

摘要

Monitoring the concentration dynamics of neurochemicals in the brain is a key tool in the effort to understand brain function, diseases, and treatments. A versatile and effective approach for in vivo monitoring of chemical messages is to couple sampling methods, such as microdialysis, to analytical measurements. Although this approach has proven invaluable, it is limited by poor temporal and spatial resolution. Temporal resolution is limited to about 60 s by the dispersion of sampled concentration zones during transportation. Spatial resolution is limited by the size of microdialysis probes which are typically 200 to 500 microm diameter and 1--4 mm long. As a result, some rapid chemical changes cannot be detected and the method cannot be used to probe smaller brain nuclei. To address the temporal resolution problem, a microfluidic device was developed that segmented a sample stream into plugs separated by fluorinated oil to prevent temporal distortion during transport. To quantify the chemical contents of the plugs, systems were developed for enzymatic and electrophoretic analysis. For enzyme assay, reagents were added to the plugs as they were formed and detected by fluorescence. For electrophoresis, a microfluidic device was developed that extracted plugs from the segmented flow stream and then injected them into a narrow channel for electrophoretic separation. The sampling and analysis systems allowed temporal resolution as good as 2 s. Alternatively, a capillary-based electrophoresis system could also be used to analyze dialysate plugs through a simple interface with 14 s on-line temporal resolution and robust performances. To address the spatial resolution problem, the segmented flow analysis approach was coupled with miniauturized microdialysis probe with 0.5 mm sampling length. This system improved spatial resolution 2--8 fold over regular microdialysis while maintaining temporal resolution of 10--15 seconds. Dynamics of glucose and neuroactive amino acids were monitored in vivo with both anesthetized and freely-moving rats during pharmacological manipulations. These applications not only validated the robustness of this system, but also revealed different releasing pattern of taurine upon long term and short puffs of potassium infusion due to unprecedented spatial and temporal resolution it could offer.