120) Yang PL, Whelan RJ, Mao YW, et al. "Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis ", ANALYTICAL CHEMISTRY 79 (4): 1690-1695 FEB 15 2007. Abstract.

119) Shackman HM, Shou M, Cellar NA, et al. "Microdialysis coupled on-line to capillary liquid chromatography with tandem mass spectrometry for monitoring acetylcholine in vivo", JOURNAL OF NEUROSCIENCE METHODS 159 (1): 86-92 JAN 15 2007. Abstract.

117) Jameson EE, Pei J, Wade SM, et al. "Capillary electrophoresis assay for G protein-coupled receptor-mediated GTPase activity", ANALYTICAL CHEMISTRY 79 (3): 1158-1163 FEB 1 2007. Abstract

116) Shou MS, Ferrario CR, Schultz KN, et al. "Monitoring dopamine in vivo by microdialysis sampling and on-line CE-laser-induced fluorescence", Analytical Chemistry, 2006 78(19): 6717-6725. Abstract.

115) Celllar NA, Kennedy RT. "A capillary-PDMS hybrid chip for separations-based sensing of neurotransmitters in vivo", Lab On A Chip, 2006 6(9): 1205-1212. Abstract.

114) Wei H, Nolkrantz K, Parkin MC, et al. "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): 4342-4351. Abstract.

113)Cunliffe JM, Sunahara RK, Kennedy RT. "Detection of adenylyl cyclase activity using a fluorescent ATP substrate and capillary electrophoresis", Analytical Chemistry, 2006 78 (6): 1731-1738. Abstract.

112)Edwards JL, Chisolm CN, Shackman JG, Kennedy RT. "Negative mode sheathless capillary electrophoresis electrospray ionization-mass spectrometry for metabolite analysis of prokaryotes", Journal of Chromatography A, 2006 1106 (1-2): 80-88. Abstract.

111) Sandlin ZD, Shou MS, Shackman JG, Kennedy RT. "Microfluidic electrophoresis chip coupled to microdialysis for in vivo monitoring of amino acid neurotransmitters", Analytical Chemistry, 2005 77 (23): 7702-7708. Abstract.

110) Cellar NA, Burns ST, Meiners JC, Chen H, Kennedy RT. "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. Abstract.

109) Wei H, Dean SL, Parkin MC, Nolkrantz K, O'Callaghan JP, Kennedy RT. "Microscale sample deposition onto hydrophobic target plates for trace level detection of neuropeptides in brain tissue by MALDI-MS", Journal of Mass Spectrometry, 2005 40 (10): 1338-1346. Abstract.

108) Parkin MC, Wei H, O'Callaghan JP, Kennedy RT. "Sample-Dependent Effects on the Neuropeptidome Detected in Rat Brain Tissue Preparations by Capillary Liquid Chromatography with Tandem Mass Spectrometry", Analytical Chemistry, 2005 77 (19): 6331-6338. Abstract.

107) Edwards JL, Kennedy RT. "Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI time-of-flight mass spectrometry", Analytical Chemistry, 2005 77 (7): 2201-2209. Abstract.

106) Yang PL, Whelan RJ, Jameson EE, Kurzer JH, Argetsinger LS, Carter-Su C, Kabir A, Malik A, Kennedy RT. "Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-B beta as a model system", Analytical Chemistry, 2005 77 (8): 2482-2489. Abstract.

105) Jameson EE, Roof RA, Whorton MR, Mosberg HI, Sunahara RK, Neubig RR, Kennedy RT. "Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues", Journal of Biological Chemistry, 2005 280 (9): 7712-7719. Abstract

104) Baseski, HM; Watson, CJ; Cellar, NA; Shackman, JG; Kennedy, RT. "Capillary Liquid Chromatography with MS³ for the Determination of Enkephalins in Microdialysis Samples from the Striatum of Anesthetized and Freely-moving Rats", Journal of Mass Spectrometry, 2005, 40 (2), 146-153. Abstract

103) Shackman, JG; Dahlgren, GM; Peters, JL; Kennedy, RT. "Perfusion and Chemical Monitoring of Living Cells on a Microfluidic Chip", Lab on a Chip, 2005, 5(1), 56-63. Abstract

102) Qian, WJ; Peters, JL; Dahlgren, GM; Gee, KR; Kennedy, RT. "Simultaneous Monitoring of Zn2+ Secretion and Intracellular Ca2+ from Islets and Islet Cells by Fluorescence Microscopy", Biotechniques, 2004, 37(6), 922-933. Abstract

101) Whelan, RJ; Sunahara, RK; Neubig, RR; Kennedy, RT. "Affinity Assays Using Fluorescence Anisotropy with Capillary Electrophoresis Separation", Analytical Chemistry, 2004, 76(24), 7380-7386. Abstract

100) Haskins, WE; Watson, CJ; Cellar, NA; Powell, DH; Kennedy, RT. "Discovery and Neurochemical Screening of Peptides in Brain Extracellular Fluid by Chemical Analysis of In Vivo Microdialysis Samples", Analytical Chemistry, 2004, 76 (18): 5523-5533. Abstract

99) Shou, M; Smith, AD; Shackman, JG; Peris, J; Kennedy, RT. "In Vivo Monitoring of Amino Acids by Microdialysis Sampling with On-line Derivatization by Naphthalene-2,3-dicarboxyaldehyde and Rapid Micellar Electrokinetic Capillary Chromatography", Journal of Neuroscience Methods, 2004, 138(1-2): 189-197. Abstract

98) Cunliffe, JM; Liu, Z; Pawliszyn, J; Kennedy, RT. "Use of a Native Affinity Ligand for the Detection of G Proteins by Capillary Isoelectric Focusing with Laser-induced Fluorescence Detection", Electrophoresis, 2004, 25(14), 2319-2325. Abstract

97) Rivera, RM; Dahlgren, GM; Paula, LADE; Kennedy, RT; Hansen, PJ. "Actions of Thermal Stress in Two-cell Bovine Embryos: Oxygen Metabolism, Glutathione and ATP Content, and the Time-course of Development", Reproduction, 2004, 128(1), 33-42. Abstract

96) Smith, A; Watson, CJ; Frantz, KJ; Eppler, B; Kennedy, RT; Peris, J. "Differential Increase in Taurine Levels by Low-dose Ethanol in the Dorsal and Ventral Striatum Revealed by Microdialysis with On-line Capillary Electrophoresis", Alcoholism - Clinical and Experimental Research, 2004, 28 (7), 1028-1038. Abstract

95) Shackman, JG; Watson, CJ; Kennedy, RT. "High-throughput Automated Post-processing of Separation Data", Journal of Chromatography A, 2004, 1040(2), 273-282. Abstract

94) Kulkarni, RN; Roper, MG; Dahlgren, G; Shih, DQ; Kauri, LM; Peters, JL; Stoffel, M; Kennedy RT. "Islet Secretory Defect in Insulin Receptor Substrate 1 Null Mice Is Linked With Reduced Calcium Signaling and Expression of Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA)-2b and -3", Diabetes, 2004, 53, 1517-1525. Abstract

93) Presti, MF; Watson, CJ; Kennedy, RT; Yang, M; Lewis, MH. "Behavior-related alterations of striatal neurochemistry in a mouse model of stereotyped movement disorder", Pharmacology Biochemistry and Behavior, 2004, 77(3), 501-507. Abstract

92) Wei, H; Nolkrantz, K; Powell, DH; Woods, JH; Ko, MC; Kennedy, RT. "Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits", Rapid Communications in Mass Spectrometry, 2004, 18(11), 1193-1200. Abstract

91) Roper, MG; Shackman, JG; Dahlgren, GM; Kennedy, RT. "Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay", Analytical Chemistry, 2003, 75(18), 4711-4717. Abstract

90) Smith, A; Watson, CJ; Kennedy, RT; Peris, J. "Ethanol-induced taurine efflux - Low dose effects and high temporal resolution", Advances in Experimental Medicine and Biology, 2003, 526, 485-492. Abstract

