Publications
297) Jun, H., Liu, S, Knights, A.J., Zhu, K., Ma, Y., Gong, J., Lenhart, A.E., Peng, X., Huang, Y., Ginder, J.P., Downie, C.H., Ramos, E.T., Kullander, K., Kennedy, R.T., Xu X.Z.S., Wu, J. “Signaling through the nicotinic acetylcholine receptor in the liver protects against the development of metabolic dysfunction-associated steatohepatitis.” PLOS Biology, 2024, in press.
296) Makey, D.M., Gadkari, V.V., Kennedy, R.T., Ruotolo, B.T. “Cylic ion mobility-mass spectrometry and tandem collision induced unfolding for quantification of elusive protein biomarkers.” Analytical Chemistry, 2024, 96 (15), 6021-6029.
295) Lenhart, A.E., Booth, P.M., Simcox, K.M., Ramos, B.A., Kennedy, R.T. “Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyze classes.” Journal of Chromatography A, 2024, 1722, 464872.
294) Anderson, B.G., Hancock, T.A., Kennedy, R.T. “Preparation of high-efficiency HILIC capillary columns utilizing slurry packing at 2,100 bar.” Journal of Chromatography A, 2024, 1722, 464856.
293) Wells, S.W., Bain, I.J., Valenta, A.C., Lenhart, A.E., Steyer, D.J., Kennedy, R.T. “Microdialysis coupled with droplet micro fluidics and mass spectrometry for determination of neurotransmitters in vivo with high temporal resolution.” Analyst, 2024, 149, 2328-2337.
292) Xin, Y., Foster, S.W., Makey, D.M., Parker, D., Bradow, J., Wang, X., Mongillo, R., Grinias, J.P., Kennedy, R.T. “High-throughput capillary liquid chromatography using a droplet injection and application to reaction screening.” Analytical Chemistry, 2024, 96 (11), 4693-4701.
291) Makey, D.M., Diehl, R.C., Xin, Y., Murray, B.E., Stoll, D.R., Ruotolo, B.T., Grinias, J.P., Narayan, A.R.H., Lopez-Carillo, V., Stark, M., Johnen, P., Kennedy, R.T. “High-throughput liquid chromatographic analysis using a segmented flow injector with a 1 s cycle time” Analytical Chemistry, 2023, 95 (46), 17028-17036.
290) Payne, E.M., Murray, B.E., Penabad, L.I., Abbate, E., Kennedy, R.T. “Mass-activated droplet sorting for the selection of lysine-producing Escheria coli“ Analytical Chemistry, 2023, 95 (42) 15716-15724.
289) Lenhart, E.A., Kennedy, R.T. “Evaluation of surface treatments of PDMS microfluidic devices for improving small-molecule recovery with application to monitoring metabolites secreted from islets of Langerhans” ACS Measurement Science Au, 2023, 3 (5), 380-389.
288) Murray, B.E., Penabad, L.I., Kennedy, R.T. “Advances in coupling droplet microfluidics to mass spectrometry” Current Opinion in Biotechnology, 2023, 82, 102962.
287) D’Amico, C.I., Robbins, G., Po, I., Fang, Z., Slaney, T.R., Tremml, G., Tao, L., Ruotolo, B.T., Kennedy, R.T. “Screening clones for monoclonal antibody production using droplet microfluidics interfaced to electrospray ionization mass spectrometry” Journal of the American Society for Mass Spectrometry, 2023, 34 (6), 1117-1124.
286) Jones, J.D., Simcox, K.M., Kennedy, R.T., Koutmou, K.S. “Direct sequencing of total S. cerevisiae tRNAs by LC-MS/MS” RNA, 2023, 29 (8), 1201-1214.
285) Sun, A.C., Steyer, D.J., Robinson, R.I., Ginsburg-Moraff, C., Plummer, S., Gao, J., Tucker, J.W., Alpers, D., Kennedy, R.T., Stephenson, C.R.J. “High-throughput optimization of photochemical reactions using segmented-flow nanoelectrospray-ionization mass spectrometry” Angewandte Chemie International Edition, 2023, 62, e202301664.
284) Payne, E.M., Taraji, M., Murray, B.E., Holland-Moritz, D.A., Moore J.C., Haddad, P.R., Kennedy, R.T. “Evaluation of analyte transfer between microfluidic droplets by mass spectrometry” Analytical Chemistry, 2023, 95 (10), 4662-4670.
