156) Mabrouk, Omar S.; Li, Qiang; Song, Peng; Kennedy, and Robert T. "Microdialysis and mass spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal reciprocal interactions that regulate movement". Journal of Neurochemistry, 2011, 118(1), 24-33.

155) Thomas R. Slaney, Jing Nie, Neil D. Hershey, Prasanna K. Thwar, Jennifer Linderman, Mark A. Burns, and Robert T. Kennedy. "Push–Pull Perfusion Sampling with Segmented Flow for High Temporal and Spatial Resolution in Vivo Chemical Monitoring". Analytical Chemistry 2011 83 (13), 5207-5213.

154) Wang, Meng; Hershey, Neil; Mabrouk, Omar; and Kennedy, Robert. "Collection, storage, and electrophoretic analysis of nanoliter microdialysis samples collected from awake animals in vivo". Analytical and Bioanalytical Chemistry, 2011, 400 (7), 2013-2023.

153) Matthew A. Lorenz, Charles F. Burant, and Robert T. Kennedy. "Reducing Time and Increasing Sensitivity in Sample Preparation for Adherent Mammalian Cell Metabolomics". Analytical Chemistry 2011 83 (9), 3406-3414

152) Gwendolyn J. Anderson, Cynthia M. Cipolla, and Robert T. Kennedy. "Western Blotting Using Capillary Electrophoresis", Analytical Chemistry 2011 83 (4), 1350-1355.

151) Claire N. Chisolm, Charles R. Evans, Colin Jennings, Will A. Black, Frederick J. Antosz, Yangqiao Qiang, Angel R. Diaz, Robert T. Kennedy, "Development and characterization of “push–pull” sampling device with fast reaction quenching coupled to high-performance liquid chromatography for pharmaceutical process analytical technologies", Journal of Chromatography A, Volume 1217, Issue 48, 26 November 2010, Pages 7471-7477.

150) Jian Pei, Jing Nie, and Robert T. Kennedy. "Parallel Electrophoretic Analysis of Segmented Samples On Chip for High-Throughput Determination of Enzyme Activities",Analytical Chemistry 2010 82 (22), 9261-9267

149) Jing Nie and Robert T. Kennedy. "Sampling from Nanoliter Plugs via Asymmetrical Splitting of Segmented Flow". Analytical Chemistry 2010 82 (18), 7852-7856

148) Clark, Anna; Sousa, Kyle; Chisolm, Claire; MacDougald, Ormond; Kennedy, Robert. "Reversibly sealed multilayer microfluidic device for integrated cell perfusion and on-line chemical analysis of cultured adipocyte secretions". Analytical and Bioanalytical Chemistry. 2010. 397 (7), 2939-2947.

147) Perry, Maura L.; Leinninger; Gina M.; Chen, Rong; Luderman, Kathryn D.; Yang, Hongyan; Gnegy, Margaret E.; Myers, Martin G.; Kennedy, Robert T. "Leptin promotes dopamine transporter and tyrosine hydroxylase activity in the nucleus accumbens of Sprague-Dawley rats". Journal of Neurochemistry. 114(3), August 2010, 666-674.

146) Jian Pei, Qiang Li, Robert T. Kennedy, "Rapid and Label-Free Screening of Enzyme Inhibitors Using Segmented Flow Electrospray Ionization Mass Spectrometry", Journal of the American Society for Mass Spectrometry, Volume 21, Issue 7, July 2010, Pages 1107-1113.

145)  Meng Wang, Thomas Slaney, Omar Mabrouk, Robert T. Kennedy, "Collection of nanoliter microdialysate fractions in plugs for off-line in vivo chemical monitoring with up to 2 s temporal resolution", Journal of Neuroscience Methods, Volume 190, Issue 1, 30 June 2010, Pages 39-48.

144) Qiang Li, Jian Pei, Peng Song, and Robert T. Kennedy. "Fraction Collection from Capillary Liquid Chromatography and Off-line Electrospray Ionization Mass Spectrometry Using Oil Segmented Flow", Analytical Chemistry 2010 82 (12), 5260-5267

143) Nunemaker CS, Dishinger JF, Dula SB, Wu R, Merrins MJ, Reid KR, Sherman A, Kennedy RT, and Satin LS. "Glucose Metabolism, Islet Architecture, and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets". PLoS ONE, 2009, 4(12): e8428.

142) Meng Wang, Gregory T. Roman, Maura L. Perry, and Robert T. Kennedy, "Microfluidic Chip for High Efficiency Electrophoretic Analysis of Segmented Flow from a Microdialysis Probe and in Vivo Chemical Monitoring", Analytical Chemistry 2009 81 (21), 9072-9078.

141) Kendra R. Reid, Charles F. Burant, and Robert T. Kennedy, "Capillary LC−MS for High Sensitivity Metabolomic Analysis of Single Islets of Langerhans", Analytical Chemistry 2009 81 (21), 9201-9201

140) Maura Perry, Qiang Li, Robert T. Kennedy, "Review of recent advances in analytical techniques for the determination of neurotransmitters", Analytica Chimica Acta, Volume 653, Issue 1, 19 October 2009, Pages 1-22.

