Normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) will be used as on-line detection techniques for ribonucleotides and other neurochemicals preconcentrated by capillary electrophoresis. Raman spectroscopy will allow for identification and quantification of compounds from cell cultures and extracts. Isotachophoresis, with subsequent electrophoresis if needed will be used to preconcentrate compounds to 10-6 - 10-4 M for analysis by SERS and 10-2 - 10-1 M concentration for analysis by normal Raman spectroscopy. Conventional capillaries and microfabricated capillaries will be employed. The SERS system will employ lithographically fabricated silver micro-electrodes, which can be placed at the exit of a capillary in a field-free region. In microfabricated capillaries, the electrode system will be integrated into the separation channel. A locally designed confocal epi-illumination microprobe optical system will be used for Raman spectroscopy. A high throughput spectrograph, charge-coupled detector and a 2 Watt 532 nm solid-state laser will complete the Raman spectroscopy system. The Gran-Schmidt orthogonalization procedure and other statistical tests will be used to identify and quantify target compounds at initial concentrations as low as 10-9 M. Synthetic test mixtures of ribonucleotides will be used for development and testing of the normal Raman spectroscopy system. When initial development of the system is completed, performance in a complex biological matrix will be demonstrated by glutamate measurements in the giant dopamine neuron of the pond snail. The system will be used to study hydrolysis kinetics of ATP to ADP and AMP in cultured chromaffin cells and in chick brain embryo extracts. Presently, only the rapidly hydrolyzed ATP can be conveniently measured and the project is expected to yield more detailed and accurate release rates than are now available. Additionally, ATP/adenosine ratios will be measured in the same matrices. Finally, the system will be used to measure c-AMP release in PC12 cells.
| Timlin, J A; Carden, A; Morris, M D et al. (2000) Raman spectroscopic imaging markers for fatigue-related microdamage in bovine bone. Anal Chem 72:2229-36 |
| Walker 3rd, P A; Morris, M D; Burns, M A et al. (1998) Isotachophoretic separations on a microchip. Normal Raman spectroscopy detection. Anal Chem 70:3766-9 |
| Walker 3rd, P A; Morris, M D (1998) Capillary isotachophoresis with fiber-optic Raman spectroscopic detection. Performance and application to ribonucleotides. J Chromatogr A 805:269-75 |
| Jestel, N L; Morris, M D; O'Brien, W J (1998) Depth-resolved Raman microprobe examination of a commercial dental porcelain exposed to a simulated oral environment. Dent Mater 14:375-82 |