283) Jones, J.D., Grassmyer, K.T., Kennedy, R.T., Koutmou, K.S., Maloney, T.D. “Nuclease P1 digestion for bottom-up RNA sequencing of modified siRNA therapeutics” Analytical Chemistry, 2023, 95 (9), 4404-4411.
282) Jones, J.D., Franco, M.K., Smith, T.J., Snyder, L.R., Anders, A.G., Ruotolo, B.T., Kennedy, R.T, Koutmou, K.S. “Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation” RSC Chem. Biol., 2023, 4, 363-378.
281) Glynos, N.G., Carter, L., Lee, S.J., Kim, Y., Kennedy, R.T., Mashour, G.A., Wang, M.M., Borjigin, J. “Indolethylamine N-methyltransferase (INMT) is not essential for endogenous tryptamine-dependent methylation activity in rats” Sci Rep, 2023, 13 (280).
280) Sorensen, M.J., Miller, K.E., Jorgenson, J.W., Kennedy, R.T. “Two-dimensional liquid chromatography-mass spectrometry for lipidomics using off-line coupling of hydrophilic interaction liquid chromatography with 50 cm long reversed phase capillary columns” Journal of Chromatography A, 2022, 1687, 463707.
279) Lenhart, A.E., Kennedy, R.T. “Monitoring hormone and small molecule secretion dynamics from islets-on-chip” Analytical and Bioanalytical Chemistry, 2022, 415, 533-544.
278) Sun, A.C, Stephenson, C.R.J., Kennedy, R.T. “High-Throughput Photochemistry Using Droplet Microfluidics“ In The Power of High-Throughput Experimentation: General Topics and Enabling Technologies for Synthesis and Catalysis (Volume 1); Emmert, M.H., Jouffroy, M., Leitch, D.C.; American Chemical Society, 2022, pp 131-143.
277) Vollbrecht P.J., Nesbitt, K.M., Addis, V.M., Boulnemour, K.M., Micheli, D.A., Smith, K.B., Sandoval, D.A., Kennedy, R.T., Ferrario, C.R. “Differential regulation of nucleus accumbens glutamate and GABA in obesity-prone and obesity-resistant rats” Journal of Neurochemistry, 2022, 164 (4), 499-511.
276) D’Amico, C.I., Polasky, D.A., Steyer, D.J., Ruotolo, B.T., Kennedy, R.T. “Ion mobility-mass spectrometry coupled to droplet microfluidics for rapid protein structure analysis and drug discovery” Analytical Chemistry, 2022, 94 (38), 13084-13091.
275) Kim, Y., Geng, L., Lenhart, A.L., Li, J., Dauer, W.T., Kennedy, R.T. “Measurement of α-synuclein dynamics in vivo using microdialysis with a novel homogeneous Immunoassay” ACS Chemical Neuroscience, 2022, 13, 2557-2564.
274) Booth, P.P.M., Lamb, D.T., Anderson, J.P., Furtaw, M.D., Kennedy, R.T. “Capillary electrophoresis Western blot using inkjet transfer to membrane” Journal of Chromatography A, 2022, 1679, 463389
273) Xu, L., Chang, K-C., Payne, E.M., Modavi, C., Liu, L., Palmer, C.M., Tao N., Alper, H.S., Kennedy, R.T., Cornett, D.S., Abate, A.R. “Mapping enzyme catalysis with metabolic biosensing” Nature Communications, 2021, 12, 6803.
272) Anderson, B.G., Raskind, A., Habra, H., Kennedy, R.T., Evans. C.R. “Modifying chromatography conditions for improved unknown feature identification in untargeted metabolomics” Analytical Chemistry, 2021, 93 (48), 15840–15849.
271) Takatani, T., Shirakawa, J., Sjibue, K., Gupta, M.K., Kim, H., Lu, S., Hu, J., White, M.F., Kennedy, R.T., Kulkarni, R.N. “Insulin receptor substrate 1, but not IRS2, plays a dominant role in regulating pancreatic alpha cell function in mice” Journal of Biological Chemistry, 2021, 296, 100646.
270) Knights, A.J., Liu, S., Ma, Y., Nudell, V.S., Perkey, E., Sorensen, M.J., Kennedy, R.T., Mailard, I., Ye, L., Jun, H., Jun, W. “Acetylcholine-synthesizing macrophages in subcutaneous fat are regulated by β2-adrenergic signaling” The EMBO Journal, 2021, e106061.