139) Kendra R. Reid and Robert T. Kennedy, "Continuous Operation of Microfabricated Electrophoresis Devices for 24 Hours and Application to Chemical Monitoring of Living Cells", Analytical Chemistry 2009 81 (16), 6837-6842

138) Jian Pei, Qiang Li, Mike S. Lee, Gary A. Valaskovic, and Robert T. Kennedy, "Analysis of Samples Stored as Individual Plugs in a Capillary by Electrospray Ionization Mass Spectrometry", Analytical Chemistry 2009 81 (15), 6558-6561

137) John F. Dishinger, Kendra R. Reid, and Robert T. Kennedy, "Quantitative Monitoring of Insulin Secretion from Single Islets of Langerhans in Parallel on a Microfluidic Chip", Analytical Chemistry 2009 81 (8), 3119-3127

136) Li, Q; Zubieta, JK; Kennedy, RT, " Practical Aspects of in Vivo Detection of Neuropeptides by Microdialysis Coupled Off-Line to Capillary LC with Multistage MS", ANALYTICAL CHEMISTRY  81 (6): 2242-2250 2009

135) Clark, AM; Sousa, KM; Jennings, C, et al. " Continuous-Flow Enzyme Assay on a Microfluidic Chip for Monitoring Glycerol Secretion from Cultured Adipocytes", ANALYTICAL CHEMISTRY 81(6): 2350-2356 2009

134) Yang, Peilin; Mao, Yingwei; Lee, Angel W-M, et al. "Measurement of dissociation rate of biomolecular complexes using CE", Electrophoresis 30(3): 457-64 2009

133) Schultz, KN; Von Esenwein, SA; Hu, M, et al. "Viral Vector-Mediated Overexpression of Estrogen Receptor-alpha in Striatum Enhances the Estradiol-Induced Motor Activity in Female Rats and Estradiol-Modulated GABA Release", JOURNAL OF NEUROSCIENCE 29(6): 1897-1903 2009

132) Schltz, KN; Kennedy, RT. "Time-Resolved Microdialysis for In Vivo Neurochemical Measurements and Other Applications", ANNUAL REVIEW OF ANALYTICAL CHEMISTRY  1: 627-661 2008

131) Roman, GT; Wang, M; Shultz, KN, et al. "Sampling and Electrophoretic Analysis of Segmented Flow Streams Using Virtual Walls in a Microfluidic Device ", ANALYTICAL CHEMISTRY 80(21): 8231-8238   Published: 2008

130) Furchak, JRW; Yang, PL; Jennings, C, et al. "Assay for Glucosamine 6-Phosphate Using a Ligand-Activated Ribozyme with Fluorescence Resonance Energy Transfer or CE-Laser-Induced Fluorescence Detection", ANALYTICAL CHEMISTRY 80 (21): 8195-82012008

129) Dishinger, JF; Kennedy, RT. "Multiplexed detection and applications for separations on parallel microchips ", ELECTROPHORESIS 29 (16): 3296-3305 2008

128) Wang, M; Roman, GT; Schultz, K, et al."Improved temporal resolution for in vivo microdialysis by using segmented flow", ANALYTICAL CHEMISTRY 80 (14): 5607-5615 2008

127) Pei, J; Dishinger, JF; Roman, DL, et al. "Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system", ANALYTICAL CHEMISTRY 80 (13): 5225-5231 2008

126) Yang, PL; Kennedy, RT. "High performance liquid chromatography coupled on-line to capillary electrophoresis with laser-induced fluorescence detection for detecting inhibitors of Src homology 2 domain-phosphopeptide binding in mixtures", JOURNAL OF CHROMATOGRAPHY A 1194 (2): 225-230 2008

125) Ni, QH; Reid, KR; Burant, CF, et al. "Capillary LC-MS for high sensitivity metabolomic analysis of single islets of Langerhans", ANALYTICAL CHEMISTRY 80 (10): 3539-3546 2008

124) Edwards, JL; Edwards, RL; Reid, KR, et al. "Effect of decreasing column inner diameter and use of off-line two-dimensional chromatography on metabolite detection in complex mixtures", JOURNAL OF CHROMATOGRAPHY A 1172 (2):127-134 2007

123) Roman, GT; Kennedy, RT. "Fully integrated microfluidic separations systems for biochemical analysis", JOURNAL OF CHROMATOGRAPHY A   1168(1-2):170-188 2007

122) Cunliffe, JM; Sunahara, RK; Kennedy, RT. "Detection of G protein coupled receptor mediated adenylyl cyclase activity by capillary electrophoresis using fluorescently labeled ATP", ANALYTICAL CHEMISTRY 79:  7534-7539 2007

121) Cunliffe, JM; Whorton, MR; Sunahara, RK, et al. "A CE assay for the detection of agonist-stimulated adenylyl cyclase activity", ELECTROPHORESIS 28 (12):1913-1920 JUN 2007

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 from simultaneous measurements equaled the sum of the mean area of insulin and 5-HT measured independently. Additionally, analyses of the number of spikes obtained for detection of insulin, 5-HT, or both compounds were similar for several common secretagogues. These data support the hypothesis that accumulated 5-HT is released from insulin containing secretory vesicles, exclusively. In addition, measurement of insulin and 5-HT from beta-cells of different species was compared to determine whether a species dependence exists for the two methods compared here. Detection of 5-HT results in a similar number of spikes that are equivalent to insulin in frequency and amplitude in human, porcine, and canine beta-cells; however, in mouse and INS-1 beta-cells, 5-HT is more readily detected than insulin.
 
 

61.
Spatially resolved measurements of exocytosis in pancreatic beta-cells were made using amperometry with 1-mum radius electrodes.  These measurements revealed that certain portions of a cell actively undergo exocytosis following stimulation with depolarizing agents, but other regions are inactive.  The amperometric measurements were compared to measurements made with the membrane indicator dye, FM1-43, which showed uneven increases in fluorescence around the surface of the cell, with amperometric secretion being detected only at the brightest regions.  In some instances, a large number of exocytotic events were detected from one electrode position.  The number of events was larger than what would be expected based on the number of vesicles that could fit under an electrode of the dimensions used.  These results suggest a mechanism of vesicle traffic that allows multiple fusions at a small membrane area.
 
 

60.
The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and beta-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22 degrees C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40degrees C before storage in liquid nitrogen (-196 degrees C). Frozen islets were rapidly thawed (200 degrees C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcry opreservation tissue culture was 81.5 +/- 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 +/- 1.5 (mean +/- SEM) for the frozen-thawed islets, compared with 12.4 +/- 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments).  Amperometric detection of secretion from single beta-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single beta-cells in real time. Secretion from single beta-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed beta-cells with an average of 7.0 +/- 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 +/- 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, beta-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that beta-cells recovered following cryopreservation are capable of secreting insulin at levels and  frequencies comparable to those of cultured nonfrozen islet preparations.
 