89) Jameson, EE; Cunliffe, JM; Neubig, RR; Sunahara, RK; Kennedy, RT. "Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue", Analytical Chemistry, 2003, 75(16), 4297-4304. Abstract

88) Qian, WJ; Gee, KR; Kennedy, RT. "Imaging of Zn2+ release from pancreatic beta-cells at the level of single exocytotic events", Analytical Chemistry, 2003, 75(14), 3468-3475. Abstract

87) Deng, Q; Kauri, LM; Qian, WJ; Dahlgren, GM; Kennedy, RT. "Microscale determination of purines in tissue samples by capillary liquid chromatography with electrochemical detection", The Analyst, 2003, 128(8), 1013-1018. Abstract

86) Deng, Q; Watson, CJ; Kennedy, RT. "Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo", Journal of Chromatography A, 2003, 1005(1-2), 123-130. Abstract

85) Buchanan, DD; Jameson, EE; Perlette, J; Malik, A; Kennedy, RT. "Effect of buffer, electric field, and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis", Electrophoresis, 2003, 24(9), 1375-1382. Abstract

84) Kauri, L.M.; Jung, S.K.; and Kennedy, R.T. "Direct Measurement of Glucose Gradients and Mass Transport Within Islets of Langerhans", Biochemical and Biophysical Research Communications, 2003, 304(2), 371-377. Abstract

83) McKenzie, J.A.M.; Watson, C.J.; Rostand, R.D.; German, I.; Witowski, S.R.; and Kennedy, R.T. "Automated Capillary Liquid Chromatography for Simultaneous Determination of Neuroactive Amines and Amino Acids", Journal of Chromatography A, 2002, 962, 105-115. Abstract

82) Kennedy, R.T.; Thompson, J.E.; and Vickroy, T.W. "In Vivo Monitoring of Amino Acids by Direct Sampling of Brain Extracellular Fluid at Ultralow Flow Rates and Capillary Electrophoresis", Journal of Neuroscience Methods, 2002, 114(1), 39-49. Abstract

81) Roper, M.G.; Qian, W.J.; Zhang, B.B.; Kulkarni, R.N.; Kahn, C.R.; and Kennedy, R.T. "Effect of the Insulin Mimetic L-783,281 on Intracellular [Ca2+] and Insulin Secretion From Pancreatic Beta-cells", Diabetes, 2002, 51, S43-S49. Abstract

80) Kennedy, R.T.; Kauri, L.M.; Dahlgren, G.M.; and Jung, S.K. "Metabolic Oscillations in Beta-cells", Diabetes, 2002, 51, S152-S161. Abstract

79) Deng, Q.; German, I.; Buchanan, D.; and Kennedy, R.T. "Retention and Separation of Adenosine and Analogues by Affinity Chromatography With an Aptamer Stationary Phase", Analytical Chemistry, 2001, 73(22), 5415-5421. Abstract

78) Haskins, W.E.; Wang, Z.Q.; Watson, C.J.; Rostand, R.R.; Witowski, S.R.; Powell, D.H.; and Kennedy, R.T. "Capillary LC-MS2 at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected In Vivo", Analytical Chemistry, 2001, 73(21), 5005-5014. Abstract

77) German, I.; Roper, M.G.; Kalra, S.P.; Rhinehart, E.; and Kennedy, R.T. " Capillary Liquid Chromatography of Multiple Peptides with On-line Capillary Electrophoresis Immunoassay Detection", Electrophoresis, 2001, 22(17), 3659-3667. Abstract

76) Qian, W.J. and Kennedy, R.T. "Spatial Organization of Ca2+ Entry and Exocytosis in Mouse Pancreatic Beta-cells", Biochemical and Biophysical Research Communications, 2001, 282(2), 315-321. Abstract

75) Behar, T.N.; Smith, S.V.; Kennedy, R.T.; Mckenzie, J.M.; Maric, I.; and Barker, A.L. "GABA(B) Receptors Mediate Motility Signals for Migrating Embryonic Cortical Cells", Cerebral Cortex, 2001, 11(8), 744-753. Abstract

74) Bowser, M.T.; and Kennedy, R.T. ”In Vivo Monitoring of Amine Neurotransmitters using Microdialysis with On-line capillary electrophoresis”, Electrophoresis, 2001, 22(17), 3668-3676.  Abstract

73) Witowski, S.R.; Vickroy, T.W.; and Kennedy, R.T. ”Regulation of Synaptic Glutamate Overflow in Hippocampus Following Perforant Path Stimulation In Vivo: Evidence for Volume Transmission”, submitted.  Abstract

72) German, I. and Kennedy, R.T. ”Reversed-phase Capillary Liquid Chromatography Coupled On-line to Capillary Electrophoresis Immunoassays”, Analytical Chemistry, 2000, 72, 5365-5372.  Abstract

71) Boyd, B.W.; Kennedy, R.T. ”Automated Capillary Liquid Chromatography for High Sensitivity Determination of Amino Acids”, Journal of Microcolumn Separations, 2001, 13, 24-32.  Abstract

70) Aspinwall, C.A.; Qian, W.; Roper, M.; Kahn, C.R.; Kulkarni, R.; and Kennedy, R.T. “Roles of insulin receptor substrate-1, phophatidyl-inositol-3-kinase, and release of intracellular Ca2+ in insulin-stimulated insulin secretion”, Journal of Biological Chemistry, 2000, 275, 22331-22338.  Abstract

69) Jung, S.-K.; Kauri, L.; Qian, W.; and Kennedy, R.T. “Correlated oscillations in glucose consumption, oxygen consumption, and intracellular Ca2+ in single islets of Langerhans”, Journal of Biological Chemistry, 2000, 275, 6642-6650. Abstract

68) German, I. and Kennedy, R.T. “Rapid simultaneous determination of glucagon and insulin by capillary electrophoresis immunoassays”, Journal of Chromatography B, 2000, 742, 353-362.  Abstract

67) Boyd, B.W.; Witowski, S. R. and Kennedy, R.T. “Trace-level amino acid analysis by capillary liquid chromatography and application to in vivo microdialysis sampling with 10-s temporal resolution”, Analytical Chemistry, 2000, 72, 865-871.  Abstract

66) Qian, W.; Aspinwall, C. A.; Kennedy, R.T. “Detection of secretion from single pancreatic beta-cells using extracellular fluorogenic reactions and confocal fluorescence microscopy” Analytical Chemistry, 2000, 72, 711-717. Abstract

65) Jhaveri, S.D.; Kirby, R.; Conrad, R.; Maglott, E.J.; Bowser, M.; Kennedy R.T.; Glick, G.; Ellington, A.D. “Designed signaling aptamers that transduce molecular recognition to changes in fluorescence intensity”, Journal of the American Chemical Society, 2000, 122, 2469-73.  Abstract

64) Heegaard, N.H.H. and Kennedy, R.T. “Identification, Quantitation, and Characterization of Biomolecules by Capillary Electrophoretic Analysis of Binding Interactions”, Electrophoresis, 1999, 20, 3122-3133.  Abstract

63) Witowski, S. R.; Kennedy, R. T. “Pressure and Electroosmotically-Driven Flow in Capillaries Packed with Non-Porous Particles for High Speed Separations” Journal of Microcolumn Separations, 1999, 11, 723-728.  Abstract

62) Huang, L.; Aspinwall, C.A., and Kennedy, R. T. “Comparison of amperometric methods for detection of exocytosis from single pancreatic beta-cells”, Analytical Chemistry, 1999, 71, 5551-5556.  Abstract

61) Paras, C.; Lakey, J.R.T.; Tan, W.H.; and Kennedy, R.T. “Localized and compound exocytosis detected by spatially-resolved amperometry at single pancreatic b-cells” Cell Biochemistry and Biophysics, 2000, 33, 227-240. Abstract

