Collection, storage, and electrophoretic analysis of nanoliter microdialysis samples collected from awake animals in vivo

Microdialysis sampling is an important tool for chemical monitoring in living systems. Temporal resolution is an important figure of merit that is determined by sampling frequency, assay sensitivity, and dispersion of chemical zones during transport from sampling device to fraction collector or analytical system. Temporal resolution has recently been improved by segmenting flow into plugs, so that nanoliter fractions are collected at intervals of 0.1-2 s, thus eliminating temporal distortion associated with dispersion in continuous flow. Such systems, however, have yet to be used with behaving subjects. Furthermore, long-term storage of nanoliter samples created by segmented flow has not been reported. In this work, we have addressed these challenges. A microdialysis probe was integrated to a plug generator that could be stably mounted onto behaving animals. Long-term storage of dialysate plugs was achieved by collecting plugs into high-purity perfluoroalkoxy tubes, placing the tube into hexane and then freezing at -80A degrees C. Slow warming with even temperatures prevented plug coalescence during sample thawing. As a demonstration of the system, plugs were collected from the striatum of behaving rats using a 0.5-mm-long microdialysis probe. Resulting plugs were analyzed 1-4 days later by chip-based electrophoresis. To improve throughput of plug analysis over previous work, the speed of electrophoretic separation was increased by using forced air cooling and 1-butyl-2,3-dimethylimidazolium tetrafluoroborate as a separation buffer additive, allowing resolution of six neuroactive amino acids in 30 s. Concentration changes induced by K+ microinjections were monitored with 10 s temporal resolution. The improvements reported in this work make it possible to apply segmented flow microdialysis to the study of behaving animals and enable experiments where the analytical system cannot be placed close to the animal.