 

59.
An oxygen microsensor with a < 3-micron tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 +/- 0.29 pA/mmHg (mean +/- SD, n = 42), signals that were minimally perturbed by convection, and response times of < 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 +/- 1.6 mmHg (mean +/- SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 +/- 2.3 mmHg (mean +/- SEM, n = 6) and oscillations with a period of 3.3 +/- 0.6 min (mean +/- SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 +/- 1.7 s (mean +/- SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.
 
 

58.
Capillary electrophoresis is unique among liquid-phase separations in its utility for fast separations. Development of technology such as optical-gating, flow-gating, and microfabrication has allowed separations on the millisecond time scale to be developed. The fast separation times place great demands on the detector systems, frequently requiring detection limits below 1 amol to be practical. The development of such fast separations has opened many new applications not previously feasible for separations-based methods. This has included real time chemical monitoring, detection of short-lived species such as protein conformers or non-covalent complexes, and rapid multi-dimensional separations. Other applications currently being developed include high-throughput assays for clinical laboratories or screening combinatorial libraries. This review covers recent developments in the instrumentation for fast CE and some of the applications.
 
 

57.
Separations are generally considered a slow step in any analytical methodology.  HPLC and electrophoresis, the mainstay of separations of nonvolatile compounds in the modern laboratory, take anywhere from tens of minutes to hours to complete depending upon the complexity of the sample, the desired information, and selectivity available.  This time requirement has limited the types of problems that separations can be asked to solve.  For instance, separation methods are not typically used in high-throughput analyses or for monitoring applications.  The time-consuming nature of separations has driven many researchers to investigate methods of improving the speed of separations techniques.  In this review, we examine recent trends in improving the speed of liquid-phase analytical-scale separations based on chromatography and electrophoresis.  While the present review is limited to liquid-phase separations, we note that recent advances have been made in the speed of gas chromatography separations as well, and this work has recently been reviewed elsewhere.  Because many of the technical advances that have improved the speed of separations have occurred since 1990, our review emphasizes the past 9 years.
 
 

56.
5-(3' '-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.
 
 

55.
Microdialysis was coupled on-line with derivatization by o-phthalaldehyde and beta-mercaptoethanol and optically gated capillary electrophoresis to determine D- and L-aspartate in tissue samples obtained from rats. The microdialysis probe was inserted into a homogenized tissue sample which allowed generation of a continuous sample stream that was filtered and deproteinated. With 7.5 mM beta-cyclodextrin (CD) in the electrophoresis buffer, the enantiomers of interest could be resolved in 3 s with an electric field of 2500 V/cm over a separation length of 15 mm. Values of D- and L-aspartate in different tissues agreed well with those obtained by an HPLC procedure that required protein precipitation, centrifugation, and extraction. The speed and compatibility with automation of the microdialysis/CE method may make it a general approach for a variety of applications involving high-throughput analysis or sensorlike operation.
 
 

54.
A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 +/- 1.17, 9.21 +/- 2.15, and 12.22 +/- 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean +/- SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 +/- 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 +/- 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca2+-channel blocker nifedipine (40 microM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca2+. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca2+ levels and are suggestive of an important role for Ca2+ in amplifying metabolic oscillation.
 
 

53.
Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca2+ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.
 
 

52.
The separation and detection of biuret complexes of neuropeptides by capillary liquid chromatography with electrochemical detection was explored. Capillaries of 25-micron inner diameter packed with base-resistant, polymer-based reversed-phase particles were used for separation, and C-fiber electrodes were used for detection. Detection at the C-fiber electrode was found to have some differences in relative sensitivity for peptides compared to glassy carbon electrodes used previously. On-column preconcentration of preformed complexes allowed up to 1-microL samples to be injected with minimal band broadening resulting in a 100-fold improvement in concentration detection limit with no effect on mass detection limit. Concentration detection limits ranged from 5 to 59 pM, depending upon the peptide, corresponding to 5-59 amol injected. The low concentration detection limit was possible because of minimal baseline disturbances, minimal formation of unwanted products, and high efficiency of complex formation associated with biuret derivatization. The method was applied to determination of vasopressin and bradykinin in dialysates collected with 5-min sampling frequency from the rat supraoptic nucleus.
 
 

51.
Laser-induced fluorescence (LIF) has been used extensively in capillary separations due to its high sensitivity and selectivity. This article highlights the history and recent advances and applications of LIF detection in capillary electrophoresis and capillary chromatography.
 
 

50.
An improved pre-column derivatization with o-phthalaldehyde/tert-butylthiol and on-column preconcentration are used with packed capillary liquid chromatography and electrochemical detection to obtain low concentration detection limits for gamma-aminobutyric acid (GABA). Using derivatization procedures from the literature, it was found that the detection limits for GABA were 380 amol in 50 microns i.d. packed capillaries, which is over 10-fold worse than the detection limit possible with the instrumentation. The higher detection limit was directly the result of electroactive interferences generated by the derivatization chemistry. Derivatization was improved by scavenging excess reagents with excess amine and iodoacetamide. With these improvements, the interfering peaks were eliminated and the detection limit was improved to 50 amol. With injection volumes of 0.5 microL, under conditions that permitted on-column preconcentration, the concentration detection limit, that is the concentration at which analytes could be derivatized and detected, was 100 pM. The technique was applied to determination of GABA release from islets of Langerhans.
 
 

49.
A DNA aptamer against IgE was labeled with fluorophore and used as a selective fluorescent tag for determining IgE by capillary electrophoresis with laser-inducedfluorescence detection (CE-LIF). CE-LIF separations of samples containing fluorescently labeled aptamer and IgE were complete in less than 60 s and revealed twozones, one corresponding to free aptamer and the other to aptamer bound to IgE. The free aptamer peak decreased and bound aptamer peak increased inproportion to the amount of IgE in the sample so that IgE could be detected with a linear dynamic range of 10(5) and a detection limit of 46 pM. The assay washighly selective as aptamer was unaffected by the presence of IgG and IgE did not bind other DNA sequences. IgE was determined in serum samples with similaranalytical figures of merit. Similar conditions using a thrombin aptamer allowed detection of thrombin.
 