60) Lakey, J.R.T.; Aspinwall, C.A.; Cavanagh, T.J.; Kennedy, R.T. “Secretion from islets and single islet cells following cryopreservation”, Cell Transplant, 1999, 8, 691-698.  Abstract

59) Jung, S. K.; Aspinwall, C. A.; Kauri, L.; Gorski, W.; Kennedy, R. T. “Oxygen microsensor and its application to single cells and single islets of Langerhans”, Analytical Chemistry, 1999, 71, 3642-3649.  Abstract

58) Kennedy, R. T. “Bioanalytical Applications of Fast Capillary Electrophoresis” Analytica Chimica Acta, 1999, 400, 163-180.  Abstract

57) Kennedy, R.T.; German, I.; Thompson, J.E.; Witwoski, S. “Fast analytical scale separations by liquid chromatography and capillary electrophoresis”, Chemical Reviews, 1999, 99, 3081.  Abstract

56) Battersby, T.R.; Darwin, Ang; Burgstaller, P.; Held, H.A.; Jurczyk, S.; Bowser, M.T.; Buchanan, D.D.; Kennedy, R.T. and Benner, S.A. “Quantitative analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog” Journal of the American Chemical Society, 1999, 121, 9781-9789.  Abstract

55) Thompson, J. E. and Kennedy, R.T. “Rapid determination of aspartate enantiomers in tissue samples by microdialysis coupled on-line with capillary electrophoresis” Analytical Chemistry, 1999, 71, 2379-2385.  Abstract

54) Sung-Kwon Jung; Aspinwall, C. A.; Kennedy, R. T. “Detection of Multiple Patterns of Oscillatory Oxygen Consumption in Single Islets of Langerhans” Biochemical and Biophysical Research Communications, 1999, 259, 331-336. Abstract

53) Aspinwall, C. A.; Lakey, J. T. and Kennedy, R.T. “Insulin stimulated insulin secretion at single pancreatic b-cells”, Journal of Biological Chemistry, 1999, 274, 6360-6365.  Abstract

52) Shen, H.; Witowski, S.; Boyd, B. W.; and Kennedy, R.T. “Detection of peptides by precolumn derivatization with biuret reagent and preconcentration on capillary liquid chromatography columns with electrochemical detection” Analytical Chemistry, 1999, 71, 987-994.  Abstract

51) Tao, L. and Kennedy, R.T. “Laser-induced fluorescence detection in microcolumn separations”, Trends in Analytical Chemistry, 1998, 17, 484-491.  Abstract

50) Boyd, B. W.; and Kennedy, R.T. “Determination of trace level g-amino butyric acid using an improved OPA pre-column derivatization and on-column preconcentration capillary liquid chromatography with electrochemical detection”, The Analyst, 1998, 123, 2119-2124.  Abstract

49) German, I.; Buchanan, D.; and Kennedy, R.T. “Aptamers as ligands in affinity probe capillary electrophoresis”, Analytical Chemistry, 1998, 70, 4540-4545.  Abstract

48) Lakey J.R.T.; Aspinwall C.A., Cavanagh T.J. and Kennedy, R.T. “Effect of cryopreservation on canine islet insulin secretion as measured by amperometric techniques”, Transplantation Proceedings, 1998 30, 382-382.  Abstract

47) Tao, L.; Thompson, J. T.; and Kennedy R.T. “Optically-Gated Capillary Electrophoresis of o-Phthaldehyde/b-Mercaptoethanol Derivatives of Amino Acids for Chemical Monitoring”, Analytical Chemistry, 1998, 70, 4015-4022. Abstract

46) Tao, L. and Kennedy, R. T. “On-line Competitive Immunoassay based on Capillary Electrophoresis Applied to Monitoring Insulin Secretion from Single Islets of Langerhans”, Electrophoresis, 1998, 19, 403-408. Abstract

45) Brooks, S. A. and Kennedy, R. T. “A Microfabricated Flow-Through Cell with Parallel-Opposed Electrodes for Recycling Amperometric Detection”, Journal of Electroanalytical Chemistry, 1997, 436, 27-34.  Abstract

44) Lada, M.W.; Vickroy, T.W.; and Kennedy, R.T. “Evidence for Neuronal Origin and Metabotropic Receptor-Mediated Regulation of Extracellular Glutamate and Aspartate in Rat Striatum in Vivo Following Electrical Stimulation of the Prefrontal Cortex”, Journal of Neurochemistry, 1998, 70, 617-625. Abstract

43) Aspinwall, C.A.; Brooks, S.A.; Lakey, J.R.T.; and Kennedy, R.T. “Effects of Intravesicular H+ and Extracellular H+ and Zn2+ on Insulin Secretion in Pancreatic b-Cells”,  Journal of Biological Chemistry, 1997, 272, 31308-31314.  Abstract

42) Lada, M.W.; Vickroy, T.W.; and Kennedy, R.T. “High Temporal Resolution Monitoring of Glutamate and Aspartate in Vivo Using Microdialysis On-line with Capillary Electrophoresis with Laser-Induced Fluorescence Detection”, Analytical Chemistry, 1997, 69, 4560-4565.  Abstract

41) Shen, H.; Lada, M.W.; and Kennedy, R.T. “Monitoring of Met-Enkephalin in Vivo with 5-Min Temporal Resolution Using Microdialysis Sampling and Capillary Liquid Chromatography with Electrochemical Detection”, Journal of Chromatography B, 1997, 704, 43-52.  Abstract

40) Tao, L. and Kennedy, R.T. “Measurement of antibody-antigen dissociation constants using fast capillary electrophoresis with laser-induced fluorescence detection”, Electrophoresis, 1997, 18, 112-117.  Abstract

39) Zhang, Q.; Tally, M.; Larsson, O.; Kennedy, R. T.; Huang, L.; Wroblewski, R.; Hall, K.; and Berggren,  P.O., “Insulin-like Growth Factor II Signalling Through the IGF-II/Mannose-6-Phosphate Receptor Directly Promotes Exocytosis in Insulin-Secreting Cells”, Proceedings of the National Academy of Sciences, 1997, 94, 6232-6237.  Abstract

38) Lada, M. W. and Kennedy, R. T. “In Vivo Monitoring of Thiols in Rat Caudate Nucleus Using Microdialysis Coupled by a Flow-Gated Interface with Capillary Electrophoresis”, Journal of Neuroscience Methods, 1997, 72, 153-159.  Abstract

37) Paras, C. and Kennedy, R. T. “Amperometric and Cyclic Voltammetric Detection of Pro-Opiocortin Peptides at the Single Cell Level using Electrochemically Pretreated Carbon Fiber Microelectrodes”, Electroanalysis, 1997, 9, 203-208. Abstract

36) Tao, L. and Kennedy, R. T. “On-line Competitive Immunoassay for Insulin Using Capillary Electrophoresis with Laser-Induced Fluorescence Detection”, Analytical Chemistry, 1996, 68, 3899-3907.  Abstract

35) Gorski, W. and Kennedy, R. T. “Electrocatalyst for Non-Enzymatic Oxidation of Glucose in Neutral Saline Solutions”, Journal of Electroanalytical Chemistry, 1997, 424, 43-48.  Abstract

34) Gorski, W.; Aspinwall, C. A. and Kennedy, R. T. “Carbon Fiber Electrodes Modified with Oxides of Ruthenium as Amperometric Detectors of Insulin Exocytosis at Single Pancreatic b-Cells”, Journal of Electroanalytical Chemistry, 1997, 425, 191-199.  Abstract

33) Lada, M. W. and Kennedy, R. T. “Quantitative In Vivo Monitoring of Primary Amines in Rat Caudate Nucleus Using Microdialysis Coupled by a Flow-Gated Interface with Capillary Electrophoresis-Laser Induced Fluorescence Detection”, Analytical Chemistry, 1996, 68, 2790-2797.  Abstract

32) Shen, H., Aspinwall, C. A. and Kennedy, R. T. “Dual Microcolumn Immunoassay Applied to Determination of Insulin Secretion from Single Islets of Langerhans and Insulin in Serum”, Journal of Chromatography B, 1997, 689, 295-303.  Abstract