269) *Payne, E.M., *Wells, S.S., Kennedy, R.T. “Continuous and automated slug flow nanoextraction for rapid partition coefficient measurement” Analyst, 2021, 146, 5722-5731.
268) Grunkemeyer, T.J., Ghosh, S., Patel, A.M., Sajja, K., Windak, J., Basrur, V., Kim, Y., Nesvizhskii, A.I., Kennedy, R.T., Marsh, E.N.G. “The antiviral enzyme viperin inhibits cholesterol biosynthesis” Journal of Biological Chemistry, 2021, 100824
267) Bartlett, M.J., Mabrouk, O.S., Szabò, L., Flores, A.J., Parent, K.L., Bidlack, J.M., Heien, M.L., Kennedy, R.T., Polt, R., Sherman, S.J., Falk, T. “The Delta-Specific Opioid Glycopeptide BBI-11008: CNS Penetration and Behavioral Analysis in a Preclinical Model of Levodopa-Induced Dyskinesia,” International Journal of Molecular Sciences, 2021, 22(1), 20
266) Valenta, A.C., D’Amico, C.I., Dugan, C.E., Grinias, J.P., Kennedy, R.T. “A microfluidic chip for on-line derivatization and application to in vivo neurochemical monitoring,” Analyst, 2021, 146, 825-834
265) Sorensen, M.J., Kennedy, R.T. “Capillary ultrahigh-pressure liquid chromatography-mass spectrometry for fast and high resolution metabolomics separations,” Journal of Chromatography A, 2021, 1635, 461706
264) *Sun, A.C., *Steyer, D.J., Allen, A.R., Payne, E.M., Kennedy, R.T., Stephenson, C.R.J. “A droplet microfluidic platform for high-throughput photochemical reaction discovery,” Nature Communications, 2020, 11, 6202.
263) Bodner, O., Radzishevsky, I., Foltyn, V.N., Touitou, A., Valenta, A.C., Rangel, I.F., Panizzutti, R., Kennedy, R.T., Billard, J.M., Wolosker, H. “D-Serine signaling and NMDAR-mediated synaptic plasticity are regulated by system A-type of glutamine/D-serine dual transporters,” Journal of Neuroscience, 2020, 40(34), 6489-6502
262) Flak, J.N., Goforth, P.B., Dell’Orco, J., Sabatini, P.V., Li, C., Bozadjieva, N., Sorensen, M.J., Valenta, A.C., Rupp, A., Affinati, A.H., Cras-Méneur, C., Ansari, A., Sacksner, J., Kodur, N., Sandoval, D.A., Kennedy, R.T., Olsen, D.P., Myers, M.G. “Ventromedial hypothalamic nucleus neuronal subset regulates blood glucose independently of insulin,” Journal of Clinical Investigation, 2020, 130(6), 2943-2952
261) Payne, E.M., Holland-Moritz, D.A., Sun, S., Kennedy, R.T. “High-throughput screening by droplet microfluidics: perspective into key challenges and future prospects,” Lab on a Chip, 2020, 20, 2247-2262
260) Mascia, P., Wang, Q., Brown, J., Nesbitt, K.M., Kennedy, R.T., Vezina, P. “Maladaptive consequences of repeated intermittent exposure to uncertainty,” Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2020, 99, 109864
259) Arvin, N.E., Dawod, M., Lamb, D., Furtaw, M.D., Anderson, J., Kennedy, R.T. “Fast Immunoassay for Microfluidic Western Blotting by Direct Deposition of Reagents onto Capture Membrane,” Analytical Methods, 2020, 12, 1606-1616
258) Wells, S.S., Kennedy, R.T. “High-throughput Liquid-Liquid Extractions with Nanoliter Volumes,” Analytical Chemistry, 2020, 92 (4), 3189-3197
257) Ghosh, S., Patel, A.M., Grunkemeyer, T.J., Dumbrepatil, A.B., Zegalia, K.A., Kennedy, R.T., Marsh, E.N.G. “Interactions between Viperin, Vesicle-Associated Membrane Protein A, and Hepatitis C Virus Protein NS5A Modulate Viperin Activity and NS5A Degradation,” Biochemistry, 2020, 59 (6) 780-789