 

48.
The ability to cryopreserve pancreatic islets has allowed the development of low temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing.  However, previous studies have shown that during cryopreservation and thawing there is both a loss of islet mass and a reduction in islet function.  Recently developed amperometric techniques for detection of hormonal secretion from individual cells in vitro have the sensitivity and temporal resolution to detect single exocytotoxic events.  The aim of this study was to measure and compare insulin secretion from freshly isolated and frozen/thawed canine islets using amperometry.
 
 

47.
Optically gated capillary electrophoresis (CE) of amino acids derivatized with o-phthalaldehyde/beta-mercaptoethanol (OPA/-betaME) was explored as a means to monitor amino acids with high temporal resolution. In agreement with a theoretical model described herein, 98% of a given concentration of OPA/beta-ME derivatives can be photobleached by a few milliwatts of the 350-nm line of an argon ion laser with just 0.7-ms exposure times in 5-micron-i.d. capillaries. The low background from such high photobleaching efficiency allows detection limits in the low-nanomolar range for all amino acids tested. The short injection times possible with optical gating allow separation efficiencies of nearly 200 000 plates to be achieved in less than 1 s under ideal conditions. Under mock in vivo conditions, separations were slower and had lower efficiency due to reduced electroosmotic flow associated with the high salt content. To demonstrate chemical monitoring, the optically gated CE system was interfaced to two different sampling probes with on-line derivatization with OPA/beta-ME. With microdialysis sampling, the optically gated CE system could assay the sample stream every 2 s but actual temporal resolution for monitoring was limited by band broadening in the dialysis probe to ~12 s. Optically gated CE was also interfaced to a 10-micron-i.d. sampling capillary that continuously pulled samples into the separation capillary at 6.5 nL/min. This direct sampling probe allowed monitoring of multiple amino acids with 10-s temporal resolution with several advantages compared to microdialysis including improved detection limits and spatial resolution.
 
 

46.
An automated on-line competitive immunoassay based on capillary electrophoresis (CE) was utilized to monitor secretion of insulin from single islets of Langerhans stimulated by glucose and tolbutamide. In the instrument, fluorescein isothiocyanate-labeled insulin (FITC-insulin), monoclonal anti-insulin and perifusate of single islets were mixed on-line while islets were exposed to different levels of glucose and tolbutamide. Insulin released from single islets competed with FITC-insulin for antibody binding sites. Therefore, the amounts of bound and free FITC-insulin were modulated by insulin released from islets. The bound and the free FITC-insulin were separated by CE every 3 s and the bound over free ratio (B/F) was measured. Insulin levels were obtained by comparing B/F with calibration curves obtained under the same conditions except that the islet perfusate was replaced with various concentrations of insulin. Patterns of insulin secretion stimulated by glucose and tolbutamide observed were comparable to what has been seen previously using radioimmunoassay or enzyme-linked immunoassay. This on-line competitive immunoassay system provided a fast and direct way to measure insulin release from single islets. The effects of temperature on antibody-antigen reaction rate and binding equilibrium were also studied.
 
 

45.
Flow-through recycling electrochemical detectors with dual Au electrodes were fabricated using micromachining techniques. Detector cells contained two microband electrodes that were 50 mu m wide by 3-5 mm long separated by a 5 or 10 mu m gap in a parallel-opposed configuration. One electrode was defined in the bottom of a channel which was etched into the surface of a Si wafer while the other electrode was defined on a glass substrate. The depth of the channel defined the gap distance between the electrodes. The Si and glass pieces were anodically bonded together to create a flow cell. The stability and reproducibility of single and dual electrode cells were characterized with cyclic voltammetry and amperometry during flow injection analysis (FIA). Single electrode detectors had detection limits of 50 nM with root mean square noise in the 3-10 pA range. The dual electrode cells showed enhanced sensitivity over single electrode cells through detection of analyte molecules multiple times as they were transported through the cell. Signal enhancements up to 60-fold were obtained with dual electrode cells during FIA at a flow rate of 50 nL/min. Simulations predict that signal-to-noise ratio enhancements > 100-fold may be possible with appropriate designs.
 
 

44.
Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200-300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 microM tetrodotoxin or depletion of extracellular Ca2+, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (200 microM) increased resting Glu and Asp levels by 300-500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1S,3R)-1-aminocyclopentane-trans-1,3-dicarboxylate (200 microM). This effect was blocked by the metabotropic Glu receptor antagonist (RS)-alpha-methylcarboxyphenylglycine (MCPG; 200 microM). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of L-trans-pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.
 
 

43.
The effects of extracellular Zn2+ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H+-ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn2+ concentration from 0 to 25 microM increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn2+ effects were likely exerted through a common-ion effect on Zn2+-insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.
 
 

42.
An automated method for high temporal resolution monitoring of the neurotransmitters glutamate and aspartate in vivo using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was developed. Microdialysis probes placed in the striatum of anesthetized rats were coupled on-line with the CE system by an automated flow-gated interface. Analytes were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and detected by LIF using the 354 nm line (7 mW) of a He-Cd laser for excitation. With dialysis flow rates of 1.2 microL/ min, the detection limit at the dialysis probe was 200 nM for glutamate and aspartate. Glutamate and aspartate could be resolved in less than 5 s with over 200,000 theoretical plates. The sampling time was limited by the separation time while the temporal resolution was limited to approximately 12 s because of band broadening that occurs within the probe and its associated tubing. The high temporal resolution allowed the first simultaneous monitoring of glutamate and aspartate overflow during acute electrical stimulation in the rat brain.
 