31) Finnegan, J. M.; Pihel, K.; Cahill, P. S.; Huang, L.; Zerby, S. E.; Ewing, A. G.; Kennedy, R. T.; and Wightman, R. M., “Vesicular Quantal Size Measured by Amperometry at Chromaffin, Mast, Pheochromocytoma, and Pancreatic b-Cells”, Journal of Neurochemistry, 1996, 66, 1914-1923. Abstract

30) Kennedy, R.T.; Schultz, N.M.; Rose, D.R., “Immunoassays and Enzyme Assays Using CE” in CRC Handbook of Capillary Electrophoresis, (ed. Landers, J.) , CRC Press, 1997, Chapter 22, pp. 523-545.  Abstract

29) Kennedy, R. T.; Aspinwall, C. A., and Huang, L. “Extracellular pH Causes Rapid Release of Insulin from Zn-Insulin Precipitates During Exocytosis”, Journal of the American Chemical Society, 1996, 118, 1795-1796.  Abstract

28) Paras, C. D. and Kennedy, R.T. “Electrochemical Detection of Exocytosis at Single Rat Melanotrophs”, Analytical Chemistry, 1995, 67, 3633-3637. Abstract

27) Huang, L.; Shen, H., Atkinson, M.A.; and Kennedy, R.T. “Electrochemical Detection of Exocytosis at Pancreatic b-cells”, Proceedings of the National Academy of Sciences, 1995, 92, 9608-9612.  Abstract

26) Lada, M. W. and Kennedy, R. T. “Quantitative In Vivo Measurements Using Microdialysis On-line with Capillary Zone Electrophoresis”, Journal of Neuroscience Methods, 1995, 63, 147-152.  Abstract

25) Cole, L.J. and Kennedy, R.T. “Selective Preconcentration for Capillary Zone Electrophoresis Using Protein G Immunoaffinity Capillary Chromatography”, Electrophoresis, 1995, 16, 549-556.  Abstract

24) Huang, L. and Kennedy, R.T. “Exploring Single Cell Dynamics using Chemically Modified Microelectrodes”, Trends in Analytical Chemistry, 1995, 14, 158-164. (invited paper)  Abstract

23) Lada, M.W.; Schaller, G.M.; Carriger, M.; Vickroy, T.M.; and Kennedy, R.T. “An On-Line Interface between Capillary Electrophoresis and Microdialysis”, Analytica Chimica Acta, 1995, 307, 217-225. (invited paper)  Abstract

22) Schultz, N.M.; Huang, L.; Kennedy, R.T.; “Capillary Electrophoresis-Based Immunoassay to Determine Insulin Content and Insulin Secretion from Single Islets of Langerhans”, Analytical Chemistry, 1995, 67, 924-929.  Abstract

21) Monnig, C.A.; Kennedy, R.T. “Capillary Electrophoresis”, Analytical Chemistry 1994, 66, 280R-314R (review article).  Abstract

20) Pyo, M.; Maeder, G. M.; Kennedy, R. T.; Reynolds, J. R., “Controlled Release of Biological Molecules from Conducting Polymer Modified Electrodes”, Journal of Electroanalytical Chemistry, 1994, 368, 329-332.  Abstract

19) Cole, L. J.; Schultz, N. M.; Kennedy, R.T., “The Effect of Column Diameter on Plate Height in High Speed Liquid Chromatography Using Pellicular and Perfused Particles in Packed Capillaries”, Journal of Microcolumn Separations, 1993, 5, 433-439.  Abstract

18) Schultz, N. M.; Kennedy, R. T., “Rapid Immunoassays using Capillary Electrophoresis with Fluorescence Detection”, Analytical Chemistry, 1993, 65, 3161-3165.  Abstract

17) Kennedy, R.T.; Huang, L.; Atkinson, M.A.; Dush, P., “Amperometric Monitoring of Chemical Secretions from Individual Pancreatic b-cells”, Analytical Chemistry, 1993, 65, 1882-1887.  Abstract

16) Kennedy, R.T.; Jones, S.J.; Wightman, R.M., “Dynamic Observation of Dopamine Autoreceptor Effects in Rat Striatal Slices”, Journal of  Neurochemistry, 1992, 59, 449-455.  Abstract

15) Zimmerman, J.B.; Kennedy, R.T.; Wightman, R.M., “Rapid O2 Measurements in Rat Caudate Nucleus During Stimulation of the Medial Forebrain Bundle Reflect Changes in Local Cerebral Blood Flow”, Journal of Cerebral Blood Flow and Metabolism, 1992, 12, 629-637.  Abstract

14) Kennedy, R.T.; Jones, S.J.; Wightman, R.M., “Simultaneous Measurement of Dopamine and Oxygen: Coupling of Oxygen Consumption and Neurotransmission”, Neuroscience, 1992, 47, 603-612.  Abstract

13) Wightman, R.M.; Jankowski, J.A.; Kennedy, R.T.; Kawagoe, K.T.; Schroeder, T.J.; Leszczyszyn, D.J.; Near, J.A.; Diliberto, E.J., Jr.; Viveros, O.H. “Resolved Catecholamine Concentration Spikes Correspond to Vesicular Release from Individual Chromaffin Cells”, Proceedings of the National Academy of Sciences USA, 1991, 88, 10754-10758.  Abstract

12) Wiedemann, D.J.; Kawagoe, K.T.; Kennedy, R.T.; Ciolkowski, E.L.; Wightman, R.M., “Strategies for Low Detection Limit Measurements  with Cyclic Voltammetry”, Analytical Chemistry, 1991, 63, 2965-2970. Abstract

11) Wightman, R.M.; Kennedy, R.T.; Wiedemann, D.J.; Kawagoe, K.T.; Zimmerman, J.B.; Leszczyszyn, D.J. “Microelectrodes in Biological Systems”, in Microelectrodes: Theory and Applications. M.I. Montenegro, et al. (eds.) Kluwer Academic Publishers: Netherlands (1991) pp. 453-462.  Abstract

10) Kennedy, R. T.; Jorgenson, J.W., “Efficiency of Packed Microcolumns Compared to Large Bore Packed Columns in Size Exclusion Chromatography”, Journal of Microcolumn Separations, 1990, 2, 120-127.  Abstract

9) Kennedy, R.T.; Oates, M.D.; Cooper, B.R.; Nickerson, B.; Jorgenson, J.W. “Microcolumn Separations and the Analysis of Single Cells”, Science, 1989, 246, 57-63.  Abstract

8) Moseley, A.M.; Deterding, L.; de Wit, J.M., Tomer, K.; Kennedy, R.T.; Jorgenson, J.W., “Optimization of a Coaxial Continuous Flow Fast Atom Bombardment Interface between Capillary Liquid Chromatography and Magnetic Sector Mass Spectrometry for the Analysis of Biomolecules”, Analytical Chemistry, 1989, 61, 1577-1584.  Abstract

7) Kennedy, R.T.; Jorgenson, J.W., “Preparation and Evaluation of Packed Capillary Liquid Chromatography Columns with Inner Diameters of 20 to 50 Microns”, Analytical Chemistry, 1989, 61, 1128-1135.  Abstract

6) Kennedy, R.T.; Jorgenson, J.W., “Quantitative Analysis of Single Neurons by Open Tubular Liquid Chromatography with Voltammetric Detection”, Analytical Chemistry, 1989, 61, 441-446.  Abstract

5) Geng, L; Reed, R.A.; Kim, M.-H.; Wooster, T.T.; Oliver, B.N.; Egekeze, J.; Kennedy, R.T.; Jorgenson, J.W.; Parcher, J.F.; Murray, R.W., “Chemical Phenomena in Solid State Voltammetry in Polymer Solvents”, Journal of the American Chemical Society, 1989, 111, 1614-1619.  Abstract

