Development of an On-Animal Separation Based Sensor using On-line Microdialysis Sampling Coupled to Microchip Electrophoresis with Electrochemical Detection

Abstract

Microdialysis is a sampling technique that can be employed for the continuous monitoring of compounds both in vivo and in vitro. The online-coupling of microdialysis to microchip electrophoresis provides an attractive technology for near real time monitoring of drugs and neurotransmitters in pharmacokinetic and behavioral studies. These on-line systems for the analysis of microdialysis samples allow for the development of selective and sensitive separation based sensors with the capability of preserving high temporal resolution. Electrochemical detection is well suited for these separation-based sensors due to the possibility of integrating the working and reference electrodes directly into the chip as well as the availability of a miniaturized isolated potentiostat. This dissertation primarily focuses on the development of an on-animal separation-based sensor using microdialysis coupled to microchip electrophoresis with amperometric detection. The system consists of an on-line interface to couple the microdialysis to microchip electrophoresis, high voltage power supplies, and an electrically isolated potentiostat. Initial studies were focused on developing and fabricating an all glass microfluidic device. This system was evaluated in vitro for the continuous monitoring of the enzymatic production of hydrogen peroxide. The system was optimized for the in vivo analysis of nitrite on a freely roaming animal. The system incorporates telemetry for remote control, data acquisition, and has been optimized for the continuous on-line analysis of microdialysis samples obtained using a linear probe following nitroglycerin administration. The on-line microdialysis-microchip electrophoresis system was fabricated using an all glass substrate that includes an electrophoresis separation channel, integrated platinum working and reference electrodes, as well as an interface for direct coupling of the chip to the microdialysis probe. This dissertation describes the development of the integrated system including optimization of the electrophoresis conditions, injection of samples into the chip using the on-line microdialysis-microchip electrophoresis interface, and evaluation of the overall ruggedness of the system. The ultimate goal is to use the separation based sensor for on-animal in vivo analysis of drugs and neurotransmitters in order to correlate neurochemistry and/or metabolism with behavior in freely roaming animals.