 

41.
A method based on microdialysis sampling and capillary liquid chromatography with electrochemical detection that allows in vivo monitoring of met-enkephalin with 5-min temporal resolution is described. Sampling was achieved using a concentric microdialysis probe made from polycarbonate membrane material with a 20 kDa cut-off. This probe had an in vitro relative recovery for met-enkephalin of 63% at a dialysis flow-rate of 0.6 microl/min. Separations were performed using 7 cm x 25 microm I.D. fused-silica capillary columns packed with 5 microm Alltima C18 particles. A carbon fiber microelectrode was used as the detector electrode. The mass detection limit for met-enkephalin with this system was 40 amol. With on-column preconcentration, up to 2 microl of sample could be loaded onto the column resulting in concentration detection limits as low as 20 pM for met-enkephalin. Direct injection of dialysate, collected at 5-min intervals, allowed determination of met-enkephalin concentrations in the rat globus pallidus under basal and K+-induced depolarization conditions.
 
 

40.
The dissociation constant (K-d) of a monoclonal antibody with fluorescein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). K-d determinations were made by separating free FITC-insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC-insulin in the picomolar range, as is required to measure K-d's below 1 nM. The K-d of FITC-insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The K-d's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC-insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. K-d's for the different insulins were between 0.34 and 0.64 nM.
 
 

39.
The insulin-like growth factor II (IGF-II)/mannose-6-phosphate (M-6-P) receptor is known to participate in endocytosis as well as sorting of lysosomal enzymes and is involved in membrane trafficking through rapid cycling between cytosolic membrane compartments and the plasma membrane. Here we demonstrate that IGF-II, acting through the IGF-II/M-6-P receptor, promotes exocytosis of insulin in the pancreatic beta cell. The effect of IGF-II was evoked at nonstimulatory concentrations of glucose, was mediated by a pertussis toxin sensitive GTP-binding protein, was dependent on protein kinase C-induced phosphorylation, and was independent of changes in cytoplasmic free Ca2+ concentration. Since the applied concentration of IGF-II is within the range normally found free in circulation in humans, this novel signaling pathway for the IGF-II/M-6-P receptor is likely to be involved in modulation of insulin exocytosis under physiological conditions.
 
 

38.
A fully-automated method for monitoring thiols in vivo using microdialysis coupled on-line with capillary zone electrophoresis with laser-induced fluorescence detection was developed. Dialysates were derivatized on-line with monobromobimane and automatically transferred to the separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nl/min resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits for these analytes were in the 20-40 nM range and the response was linear up to 20 microM. The system was applied to the measurement of glutathione and cysteine in the extracellular space of the caudate nucleus of anesthetized rats. The measured basal concentrations of glutathione and cysteine were 2.0 +/- 0.1 microM and 2.3 +/- 0.3 microM, respectively which agree well with literature values. Increases in glutathione and cysteine were monitored with 180 s temporal resolution during stimulation by infusion of potassium. The average concentration of glutathione and cysteine during stimulation was 3.0 +/- 0.9 and 3.3 +/- 0.5 microM (n = 3), respectively. This system is the first to obtain high relative recoveries and high temporal resolution simultaneously for multiple thiols with microdialysis sampling in the brain.
 
 

37.
The amperometric and cyclic voltammetric detection of alpha-melanocyte stimulating hormone (MSH), beta-endorphin, and corticotropin-like intermediate lobe peptide (CLIP), all proopiocortin (POC) derived peptides secreted from melanocytes of the pituitary intermediate lobe, at carbon fiber microelectrodes was investigated. For amperometry, it was found that all of these peptides could be detected; however, fouling of the electrodes reduced the response of the electrode after successive application of the peptide in flow injection experiments. The fouling was apparently due to oxidation of tyrosine in the peptides as similar results were found for tyrosine but not tryptophan. The effect of fouling could be reversed if the electrode was electrochemically treated by scanning from -1.0V to +1.0V at 300 Vis for 2 min between application of the peptides. Using cyclic voltammtery at 800 V/s, it was possible to distinguish MSH, which had a peak shaped voltammogram, from the other POC peptides, which had relatively flat voltammetric waves at this scan rate. The scan rate dependence of the peak current for MSH revealed that the voltammetry was adsorption controlled. As a result, in a monitoring application, where voltammograms are continuously obtained with a fixed interval between them, decreasing the interval increases the temporal resolution but decreases the sensitivity for MSH. It was found that when monitoring the current in the potential range of 0.90 to 1.00 V, the temporal response to MSH was dependent upon the potential window used for scanning. Using high scan rates and a potential window of 0 to 1.2V, it was possible to monitor exocytosis from single melanocytes and use the voltammogram to demonstrate detection of MSH from the cells.
 
 

36.
An on-line competitive immunoassay for insulin has been developed and applied to monitoring insulin concentration in a flowing stream. In the assay, solutions of fluorescein-labeled insulin (FITC-insulin), monoclonal anti-insulin, and sample containing insulin are pumped into a cross where they begin to mix. The mixture flows through a fused silica reactor capillary to a flow-gated interface. During transfer to the interface, insulin and FITC-insulin compete to form a complex with the antibody. At the interface, a plug of the mixture is injected into a separation capillary, where the bound and free FITC-insulin are separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The amount of bound FITC-insulin, amount of free FITC-insulin, or bound/free ratio can be used to quantify insulin concentration. Typical relative standard deviations of bound over free ratio are 5%. The detection limit of the immunoassay in the on-line mode is < 0.3 nM. Each separation requires as little as 3 s, and over 1600 consecutive assays can be acquired with no need to rinse the separation capillary. Thus, the system can be used to monitor insulin in a flowing stream for flow injection analysis or for sensor-like monitoring. Dilution and zone broadening during transfer of sample to the interface limit the response time of the on-line system to about 25 s. As a demonstration of the on-line immunoassay, the insulin content of single islets of Langerhans was determined by flow injection analysis.
 
