Integration of microdialysis sampling and microchip electrophoresis with electrochemical detection for monitoring cellular release

In this dissertation, the fabrication, optimization, and application of a microfluidic device that integrates microdialysis (MD) sampling, microchip electrophoresis (ME), and electrochemical detection (EC) in an on-line fashion is described and this device is used to monitor neurotransmitter release from PC 12 cells. Dual carbon ink microelectrodes were first created and fully characterized for integration with microchip electrophoresis. It was then shown that microdialysis sampling could be reproducibly coupled with ME through the use of a bilayer poly(dimethylsiloxane)-based microchip that offered fixed alignment of MD/ME and ME/EC interfaces. Pneumatic valves were used for the discrete injection of sample from the hydrodynamic MD dialysate stream into a ME channel. The system was used with dual electrodes allowing detection of both species of a neurotransmitter redox couple. To enable the integration of ME with EC detection, a palladium decoupler was used to isolate the high voltages associated with electrophoresis from micron-sized carbon ink electrodes. Using this system with high salt content biological buffers proved to be a challenge. Multiple optimization procedures of the ME/EC interface were performed to enable the use of biologically appropriate perfusate buffers. The ability of the MD/ME/EC device to sample a biological system was demonstrated by using a linear probe to monitor the stimulated release of dopamine from a confluent layer of PC 12 cells. This was the first known report of the successful coupling of microdialysis and microchip electrophoresis with electrochemical detection. Norepinephrine release, however, was not detected from the cells, as the limit of detection and resolution needed improvement. Consequently, the use of multiple electrodes in series and a lengthened separation channel were investigated in order to enhance the electrochemical signal and separation efficiency so that dopamine and norepinephrine release from PC 12 cells can be monitored. While these studies have focused on in vitro applications, the same system can also be adapted for in vivo analysis in the future.