4) Kennedy, R.T.; Jorgenson, J.W., “Pneumatic Microsyringe for Use as a Injector in Open Tubular Liquid Chromatography and as a Dispenser in Microanalysis”, Analytical Chemistry, 1988, 60, 1521-1524.  Abstract

3) Jorgenson, J.W.; Kennedy, R.T.; St. Claire, R.L.; White, J.G.; Dluzneski, P.R.; de Wit, J.S.M. “Open Tubular Liquid Chromatography and the Analysis of Single Neurons”, Journal of Research of the National Bureau of Standards, 1988, 93, 403-406.  Abstract

2) Jorgenson, J.W.; Rose, D.J.; Kennedy, R.T., “Nanoscale Separations and Biotechnology”, American Laboratory, 1988, April, 33-41.  Abstract

1) Kennedy, R.T.; St. Claire, R.L.; White, J.G.; Jorgenson, J.W., “Chemical Analysis of Single Neurons by Open Tubular Liquid Chromatography”, Mikrochimica Acta, 1987, II, 37-45.  Abstract
 
 
 
 

93.
Motor stereotypy is a common component of several developmental, genetic, and neuropsychiatric disorders. In animals, these behaviors can be induced or attenuated via pharmacological manipulation of specific neural loci comprising cortico basal ganglia-cortical feedback circuits, including the striatum. The present study employed the deer mouse model of spontaneous and persistent stereotypy to assess the involvement of several endogenous neurotransmitters and neuromodulators in mediating the expression of the stereotypic behaviors (i.e., repetitive hindlimb jumping) exhibited by these mice. This was accomplished by employing a microdialysis sampling system coupled on-line to capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection apparatus. Given the 13-s temporal resolution for analyte measurement afforded by this system, discrete behavior-related alterations in striatal neurochemical concentrations were detected. Rearing behavior was found to be associated with significant and selective elevations of striatal glutamate (Glu) and aspartate (Asp) concentrations. Moreover, rearing was found to most frequently precede repetitive jumping. The results also indicated that alterations in striatal serine (Ser) concentrations were involved in the modulation of locomotor activity. The present findings support a role of the striatal glutamatergic system in the mediation of spontaneous stereotypic behavior and suggest a potential neuronal mechanism by which transition to stereotypy occurs in these mice. Moreover, the present findings demonstrate the usefulness of the microdialysis system employed in studying the neurochemical substrates of rapidly transitioning behavior.

92.
Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 µm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 µm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for 7-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 µm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was similar to seven-fold worse

91.
A microfluidic device has been developed for the determination of insulin secreted from islets of Langerhans by a capillary electrophoresis competitive immunoassay. Online assays were performed by electrophoretically sampling anti-insulin antibody (Ab), fluorescein isothiocyanate-labeled insulin (FITC-insufin), and insulin from separate reservoirs and allowing them to mix as they traveled through a 4-cm reaction channel heated to 38 °C. From the reaction channel, samples were injected onto a 1.5-cm-long electrophoresis channel where the FITC-insulin and FITC-insulin-Ab complex were separated in 5 s using an electric field of 500 V/cm. Detection limits for insulin were 3 nM in this mode of operation. Assays could be collected at 15-s intervals with continuous sampling and online mixing for up to 30 min with no intervention. Relative standard deviation was 2-6% depending on the insulin concentration. Response time to a step change in insulin concentration was 30 s. For live cell monitoring, single islets were placed into a reservoir on the chip and fluid in the immediate vicinity was continuously sampled to detect insulin secretion from the islet. Monitoring of insulin secretion with electropherograms taken at 15-s intervals resolved secretory profiles characteristic of first- and second-phase insulin secretion. The method should be amenable to other cell or tissue types for measurements of release with high temporal resolution.

90.
Not Available

89.
An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTP?S (BGTP?S), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTP?S was incubated with samples containing G proteins and the resulting mixtures of BGTP?S-G protein complexes and free BGTP?S were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTP?S-G(ao) peak heights increased linearly with G(ao) up to similar to200 nM using a 50 nM BGTP?S probe. The detection limit for G(ao) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (K-d) to be determined. The on-rate and off-rate of BGTP?S to G(ao) were 0.0068+/-0.0004 and 0.00023+/-0.00001 s(-1), respectively. The half-life of the BGTP?S-G(ao) complex was 3060+/-240 s and K-d was 8.6+/-0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTP?S was demonstrated with assays for G(a) and G(a1) and for Ras and Rab3A.

88.
Regulated secretion of Zn2+ from isolated pancreatic beta-cells was imaged using laser-scanning confocal microscopy. In the method, beta-cells were incubated in a solution containing the novel fluorescent Zn2+ indicator FluoZin-3. Zn2+ released from the cells reacted with the dye to form a fluorescent product, which was detected by the confocal microscope. The new dye is much brighter than Zinquin, previously used for this application, allowing detection limits of 10-40 nM and temporal resolution of 16 ms/image. The high temporal resolution allowed imaging of isolated fluorescent transients that occurred at the edge of the cells following stimulation with 20 mM glucose or 40 mM K+. Fluorescent transients took 1650 ms to reach a peak from the initial rise and returned to baseline after 170 +/- 50 ms (n = 78 transients from 15 cells). It was concluded that the transients correspond to detection of exocytotic release of Zn2+. Analysis of the temporal and spatial dispersion of the transients indicates that the release of Zn2+ is not diffusion limited but is instead kinetically controlled in agreement with previous observations of insulin release detected by amperometry.

87.
A microscale method for purines involved in intracellular signaling and energy metabolism, including ADP, ATP, cyclic-AMP, NADH and GTP, was developed. The analytes were separated on a fused-silica capillary liquid chromatography column (50 µm inner diameter by 25 cm long) packed with 7 µm reversed-phase particles and detected with a carbon fiber cylinder microelectrode at +1.50 V versus Ag/AgCl reference electrode. With an acetonitrile gradient, the separation was carried out within 15 min. With a 100 nL injection volume, the detection limits varied from 0.9 to 8 fmol depending upon the analyte. The low detection limits make the method suitable for analysis of small tissue samples. As a demonstration of the method, islets of Langerhans were analyzed for their adenosine-related messenger content.

86.
An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an aqueous mobile phase containing 20 mM Mg2+, adenosine was strongly retained on the column. A gradient of increasing Ni2+ (to 18 mM), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. Up to 6 µl of 1.2 µM adenosine could be injected onto the 150-µm I.D. X 7 cm long column without loss of adenosine. With UV absorbance detection, the detection limit was 30 nM or 120 fmol (4 µl injected). Samples could be repetitively injected with 4.6% relative standard deviation in peak area. Columns were stable to at least 200 injections. The adenosine assay, which required no sample preparation, was used on microdialysis samples collected from the somatosensory cortex of chloral hydrate anesthetized rats. Total analysis times were short enough that dialysate samples could be injected every 5 min. Basal dialysate concentrations of adenosine stabilized at 87 +/- 10 nM (n = 5) with the probe operated at 0.6 µl/min.

85.
The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamerthrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of non-covalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.

84.
A glucose oxidase-based glucose microsensor (<10 mm tip diameter) was used to measure the glucose concentration within single islets under static conditions and during step changes in glucose level. The sensors had response times of 4.1 +/- 0.5 s (n = 7) and sensitivities of 8.7 +/- 1.8 pA/mM (n = 11). The sensors performed independent of oxygen up to 15 mM glucose as long as the oxygen level was >70mm Hg. Spatially resolved glucose measurements revealed a glucose gradient around and inside single islets. From measurement of the glucose gradient, a glucose consumption rate of 0.48 +/- 0.14 pmol/nL islet/min (n = 6) and an intraislet glucose diffusion coefficient of 3.8 x 10(-7) cm(2)/s were determined. The measurement of the gradient demonstrates that not all cells within an islet in culture are exposed to the same glucose concentration. The sensor was also used to measure the time required for intraislet glucose concentration to reach steady state following a step increase in glucose concentration from 3 to 10 mM at the islet surface. At a depth of 70 mum inside an islet, glucose reached steady state in 180 +/- 7 s (n = 7) for islets with a diameter of 180-220 mm (smaller islets reach steady-state faster). In the presence of 10 mM mannoheptulose, an inhibitor of glucokinase, the equilibration time was reduced to 122 +/- 11 s (n = 6), indicating that glucose utilization by glycolysis limited the time required for glucose to diffuse into the islets. The long times to reach steady state and presence of glucose gradients are important in interpreting data from experiments involving islets in culture.