256) Dumbrepatil, A.B., Zegalia, K.A., Sajja, K., Kennedy, R.T., Marsh, E.N.G. “Targeting viperin to the mitochondrion inhibits the thiolase activity of the trifunctional enzyme complex,” Journal of Biological Chemistry, 2020, 295 (9), 2839-2949
255) Sorensen, M.J., Anderson, B.G., Kennedy, R.T. “Liquid chromatography above 20,000 PSI,” Trends in Analytical Chemistry, 2020, 124, 115810
254) Evers, S.S., Kim, K-S., Bozadjieva, N., Lewis, A.G., Farris, D., Sorensen, M.J., Kim, Y., Whitesall, S.E., Kennedy, R.T., Michele, D.E., Seeley, R.J., Sandoval, D.A. “Continuous glucose monitoring reveals glycemic variability and hypoglycemia after vertical sleeve gastrectomy in rats,” Molecular Metabolism, 2020, 32, 148-159
253) Holland-Moritz, D.A., Wismer, M., Mann, B.F., Farasat, I., Devine, P., Guetschow, E.D., Mangion, I., Welch, C.J., Moore, J.C., Sun, S., Kennedy, R.T. “Mass Activated Droplet Sorting (MADS) Enables High Throughput Screening of Enzymatic Reactions at Nanoliter Scale,” Angewandte Chemie International Edition, 2020, 59, 4470–4477
252) Sorensen, M.J., Miller, K.E., Jorgenson. J.W., Kennedy, R.T. “Ultrahigh-Performance capillary liquid chromatography-mass spectrometry at 35 kpsi for separation of lipids,” Journal of Chromatography A, 2020, 1611, 460575
251) Wells, S.S., Dawod, M., Kennedy, R.T. “CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters,” Electrophoresis, 2019, 40(22), 2946–2953
250) Ouimet, C.M., D’Amico, C.I., Kennedy, R.T. “Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions,” Analytical and Bioanalytical Chemistry, 2019, 411(23), 6155–6163
249) Tardu, M., Jones, J.D., Kennedy, R.T., Lin, Q., Koutmou, K.S. “Identification and Quantification of Modified Nucleosides in Saccharomyces cerevisiae mRNAs,” ACS Chemical Biology, 2019, 14(7), 1403-1409
248) Kawa, A.B., Valenta, A.C., Kennedy, R.T., Robinson, T.E. “Incentive and dopamine sensitization produced by intermittent but not long access cocaine self‐administration,” European Journal of Neuroscience, 2019, 50 (4), 2663-2682
247) Mohebi, A., Pettibone, J.R., Hamid, A.A., Wong, J-M.T., Vinson, L.T., Patriarchi, T., Tian, L., Kennedy, R.T., Berke, J.D. “Dissociable dopamine dynamics for learning and motivation,” Nature, 2019, 570, 65–70
246) Minogianis, E-A., Shams, W.M., Mabrouk, O.S., Wong, J-M.T., Brake, W.G., Kennedy, R.T., du Souich, P., Samaha, S-N. “Varying the rate of intravenous cocaine infusion influences the temporal dynamics of both drug and dopamine concentrations in the striatum,” European Journal of Neuroscience, 2019, 50 (3), 2054-2064
245) Steyer, D.J., Kennedy, R.T. “High-Throughput Nanoelectrospray Ionization-Mass Spectrometry Analysis of Microfluidic Droplet Samples,” Analytical Chemistry, 2019, 91(10), 6645-6651
244) Dumbrepatil, A.B., Ghosh, S., Zegalia, K.A., Malec, P.A. Hoff, J.D. Kennedy, R.T. Marsh, E.N.G. “Viperin interacts with the kinase IRAK1 and the E3 ubiquitin ligase TRAF6, coupling innate immune signaling to antiviral ribonucleotide synthesis,” Journal of Biological Chemistry, 2019, 294, 6888-6898
243) Zestos, A.G., Luna-Munguia, H., Stacey, W.C., Kennedy, R.T. “Use and Future Prospects of in Vivo Microdialysis for Epilepsy Studies,” ACS Chemical Neuroscience, 2019, 10, 1875-1883
