We have pioneered the development of amperometric detection in narrow-bore capillary electrophoresis and the use of this technique to detect neurotransmitters in the cytoplasm of single nerve cells. The goals of this proposal involve the miniaturization of capillary electrophoresis and development of detection schemes for separation and detection of neurochemicals removed from the cytoplasm of single nerve cells. A major portion of this proposal deals with methods development for the neurochemical experiments. 5-micro(m) i.d. capillaries will be used with amperometric detection and a novel injection scheme to sample the cytoplasm of single invertebrate neurons. The use of 2-micro(m) and even 1-micro(m) i.d. capillaries will be investigated for sampling the cytoplasm of single mammalian cells. Detection schemes based on pulsed amperometry and copper/copper oxide electrodes will be investigated for detecting removed intracellular second messengers. In addition to systems involving electrochemical detection, schemes based on laser fluorescence and electrogenerated chemiluminescence will be examined for detection of neuropeptides. Finally, low-volume capillary electrophoresis will be coupled to flow SIMS TOF MS for acquisition, separation and detection of the major components of cell membranes. This will lead to the development of technology to sample a small segment (2-5 micro(m) diameter) of membrane and provide analysis of localized membrane regions of single cells. The neurochemical goals of this proposal emphasize the study of neurotransmitter and the intracellular second messenger levels in single cell cytoplasm. These experiments are meant to complement and expand our experiments involving dynamic monitoring of neurotransmitters and intracellular messengers in cytoplasm with voltammetry.