 

35.
An electroactive coating was deposited on the surface of carbon microelectrodes from solutions containing Pb(NO3)(2), Na2IrCl6 and KNO3 by scanning their potentials from 0.0 to 1.15 V. The coating, believed to be IrO2 entrapped inside a PbO2 matrix, catalyzes oxidation of glucose, and other carbohydrates (e.g. mannose), in a phosphate buffered saline solution at pH 7.4. The microelectrodes coated with iridium and lead oxides were used as amperometric detectors at 0.75 V (versus a sodium saturated calomel electrode) for flow injection analysis of glucose samples. The linear dynamic range extended from 0.1 mM up to at least 20 mM, and the detection limit was 50 mu M for glucose. The stability and selectivity of the oxide-modified electrodes were improved by application of Nation(R) coating. The response of the Nafion(R)-coated electrode to 15 mM glucose was stable for four days.
 
 

34.
A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and -0.85 V vs. SSCE at 100 V s(-1) in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH 7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 mu M (five points) had slopes of 72+/-2 pA mu M-1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23nM and response time (t(90%)) was 40ms or less. The electrode response to 0.2 mu M insulin was stable for 3 days, The modified electrode was used for amperometric detection of exocytosis from individual pancreatic beta-cells.
 
 

33.
A method for monitoring primary amines in vivo using microdialysis coupled on-line with capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence detection was explored. Dialysates were derivatized on-line with o-phthaldialdehyde/beta-mercaptoethanol and automatically transferred to a separation capillary by a flow-gated interface. Analytes were detected on-column using the 2 mW, 354 nm line of a He-Cd laser for excitation. Dialysis probes were perfused at 79 nL/min, resulting in relative recoveries of nearly 100%, which allowed quantitative monitoring. On-line detection limits were in the 20-50 nM range, and the response was linear up to 50 microM. Temporal resolution was between 45 s and 3 min and was limited by separation time or broadening of sample zones during transfer to the separation capillary, depending on the operational parameters. The system was applied to measurement of primary amines in the caudate nucleus of anesthetized rats. Using CZE for separation, it was possible to resolve and monitor several compounds, including aspartate and glutamate. The measured basal concentrations of aspartate and glutamate were 1.2 +/- 0.1 and 5.0 +/- 0.4 microM, respectively, which agrees well with literature values. Increases in in vivo aspartate and glutamate were monitored with 90 s temporal resolution during K+ depolarization using dialysis flow rates of 79 nL/min; however, temporal resolution of 45 s was possible at the expense of lower relative recovery if the dialysis flow rate was increased to 155 nL/min. The use of MEKC as the separation mode significantly increased the number of compounds that could be resolved and detected. Using MEKC to separate the dialysate samples allowed aspartate, glutamate, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, tyrosine, and valine to be resolved and detected. The basal concentrations for these compounds using MEKC were 1.9 +/- 0.2, 4.1 +/- 0.2, 4.6 +/- 0.7, 2.6 +/- 0.3, 5.4 +/- 0.4, 1.8 +/- 0.2, 2.0 +/- 0.2, 11.3 +/- 1.3, 3.3 +/- 0.9, and 5.3 +/- 0.3 microM, respectively. The concentrations of these primary amines in the striatum were monitored after K+ depolarization with 3 min temporal resolution. This is the first microdialysis system to generate high relative recoveries and good temporal resolution simultaneously for multiple neurotransmitters.
 
 

32.
A dual microcolumn immunoassay (DMIA) was developed and applied to determination of insulin in biological samples. The DMIA utilized a protein G capillary column (150 microns I.D.) with covalently attached anti-insulin to selectively capture and concentrate insulins in a sample. Insulins retained in the capillary immunoaffinity column were desorbed and injected onto a reversed-phase capillary column (150 microns I.D.) for further separation from interferences such as cross-reactive antigens and non-specifically adsorbed sample components. Bovine, porcine and rat insulin all cross-reacted with the antibody and could be determined simultaneously. Using a UV absorbance detector, the dual microcolumn system had a detection limit of 10 fmol or 20 pM for 500-microliter sample volumes. The DMIA system was used to measure glucose-stimulated insulin secretion from single rat islets of Langerhans. Because of the separation in the second dimension, both rat I and rat II insulin could be independently determined. The system was also evaluated for determination of insulin in serum. Using microcolumns instead of conventional HPLC columns resulted in several advantages including use of less chromatographic material and improved mass detection limit.
 
 

31
Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic beta-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for < 6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.
 
 

30.
Not Available
 
 

29.
Not Available
 
 

28.
Amperometry at a carbon fiber microelectrode was used to monitor secretion of peptide hormone from single melanotrophs of the intermediate lobe of the rat pituitary. The method is based on electrochemical oxidation of tryptophan and tyrosine residues of small proopiocortin-derived peptides secreted from these cells. For single-cell measurements, the electrode, which had a sensing diameter of approximately 9 microns and a total tip diameter of 30 microns, was positioned approximately 1 micron away from single melanotrophs. When cells were stimulated by application of 64 mM K+, a series of randomly occurring current spikes with an average area of 34 +/- 6 fC was observed. The current spikes were strongly dependent on the presence of Ca2+. Current spikes of nearly identical area and shape were also elicited by mechanical stimulation. Cyclic voltammograms obtained from cell releasates confirmed that the substance detected was a tryptophan- or tyrosine-containing peptide. On the basis of amperometric tests of the most abundant peptides in melanotrophs, it is concluded that the current spikes are due to detection of primarily alpha-melanocyte stimulating hormone. The spike area corresponds to 0.32 amol of alpha-melanocyte stimulating hormone. It is concluded that the current spikes represent detection of concentration pulses that are expected following exocytosis events.
 
 

27.
Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic ß cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in ß cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.
 
 

26.
 A system which couples microdialysis with capillary zone electrophoresis (CZE) on-line is used to monitor ascorbate and lactate in the caudate nucleus of rat brain. On-line interface of microdialysis probe and electrophoresis capillary, along with the high mass sensitivity of CZE, allows the probe to be operated at flow rates as low as 40 nl/min. Under these conditions, the relative recovery is nearly 100% and quantitative monitoring is possible. The microscale system also facilitates calibration by the low flow rate method. In spite of the low flow rate, temporal resolution in the 45-125 s range is possible for these compounds. The system is demonstrated by observing changes in ascorbate due to infusions of elevated K+ through the dialysis probe and systemic injections of amphetamine and an anesthetic (ketamine/xylazine/acepromazine mixture). Lactate is monitored in response to elevated K+ infusions.
 