83.
A method for the separation and quantitative determination of neuroactive amino acids (aspartate, glutamate, citrulline, arginine, glycine, taurine, gamma-aminobutyric acid) and neuroactive amines (noradrenaline, dopamine and serotonin) in a single chromatographic analysis is presented. The method is based on pre-column derivatization with o-phthalaldehyde and tert.-butyl thiol, on-column preconcentration and separation using 50 mm I.D. packed capillary columns, and detection by amperometry. Mass limits of detection are 80-900 amol for all neurotransmitters with RSDs of 0.71 and 4.6% or better for retention time and peak area, respectively. The method was demonstrated by application to the determination of neurotransmitters in microdialysis samples collected from striatum of live rats and tissue samples extracted from butterfly brains.

82.
Extracellular levels of glutamate (GLU), aspartate (ASP), glycine (GLY), phosphoethanolarnine (PEA), and gamma-aminobutyric acid (GABA) were measured in the striatum of anesthetized rats using a novel sampling approach in which extracellular fluid (ECF) was removed at 1-50 nl/min using a fused silica capillary tube with 18-40 mm inner diameter and a outer diameter of 90 mm. The samples of ECF were analyzed by capillary electrophoresis with laser-induced fluorescence detection. Basal levels for GABA, GLY, and GLU measured using direct sampling at 1 nl/min were 270 +/- 40, 4950 +/- 1100, and 1760 +/- 150 nM, respectively in good agreement with the values obtained using microdialysis sampling calibrated by the low-flow rate method. ASP levels were approximately four-fold higher in directly sampled fluid than in dialysate. At higher direct sampling flow rates (10-50 nl/min), detected levels of the amino acids were lower by 70-90% indicating depiction of analyte under these conditions. PEA, an indicator of membrane disruption, was 5.5-fold higher in dialysate than in directly sampled ECF indicating greater tissue damage associated with microdialysis. In addition to the basal measurements, the direct sampling technique was applied to monitoring concentration changes of GLU and ASP in the striatum with better than 90 s temporal resolution after perfusion of either 120 mM K+ or 400 muM L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) through a microdialysis probe immediately adjacent to the direct sampling capillary. Levels of GLU and ASP increased 615 +/- 95 and 542 +/- 96%, respectively (n = 4) upon addition of 120 mM K I to the perfusate and 622 +/- 234 and 672 +/- 218% (n = 5) for PDC. It is concluded that direct sampling at low-flow rates allows determination of extracellular levels of the amino acids with spatial resolution that is at least 500-fold better than microdialysis.

81.
L-783,281, an antidiabetic fungal metabolite than has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca2+ ([Ca2+](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 mmol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca2+](i) to 178 +/- 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on beta-cell [Ca2+](i). The L-783,281-evoked [Ca2+](i) increase was reduced by 82 +/- 4% (n = 6, P < 0.001) in cells incubated with 1 mmol/l of the SERCA (sarco/ endoplasmic reticulum calcium ATPase) pump inhibitor thapsigargin and reduced by 33 +/- 6% (n = 6, P < 0.05) in cells incubated with 20 mmol/l of the L-type Ca2+-channel blocker nifedipine. L-783,281-stimulated [Ca2+](i) increases were reduced to 31 +/- 3% (n = 9, P <: 0.05) and 48 +/- 10% (n = 6, P < 0.05) of control values by the phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 (25 mumol/l) and wortmannin (100 nmol/l), respectively. In beta-cells from IRS-1(-/-) mice, 10 mmol/l L-783,281 had no significant effect on [Ca2+](i) (n = 5). L-783,281 also resulted in insulin secretion at single beta-cells. Application of 10 mmol/l L-783,281 for 40 s resulted in 12.2 2.1 (n = 14) exocytotic events as measured by amperometry, whereas the inactive structural analog had no stimulatory effect on secretion. Virtually no secretion was evoked by L-783,281 in IRS-1(-/-) beta-cells. LY294002 (25 mmol/l) significantly reduced the effect of the insulin mimetic on beta-cell exocytosis. It is concluded that L-783,281 evokes [Ca2+](i) increases and exocytosis in beta-cells via an IRS-1/PI3-K-dependent pathway and that the [Ca2+](i) increase involves release of Ca2+ from intracellular stores.

80.
Whereas the mechanisms underlying oscillatory insulin secretion remain unknown, several models have been advanced to explain if they involve generation of metabolic oscillations in beta-cells. Evidence, including measurements of oxygen consumption, glucose consumption, NADH, and ATP/ADP ratio, has accumulated to support the hypothesis that energy metabolism in beta-cells can oscillate. Where simultaneous measurements have been made, these oscillations are well correlated with oscillations in intracellular [Ca2+] and insulin secretion. Considerable evidence has been accumulated to suggest that entry of Ca2+ into cells can modulate metabolism both positively and negatively. The main positive effect of Ca2+ is an increase in oxygen consumption, believed to involve activation of mitochondrial dehydrogenases. Negative feedback by Ca2+ includes decreases in glucose consumption and decreases in the mitochondrial membrane potential. Ca2+ also provides negative feedback by increasing consumption of ATP. The negative feedback provided by Ca2+ provides a mechanism for generating oscillations based on a model in which glucose stimulates a rise in ATP/ADP ratio that closes ATP-sensitive K+ (K-ATP) channels, thus depolarizing the cell membrane and allowing Ca(2+)entry through voltage-sensitive channels. Ca2+ entry reduces the ATP/ ADP ratio and allows reopening of the KATP channel.

79.
A biotinylated-DNA aptamer (molecular weight 16,600) that binds adenosine and related compounds in solution was immobilized by reaction with streptavidin, which had been covalently attached to porous chromatographic supports. The aptamer medium was packed into fused-silica capillaries (50-150-mm i.d.) to form affinity chromatography columns. Frontal chromatography analysis indicated that the dissociation constants (Kd) of cyclic-AMP, AMP, ATP, ADP, and adenosine were 138 +/- 18, 58 +/- 2, 38 +/- 2, 28 +/- 6 and 3 +/- 1 mM, respectively, for aptamer immobilized on a controlled pore glass support. Similar values were obtained for aptamer immobilized on a polystyrene support except for a slightly higher Kd for adenosine. The Kd for adenosine is similar to the previously reported value of 6 +/- 3 mM for adenosine-aptamer in solution indicating that immobilized aptamers can have affinity similar to that of the solution forms. Columns had 20 nmol of binding sites/100 mL of support media, which is 3.3-fold higher than that previously reported for immobilization of IgG on similar media, indicating that the aptamer can be immobilized with higher density than antibodies. Variation of mobile-phase conditions revealed that ionic strength and Mg2+ level had strong effects on retention of analytes while pH and buffer composition had less of an effect. It was demonstrated that the column could selectively retain and separate cyclic-AMP, NAD(+), AMP, ADP, ATP, and adenosine, even in complex mixtures such as tissue extracts.

