Our primary goal is to develop nanoscale analytical techniques and explore their use in the measurement of neurotransmitters and hormones both in vivo and at single cells. This goal requires development of techniques capable of measuring zeptomole (10-21 mole) quantities in chemically complex nanoliter samples.
For in vivo measurements, we are coupling capillary zone electrophoresis (CZE) and capillary liquid chromatography (LC) with in vivo sampling methods. The sampling probes continuously remove samples which are periodically and automatically injected into the separation column. In one project, we have developed a 1 s CZE separation of several neurotransmitters which allows high resolution monitoring of neurotransmitter activity. Another project, just underway, involves combining capillary LC with mass spectrometry to discover novel neurotransmitters.
In the area of single cell analysis, we have focused on development of microsensors and confocal fluorescence microscopy for measurement of insulin secreted from cells with millisecond resolution. We are also pursuing analytical methods that allow simultaneous detection of intracellular messengers such as Zn2+, Ca2+, oxygen, and glucose at the same cells. The combination of these methods will provide a complete chemical picture of the cell as it functions.
As we begin to understand the dynamics of extracellular signaling molecules, it also becomes important to understand the intracellular chemical changes that result from the interaction of such molecules with receptors. To facilitate these studies, we are developing techniques based on capillary electrophoresis, fluorescence detection, and mass spectrometry to detect and modern intracellular signal transduction. Applications include detection of G-proteins, proteins with SH2 domains, and phosphorylated proteins. Such measurements can be considered directed proteomics measurements.
All of these techniques involve development of novel analytical chemistry as well as instrumentation. Recently, a significant effort has been made in developing microfabricated instruments for micro-total analytical systems (mu TAS) to perform the analyses described above. In addition to the fundamental analytical work, application of these methods to studies of brain and endocrine function as well as diseases such as diabetes and Alzheimer's are underway in various collaborations.
Roman GT, Wang M, Shultz KN, Jennings C, Kennedy RT. "Sampling and Electrophoretic Analysis of Segmented Flow Streams Using Virtual Walls in a Microfluidic Device." Analytical Chemistry, 2008, 80, 8231-8.
Dishinger JF, Kennedy RT. Multiplexed detection and applications for separations on parallel microchips. Electrophoresis. 2008, 29, 3296-305.
Zhang H, Saha J, Byun J, Schin M, Lorenz M, Kennedy RT, Kretzler M, Feldman EL, Pennathur S, Brosius FC 3rd. "Rosiglitazone reduces renal and plasma markers of oxidative injury and reverses urinary metabolite abnormalities in the amelioration of diabetic nephropathy." American Journal of Physiology, Renal Physiol. 2008, 295, F1071-81.
Wang M, Roman GT, Schultz K, Jennings C, Kennedy RT. "Improved temporal resolution for in vivo microdialysis by using segmented flow." Analytical Chemistry, 2008 80, 5607-15.
Pei J, Dishinger JF, Roman DL, Rungwanitcha C, Neubig RR, Kennedy RT. "Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system." Analytical Chemistry, 2008, 80, 5225-31.
Miller BR, Dorner JL, Shou M, Sari Y, Barton SJ, Sengelaub DR, Kennedy RT, Rebec GV. "Up-regulation of GLT1 expression increases glutamate uptake and attenuates the Huntington's disease phenotype in the R6/2 mouse." Neuroscience. 2008, 153, 329-37.
Morioka T., Asilmaz E., Hu J., Dishinger J.F., Kurpad A.J., Elias C.F., Li H., Elmquist J.K., Kennedy R.T., Kulkarni R.N. "Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice." J Clin Invest. 2007, 117, 2860-8.
Roman GT, Kennedy RT. "Fully integrated microfluidic separations systems for biochemical analysis." J Chromatogr A. 2007, 1168, 170-88.
Dishinger J. F. and Kennedy R. T. "Serial immunoassays in parallel on a microfluidic chip for monitoring hormone secretion from living cells". Analytical Chemistry, 2007, 79, 947-954.
Cellar NA, Kennedy RT. "A capillary-PDMS hybrid chip for separations-based sensing of neurotransmitters in vivo." Lab Chip. 2006, 6(9):1205-12.
Shou M, Ferrario CR, Schultz KN, Robinson TE, Kennedy RT. "Monitoring Dopamine in Vivo by Microdialysis Sampling and On-Line CE-Laser-Induced Fluorescence." Analytical Chemistry 2006, 78(19):6717-25.
Wei H, Nolkrantz K, Parkin MC, Chisolm CN, O'callaghan JP, Kennedy RT. "Identification and Quantification of Neuropeptides in Brain Tissue by Capillary Liquid Chromatography Coupled Off-Line to MALDI-TOF and MALDI-TOF/TOF-MS." Analytical Chemistry. 2006 . 78(13):4342-51.
Ueki, K., T. Okada, J. Hu, L.C. W., A. Assmann, G.M. Dahgren, J.L. Peters, J.G. Shackman, M. Zhang, I. Artner, L.S. Satin, R. Stein, M. Holzenberger, R.T. Kennedy, C.R. Kahn, and R.N. Kulkarni, "Total insulin and IGF-1 resistance in pancreatic beta-cells causes overt diabetes. " Nature Genetics, 2006. 38(5):583-8.
Cunliffe, J.M., R.K. Sunahara, and R.T. Kennedy, "Detection of adenylyl cyclase activity using a fluorescent atp substrate and capillary electrophoresis. " Analytical Chemistry, 2006. 78 (6): 1731-1738.
Venton, B.J., T.E. Robinson, and R.T. Kennedy, "Transient changes in nucleus accumbens amino acid concentrations correlate with individual responsivity to the predator fox odor 2,5-dihydro-2,4,5-trimethylthiazoline. " Journal of Neurochemistry, 2006, 96 (1): 236-246.
Sandlin, Z.D., M.S. Shou, J.G. Shackman, and R.T. Kennedy, "Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters. " Analytical Chemistry, 2005. 77 (23): 7702-7708.
Cellar, N.A., S.T. Burns, J.C. Meiners, H. Chen, and R.T. Kennedy, "Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids . " Analytical Chemistry, 2005. 77 (21): 7067-7073.