 

25.
Capillaries with 150 microns inner diameter were packed with a perfused protein G chromatographic support and used as immunoaffinity preconcentrators for capillary zone electrophoresis. Antibody was loaded onto the protein G support to form an immunoaffinity stationary phase. Injection of samples onto the column caused selective retention and preconcentration of antigen.  Injection of appropriate buffers onto the column caused desorption of the antibody and antigen which were then separated by capillary zone electrophoresis. The combination was used on-line and off-line. For on-line combination, a flow-gated interface coupled the two columns and allowed injection of desorbed zones onto the electrophoresis system. Off-line coupling required collection of desorbed fractions and then injection onto the electrophoresis system. Flow rates as high as 100 microL/min were used to load sample onto the affinity column. Desorbing flow rates had to be 1 microL/min or less to prevent excessive dilution during desorption. Using the system,1 mL insulin samples could be loaded onto the affinity column and desorbed in volumes as small as 1 microL for 1000-fold preconcentration. The use of the preconcentrator with serum samples spiked with insulin was demonstrated.
 
 

24.
 
 

23.
A now-gated, on-line interface between a microdialysis sampling probe and capillary zone electrophoresis with UV absorption detection was characterized and applied. Electrophoresis conditions were chosen so that ascorbic acid migrated in 42-100 s with 65 000-120 000 theoretical plates. These results were obtained using a 25 mu m inner diameter capillary with an inlet to detector length of 15 cm and electric field strength of 400-600 V cm(-1). Theoretical plates, migration times, and peak areas for ascorbic acid injected on-line from the interface were the same as those obtained for off-line injections. The system allowed step changes in ascorbic acid concentration to be followed with 85 s time resolution when the dialysis now-rate was 79 nl min(-1). The time resolution was improved to 65 s when the dialysis flow-rate was 155 nl min(-1). The relative standard deviation for peak heights was 3.8% and a linear response over the physiologically relevant range for ascorbic acid was observed. At 79 nl min(-1) the relative recovery of the probe was 98%. The high relative recoveries improved detection limits, simplified quantification, and resulted in decreased disturbance to the system being studied when compared to the more conventional dialysis flow-rates of 0.5-1 mu l min(-1). As a demonstration of the system, ascorbic acid in the caudate nucleus of rat brain was detected and monitored in response to systemic amphetamine injections and anaesthetic overdoses. This system is the first to demonstrate high relative recoveries and high time resolution simultaneously with microdialysis sampling.
 
 

22.
The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described. The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent. Reproducibility and reliability of the assay are  improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected. Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets. During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release. These values are also in good agreement with previously reported results. These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.
 
 

21.
 
 

20.
 
 

19.
The purpose of this work was to examine column designs that allow for rapid separations by packed capillary liquid chromatography. Previous work with packed capillary columns with inner diameters (i's) of 20 to 150 mum revealed significantly reduced eddy diffusion broadening in these columns compared to larger bore columns. These results suggested that resistance to mass transfer dominates band broadening in microcolumns at high flow rates. Thus, it should be possible to attain rapid separations in capillaries by using pellicular or perfused particles which reduce resistance to mass transfer in the stagnant mobile phase. Furthermore, the use of capillary columns with small eddy diffusion effects should give better performance than larger bore columns packed with similar particles. To test these possibilities, the plate height of an unretained solute as a function of flow velocity was evaluated in columns with internal diameters of 50 to 250 mum packed with pellicular particles (8 and 15 mum diameter) and perfusion particles (10 and 20 mum diameter). Decreasing both column i and column i to particle diameter ratio (rho) were found to improve the performance of columns packed with pellicular particles; however, rho was the most important factor. Column i and rho had no effect on the performance of perfusion particles within the range tested. However, the capillary perfusion columns had better performance than previously reported for larger bore perfusion columns. The best results were obtained for 8 mum pellicular particles inside 50 mum i columns. These columns generated nearly 7,000 theoretical plates in 10 s. Further improvements should be possible by using smaller particles and smaller column internal diameters.
 
 

18.
 
 

17.
The goal of the work was to develop and test an amperometric method for measuring insulin secretion from individual pancreatic beta-cells. The electrode used was a carbon fiber microelectrode modified with a polynuclear ruthenium oxide/cyanoruthenate film. The chemically modified electrode allowed anodic detection of insulin in physiological buffers with a detection limit of 0.5 microM. To measure secretion, an electrode was positioned 1 micron away from a beta-cell that had been stimulated with K+ or glucose. Recordings made from the cells consisted of a series of current spikes averaging 38 ms full width at half-height. The spikes decreased in height and increased in width as the electrode was pulled away from the cell. Spikes were only observed if a modified electrode was used and its potential was sufficient to oxidize insulin. The area under the spikes correspond to approximately 600 zmol of insulin, which is within the expected range for vesicular insulin content. Spike area was independent of stimulation method. The results support the hypothesis that the electrode was anodically detecting a substance secreted from the cells by exocytosis. The results support, but do not prove, that insulin was the primary substance detected.
 
 

16.
Fast-scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one-, two-, and 50-pulse, 10-Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 microM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 microM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 microM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 microM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one-pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half-time of approximately 17 s was found. The addition of picrotoxin (100 microM) and glutamate (10 microM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 microM) attenuated overflow for all the trains tested.
 