78.
Fused-silica capillary LC columns (25-mm i.d.) with 3-mm-i.d. integrated electrospray emitters interfaced to a quadrupole ion trap mass spectrometer were evaluated for high-sensitivity LC-MS2. Column preparation involved constructing frits by in situ photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate, preparing the electrospray emitter by pulling the column outlet to a fine tip with a CO2 laser puller, and slurry-packing the column with 5-mm reversed-phase particles. Large-volume injections were facilitated by an automated two-pump system that allowed high-flow rates for sample loading and low-flow rates for elution. Small electrospray emitters, low elution flow rates, and optimization of gradient steepness allowed a detection limit of 4 amol, corresponding to 2 pM for 1.8 mL injected on-column, for a mixture of peptides dissolved in artificial cerebral spinal fluid. The system was coupled on-line to microdialysis sampling and was used to monitor and discover endogenous neuropeptides from the globus pallidus of anesthetized male Sprague-Dawley rats. Time-segmented MS2 scans enabled simultaneous monitoring of Met-enkephalin, Leu-enkephalin, and unknown peptides. Basal dialysate levels of Met-enkephalin and Leu-enkephalin were 60 +/- 30 and 70 +/- 20 pM while K-(+)-stimulated levels were 1900 +/- 500 and 1300 +/- 300 pM, respectively (n = 7). Data-dependent and time-segmented MS2 scans revealed several unknown peptides that were present in dialysate. One of the unknowns was identified as peptide I1-10 (SPQLEDEAKE), a novel product of preproenkephalin A processing, using MS2, MS3, and database searching.

77.
A competitive immunoassay for neuropeptide Y (NPY) based on capillary electrophoresis (CE) with laser-induced fluorescence detection was developed utilizing polyclonal antisera as the immunoreagent and fluorescein-labeled NPY as the tracer. The assay was performed with on-line mixing of reagents, automated injections, and a 3 s separation time. The assay had a detection limit of 850 pm. To detect NPY at lower concentrations, the assay was coupled on-line to reversed-phase capillary liquid chromatography (LC). In this arrangement, 5 mL samples were preconcentrated by capillary LC and eluted by a gradient of isopropanol-containing mobile phase. The resulting chromatographic peaks were monitored by the CE immunoassay. With preconcentration, the concentration detection limit was improved to 40 pm and NPY could be measured in push-pull perfusion samples collected from the paraventricular nucleus of freely moving rats. The technique was extended to simultaneous detection of NPY and glucagon secretion from islets of Langerhans.

76.
Secretion from single pancreatic beta -cells was imaged using a novel technique in which Zn2+, costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta -cells was limited to an active region that comprised similar to 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca2+ concentration ([Ca2+](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca2+](i) at single cells, colocalization of exocytotic release sites and Ca2+ entry was observed when cells were stimulated by glucose or K+. Treatment of cells with the Ca2+ ionophore 4-Br-A23187 induced large Ca2+ influx around the entire cell circumference. Despite the non-localized increase in [Ca2+](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca2+ channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca2+](i), but instead involves spatial localization of other components of the exocytotic machinery

75.
During development, postmitotic neurons migrate from germinal regions into the cortical plate (cp), where lamination occurs. In rats, GABA is transiently expressed in the cp. near target destinations for migrating neurons. In vitro GABA stimulates neuronal motility, suggesting cp cells release GABA, which acts as a chemoattractant during corticogenesis. Pharmacological studies indicate GABA stimulates migration via GABA(B)-receptor (GABA(B)-R) activation. Using immunohistochemistry. RT-PCR and Western blotting, we examined embryonic cortical cell expression of GABA(B)-Rs in vivo. At E17, GABA(B)-R1(+) cells were identified in the ventricular zone (vz) and cp. RT-PCR and Western blotting demonstrated the presence of GABA(B)-R1a and GABA(B)-R1b mRNA and proteins. Using immunocytochemistry, GABA(B)-R expression was examined in vz and cp cell dissociates before and after migration to GABA in an in vitro chemotaxis assay. GABA-induced migration resulted in an increase of GABAs-R+ cells in the migrated population. While <20% of each starting dissociate was GABA(B)-R+. >70% of migrated cells were immunopositive. We used a microchemotaxis assay to analyze cp cell release of diffusible chemotropic factor(s). In vitro, cp dissociates induced vz cell migration in a cell density-dependent manner that was blocked by micromolar saclofen (a GABA(B)-R antagonist). HPLC demonstrated cp cells release micromolar levels of GABA and taurine in several hours. Micromolar levels of both molecules stimulated cell migration that was blocked by micromolar saclofen. Thus, migratory cortical cells express GABA(B)-Rs, cp cells release GABA and taurine, and both molecules stimulate cortical cell movement. Together these findings suggest GABA and/or taurine act as chemoattractants for neurons during rat cortical histogenesis via mechanisms involving GABA(B)-Rs.

74.
Microdialysis sampling was coupled via a flow-gated interface on-line to capillary electrophoresis with laser-induced fluorescence (LIF) detection for in vivo monitoring of neuroactive amino acids and amines. In the instrument, analytes are derivatized precolumn with o-phthaldehyde and beta-mercaptoethanol to form fluorescent isoindole products. The instrument was improved over previous designs by incorporating a sheath-flow cuvette for reduced background in LIF detection which improved sensitivity by 15-fold. The methodology was improved by utilizing a voltage ramped injection which allowed generation of 500,000 theoretical plates with 20 s separations. Resolution of the isoindole derivatives was further improved by addition of hydroxypropyl-modified beta -cyclodextrin to the electrophoresis buffer. The new instrumentation and methods allow resolution and detection of glutamate, gamma -aminobutyric acid, glycine, aspartate, serine, taurine, glutamine and dopamine (if levels are elevated) collected from in vivo sampling probes every 20 s. The technique is suited to continuous monitoring for dynamic measurements of these compounds in vivo.

73.
 
 

72.
Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody.  A competitive CEIA for glucagon was used for demonstration of this technique.  Five-microliter samples were injected onto a 4-cm-long by 50-mum-i.d. RPLC column.  Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and antiglucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection.  Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation.  Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM.  Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay.  The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.
 
 

71.
An automated method for determination of trace level amino acids in 2-muL samples using on-column sample; preconcentration, gradient elution on 50-mum inner diameter (i.d.) capillary columns packed with 5-mum reversed-phase particles, and electrochemical detection is described.  The 50-mum i.d. capillary columns were efficiently coupled directly to an autosampler without increasing zone dispersion by preconcentrating derivatized amino acids at the head of the capillary column and minimizing gradient dwell volume.  Using this system, the relative standard deviations (RSDs) of retention time for a 16 component amino acid mixture were between 0.2 and 0.8%.  Using an automated microinjection method, as little as 0.3 muL of derivatized sample was consumed to perform a 0.25-muL injection with peak area RSDs of 3.0-8.4%, allowing conservation of a majority of the derivatized sample for future analysis.  Precision was improved to peak area RSDs of 1.8-4.1% when consuming 1.0 muL of sample per injection.  Detection limits were < 0.2 nM for most amino acids.  The linear solvent strength theory was used to optimize the gradient and allowed resolution of 15 amino acids in 13 min.  The final gradient separation was demonstrated to be selective for the neurotransmitter amino acids in the presence of 44 primary amines commonly found in physiological samples.  The system was used to characterize amino acid secretion from single pancreatic islets of Langerhans under different physiological conditions with 2 min of temporal resolution.
 
 

70.
The signaling pathway by which insulin stimulates insulin secretion and increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in isolated mouse pancreatic beta-cells and clonal beta-cells was investigated. Application of insulin to single beta-cells resulted in increases in [Ca(2+)](i) that were of lower magnitude, slower onset, and longer lifetime than that observed with stimulation with tolbutamide. Furthermore, the increases in [Ca(2+)](i) originated from interior regions of the cell rather than from the plasma membrane as with depolarizing stimuli. The insulin-induced [Ca(2+)](i) changes and insulin secretion at single beta-cells were abolished by treatment with 100 nm wortmannin or 1 micrometer thapsigargin; however, they were unaffected by 10 micrometer U73122, 20 micrometer nifedipine, or removal of Ca(2+) from the medium. Insulin-stimulated insulin secretion was also abolished by treatment with 2 micrometer bisindolylmaleimide I, but [Ca(2+)](i) changes were unaffected. In an insulin receptor substrate-1 gene disrupted beta-cell tumor line, insulin did not evoke either [Ca(2+)](i) changes or insulin secretion. The data suggest that autocrine-activated increases in [Ca(2+)](i) are due to release of intracellular Ca(2+) stores, especially the endoplasmic reticulum, mediated by insulin receptor substrate-1 and phosphatidylinositol 3-kinase. Autocrine activation of insulin secretion is mediated by the increase in [Ca(2+)](i) and activation of protein kinase C.
 