 

15.
Dopamine and oxygen (O2) were measured in the caudate nucleus of anesthetized rats and in striatal slices during electrical stimulation. Simultaneous electrochemical detection of dopamine and O2 was accomplished with fast-scan cyclic voltammetry at a Nafion-coated carbon-fiber microelectrode. Stimulation of the medial forebrain bundle resulted in synaptic overflow of dopamine in the caudate nucleus. At the same time, O2 concentration increased in the extracellular fluid with two separate phases. The amplitude of the initial increase directly correlated with the frequency of the stimulus, with the time of maximum concentration reproducible across a range of frequencies. The second increase occurred at later times with a more random amplitude and with a broad, variable shape. Agents which blocked vasodilation affected both phases: atropine attenuated the initial increase, while the second feature was nearly absent after theophylline. Yohimbine and alpha-methyl-p-tyrosine did not affect the O2 responses. Local electrical stimulation of the slice preparation also resulted in dopamine overflow, but a prolonged decrease in O2 concentration accompanied this event. Striatal field stimulation in vivo produced changes in O2 concentration dependent on the relative position of the stimulating and working electrodes, but none of the responses resembled that seen in the caudate slice. Thus, while measurements in brain slices show O2 consumption as a result of stimulated neuronal activity, an apparent elevation of local cerebral blood flow during and after stimulation dominate the in vivo response.
 
 

14.
Fast-scan cyclic voltammetry was used to simultaneously measure increases in dopamine concentration and decreases in O2 concentration evoked by brief electrical stimulation (two pulses at 10 Hz) in slices of rat caudate nucleus. Dopamine concentration began increasing immediately after the first pulse and reached a maximum within 200 ms of stimulation. The O2 concentration began to decrease 300-700 ms after onset of stimulus. Responses for both dopamine and O2 were dependent on external Ca2+ and were Cd2+ and tetrodotoxin sensitive. Only the O2 response was sensitive to CN- (0.15 mM). At short times after exposure to 50 microM ouabain, electrically stimulated dopamine overflow was increased by 150% and electrically stimulated changes in O2 concentration were unaffected. Maximum dopamine concentration was increased 28% by sulpiride (2 microM), 78% by L-DOPA (60 microM), 105% by nomifensine (10 microM) and unaffected by nialamide (10 microM). Maximum decrease in O2 concentration was increased by 25% by sulpiride and unaffected by nialamide, L-DOPA, or nomifensine. The decreases in O2 concentration are indicative of increased O2 consumption and are a measure of oxidative energy production evoked by electrical stimulation. The increase in dopamine is due to the release of dopamine balanced by uptake and serves as an indication of neurotransmitter activity. The results indicate that increases in oxidative energy production following electrical stimulation are dependent on external Ca2+ entry through Cd(2+)-sensitive channels. Possible mechanisms for this coupling are discussed.
 
 

13.
Secretion of catecholamines from single bovine chromaffin cells in culture was elicited by brief pressure ejections from a micropipette containing nicotine, carbamoylcholine, or potassium ions or by mechanical stimulation. Release was monitored electrochemically with a carbon-fiber microelectrode placed adjacent to the cell. Cyclic voltammetry was used to identify secreted species, whereas constant potential amperometry was used for improved temporal resolution (millisecond range) of catecholamine detection. During secretion, brief current spikes were observed, which were shown to be due to detection of catecholamines by electrooxidation. The spikes have the physical characteristics of multimolecular packets of catecholamines released at random times and locations from the surface of the single cell. The half-width of the spikes was found to increase with an increase in cell-electrode spacing. The properties of the catecholamine spikes correlate well with expectations based on secretion from individual storage vesicles. Spikes do not occur in the absence of Ca 2+ in the buffer, and the majority of spikes are found to be distributed between 0.2 and 2 picocoulombs, corresponding to 1-10 attomoles of catecholamine detected. The frequency of the spikes increases with the intensity of the stimulus, but the average quantity of catecholamine in each spike is independent of the stimulus. Thus, these measurements represent time-resolved observation of quantal secretion of catecholamines and provide direct evidence for the exocytotic hypothesis.

12.
Cyclic voltammetry of Nafion-coated, carbon-fiber electrodes is used to detect trace concentrations of dopamine, both in a flow injection apparatus and in the brain of an anaesthetized rat. To improve signal-to-noise ratios, the sources of noise during cyclic voltammetry have been determined and strategies have been developed to decrease the noise. With the potentiostat employed, the measured noise is comparable to that expected for Johnson noise from the feedback resistor of the current transducer. Additional noise arises from the waveform generator employed and, in some cases, line noise. Line noise is discriminated against by starting each cyclic voltammogram either in phase or 180 degrees out of phase with the line frequency. When used in vivo, additional noise also arises from the physiological activity of the animal. Detection limits are found to closely correspond to those predicted on the basis of simulation of the voltammetric shape and the measured noise. Detection limits are improved by the use of appropriate analog and digital filtering, ensemble averaging, and appropriate timing of repetitive cyclic voltammograms. The combined use of these techniques enables the in vivo detection of approximately 100 nM of dopamine with a signal-to-noise ratio of 25.
 
 

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Capillary zone electrophoresis and open tubular liquid chromatography are two examples of an emerging area of analytical instrumentation known as microcolumn separations. The high resolution and small sample requirements of these methods make them suitable for the quantitative, multicomponent chemical analysis of single cells. Appropriate instrumentation for the analysis of nanoliter and subnanoliter samples is discussed. Data from the analysis of individual neurons are presented, including amino acid and neurotransmitter content.
 
 

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The ability to analyze individual cells is often important in biology because of the heterogeneity of tissue; this is especially true in the area of neurobiology. A method is described for the determination of trace levels of organic compounds in individual cells by open tubular liquid chromatography with voltammetric detection. In the method, a cell is isolated, an internal standard is added, the cell is homogenized and centrifuged, and the supernatant is injected directly onto the chromatography column. Since data are collected in both the electrochemical and chromatographic domains, the resolution of the method is better than that obtained by using amperometric detection. The combination of voltammetry and chromatography also aids in the identification of compounds. By use of this method three different neurons, D2, E4, and F1, from the land snail Helix aspersa are analyzed. The data show that the cells give certain unique and repeatable chemical profiles. Dopamine, serotonin, tyrosine, and tryptophan were identified and quantified in two of the cells at the femtomole level. In the third cell, only the two amino acids were observed and measured. The quantitative data indicate that the method is at least as reliable as other methods that have been applied to single cells and considerably more sensitive. The combination of qualitative and quantitative information allows for the chemical mapping of cells.
 
 

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