 

69.
Micron-sized sensors were used to monitor glucose and oxygen levels in the extracellular space of single islets of Langerhans in real-time. At 10 mM glucose, oscillations in intraislet glucose concentration were readily detected. Changes in glucose level correspond to changes in glucose consumption by glycolysis balanced by mass transport into the islet. Oscillations had a period of 3.1 +/- 0.2 min and amplitude of 0.8 +/- 0.1 mM glucose (n = 21). Superimposed on these oscillations were faster fluctuations in glucose level during the periods of low glucose consumption. Oxygen level oscillations that were out of phase with the glucose oscillations were also detected. Oscillations in both oxygen and glucose consumption were strongly dependent upon extracellular Ca(2+) and sensitive to nifedipine. Simultaneous measurements of glucose with intracellular Ca(2+) ([Ca(2+)](i)) revealed that decreases in [Ca(2+)](i) preceded increases in glucose consumption by 7.4 +/- 2.1 s during an oscillation (n = 9). Conversely, increases in [Ca(2+)](i) preceded increases in oxygen consumption by 1.5 +/- 0.2 s (n = 4). These results suggest that during oscillations, bursts of glycolysis begin after Ca(2+) has stopped entering the cell. Glycolysis stimulates further Ca(2+) entry, which in turn stimulates increases in respiration. The data during oscillation are in contrast to the time course of events during initial exposure to glucose. Under these conditions, a burst of oxygen consumption precedes the initial rise in [Ca(2+)](i). A model to explain these results is described.
 
 

68.
A rapid capillary electrophoresis (CE) with laser-induced fluorescence (LIF) competitive immunoassay has been developed for the determination of glucagon in biological mixtures. In the assay, fluorescein-conjugated glucagon is mixed with the sample followed by addition of anti-glucagon. Free and antibody-bound, tagged glucagon could be separated in 3 s using CE to obtain quantitative determination of glucagon with a concentration detection limit of 760 pM. The assay was combined with a previously developed competitive immunoassay for insulin to produce a simultaneous immunoassay for both peptides. The method was used to determine glucagon content of islets of Langerhans.
 
 

67.
A sensitive method was developed to determine 16 amino acids, including all the neurotransmitter amino acids and neuromodulators, in physiological samples. Samples were derivatized with o-phthalaldehyde/tert-butyl thiol followed by two scavenging reactions that reduced the chemical background caused by excess derivatization reagent by approximately 90%. A total of 250 nL of the derivatized sample was injected and concentrated onto a 50-micron-inner diameter capillary column packed with 5-micron reversed-phase particles and separated using gradient elution. Analytes were detected amperometrically at a cylindrical 9-micron carbon fiber microelectrode. The combination of on-column concentration, scavenging reactions after derivatization, high sensitivity electrochemical detection, and protocols to minimize amine contamination allowed detection limits of 90-350 pM (20-80 amol) for all the amino acids tested. This method was used to analyze in vivo microdialysate samples from probes implanted in the striatum of anesthetized rats. Probes were perfused at 1.2 microL/min and fractions collected every 10 s. The 200-nL fractions were diluted to 2 microL to facilitate sample handling for off-line analysis. The suitability of this method for simultaneous monitoring of all the major amino acid neurotransmitters with 10-s temporal resolution under basal conditions, during potassium stimulation, and during selective uptake inhibition of gamma-aminobutyric acid is demonstrated.
 
 

66.
Confocal microscopy with Zinquin, a fluorogenic Zn(2+)-specific indicator, was used for spatially and temporally resolved measurement of Zn2+ efflux from single pancreatic beta-cells. When cells were incubated in buffer containing Zinquin, application of insulin secretagogues evoked an increase in fluorescence around the surface of the cell, indicative of detection of Zn2+ efflux from the cell. The fluorescence increases corresponded spatially and temporally with measurements of exocytosis obtained simultaneously by amperometry. When images were taken at 266-ms intervals, the detection limit for Zn2+ was approximately 0.5 microM. With this image frequency, it was possible to observe bursts of fluorescence which were interpreted as fluctuations of Zn2+ level due to exocytosis. The average intensity of these fluorescence bursts corresponded to a Zn2+ concentration of approximately 7 microM. Since insulin is co-stored with Zn2+ in secretory vesicles, it was concluded that the Zn2+ efflux corresponded to exocytosis of insulin/Zn(2+)-containing granules from the beta-cell. Exocytosis sites identified by this technique were frequently localized to one portion of the cell, indicative of active areas of release.
 
 

65.
We have engineered aptamers that contain fluorescent reporters and that signal the presence of cognate ligands in solution. Two different anti-adenosine "signaling aptamers", one made from RNA and one from DNA, can selectively signal the presence of adenosine in solution. Increases in fluorescence intensity reproducibly follow increases in adenosine concentration, and can be used for quantitation. The facile methods we have developed can potentially be used for generating a wide variety of signaling aptamers for use in sensor arrays.
 
 

64.
The high resolving power of capillary electrophoresis combined with the specificity of binding interactions may be used with advantage to characterize the structure-function relationship of biomolecules, to quantitate specific analytes in complex sample matrices, and to determine the purity of pharmaceutical and other molecules. We here review recent and innovative methodologies and applications of high resolution affinity electrophoresis within the fields of binding constant determination, structure-activity studies, quantitative microassays, analysis of drug purity and protein conformation, and immobilized affinity ligands. Despite the virtues of these approaches with respect to applicability, resolving power, speed, and low sample consumption, problems remain with respect to analyte identification and low concentration limits of detection. The ongoing development of new detector technologies for capillary electrophoresis such as mass spectrometry, and possibly nuclear magnetic resonance and other spectroscopic methods, is therefore very promising for the continued increased use of affinity capillary electrophoresis.
 
 

63.
The chromatographic performance of capillaries with 20 and 50 micrometer inner diameter (i.d.) packed with 4.5 and 3.0 micrometer nonporous particles was evaluated under conditions of pressure- and electroosmotically-driven flow for unretained analytes with the goal of determining column configurations suitable for fast separations with both types of flow. Decreasing column diameter to particle diameter ratio (rho) enhanced performance for pressure-driven flow but not for electroosmotically-driven flow. The improved performance with decreasing rho seen for pressure-driven flow was attributed to a decrease in the A term of the Knox equation. It was concluded that decreasing rho in pressure-driven columns enhances the uniformity of the column, allowing for improved performance; however, the use of electroosmotic flow masks heterogeneous flow and thus no benefit in performance is seen from reducing rho. The best performance was with a 20 micron i.d. column packed with 3.0 micron particles. This column generated hmin of 0.30 and 3,310 plates/s at the highest electroosmotic flow rate tested (reduced velocity of 41) whereas with pressure-driven flow the same column had hmin of 0.96 and 1,200 plates/s at the maximum flow velocity tested (reduced velocity of 29).
 
 

62.
Two methods for amperometric detection of exocytosis at single pancreatic beta-cells were compared. In the first, direct detection of insulin was accomplished using an insulin-sensitive chemically modified electrode. In the second, 5-hydroxytryptamine (5-HT) that had been allowed to accumulate within the beta-cell secretory vesicles was detected with a bare carbon electrode. The goal of the comparison was to determine whether 5-HT secretion was a valid marker of insulin secretion in single beta-cells. To aid in this comparison, some experiments involved simultaneous measurement of insulin and 5-HT at cells previously allowed to accumulate 5-HT. Upon application of common insulin secretagogues, current spikes resulting from detection of 5-HT, insulin, or both compounds were obtained indicative of secretion via exocytosis. The mean area of current spikes obtained