242) Zestos, A.G., Carpenter, C., Kim, Y., Low, M.J., Kennedy, R.T., Gnegy, M.E. “Ruboxistaurin Reduces Cocaine-Stimulated Increases in Extracellular Dopamine by Modifying Dopamine-Autoreceptor Activity,” ACS Chemical Neuroscience, 2019, 10, 1960-1969
241) Mascia, P., Neugebauer, N.M., Brown, J., Bubula, N., Nesbitt, K.M., Kennedy, R.T., Vezina, P. “Exposure to conditions of uncertainty promotes the pursuit of amphetamine,” Neuropsychopharmacology, 2019, 44, 274-280
240) Luna-Munguia, H., Zestos, A.G., Gliske, S.V., Kennedy, R.T., Stacey, W.C. “Chemical biomarkers of epileptogenesis and ictogenesis in experimental epilepsy,” Neurobiology of Disease, 2019, 121, 177-186
239) Khaksari, M., Mazzoleni, L.R., Ruan, C., Song, P., Hershey, N.D., Kennedy, R.T., Burns, M.A., Minerick, A.R. “Detection and quantification of vitamins in microliter volumes of biological samples by LC‐MS for clinical screening,” AlChE, 2018, 64 (10), 3709-3718
238) Ngernsutivorakul, T., Steyer, D.J., Valenta, A.C., Kennedy, R.T. “In Vivo Chemical Monitoring at High Spatiotemporal Resolution Using Microfabricated Sampling Probes and Droplet-Based Microfluidics Coupled to Mass Spectrometry,” Analytical Chemistry, 2018, 90, 10943−10950
237) Kaplan, E., Zubedat, S., Radzishevsky, I., Valenta, A.C., Rechnitz, O., Sason, H., Sajrawi, C., Bodner, O., Konno, K., Esaki, K., Derdikman, D., Yoshikawa, T., Watanabe, M., Kennedy, R.T., Billard, J-M., Avital, A., Woloskera, H. “ASCT1 (Slc1a4) transporter is a physiologic regulator of brain d-serine and neurodevelopment,” PNAS, 2018, 115 (38), 9628-9633
236) Al-Hasani, R., Wong, J-M.T., Mabrouk, O.S., McCall, J.G., Schmitz, G.P., Porter-Stransky, K.A., Aragona, B.J., Kennedy, R.T., Bruchas, M.R. “In vivo detection of optically-evoked opioid peptide release,” eLife, 2018, 7:e36520
235) Vollbrecht, P.J., Nesbitt, K.M., Mabrouk, O.S., Chadderon, A.M., Jutkiewicz, E.M., Kennedy, R.T., Ferrario, C.R. “Cocaine and Desipramine Elicit Distinct Stiatal Noradrenergic and Behavioral Responses in Selectively Bred Obesity-Resistant and Obesity-Prone Rats,” Behavioral Brain Research, 2018, 346, 137-143
234) Jun, H., Yu, H., Gong, J., Jiang, J., Qiao, X., Perkey, E., Kim, D., Emont, M.P., Zestos, A.G., Cho, J., Liu, J., Kennedy, R.T., Maillard, I., Xu. S., Wu, J. “An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling,” Nature Medicine, 2018, 24, 814-822
233) Ngernsutivorakul, T., White, T.S., Kennedy, R.T. “Microfabricated Probes for Studying Brain Chemistry: A Review,” ChemPhysChem, 2018, 19, 1128–1142
232) Ouimet, C.M., Dawod, M., Grinias, J.P., Assimon, V.A., Lodge, J., Mapp, A.K., Gestwicki, J.E., Kennedy, R.T. “Protein Cross-Linking Capillary Electrophoresis at Increased Throughput for a Range of Protein-Protein Interactions,” Analyst, 2018, 143(8), 1805-1812
231) Mitok, K.A., Freiberger, E.C., Schueler, K.L., Rabaglia, M.E., Stapleton, D.S., Kwiecien, N.W., Malec, P.A., Herbert, A.S., Broman, A.T., Kennedy, R.T., Keller, M.P., Coon, J.J, Attie, A.D. “Islet Proteomics Reveals Genetic Variation in Dopamine Production Resulting in Altered Insulin Secretion,” JBC, 2018, 293(16), 5860-5877
230) Lu, S.S., Dugan, C.E., Kennedy, R.T. “Microfluidic Chip with Integrated Electrophoretic Immunoassay for Investigating Cell-Cell Interactions,” Anal. Chem., 2018, 90(8), 5171-5178
229) Mabrouk, O.S., Han, J.L., Wong, J-M.T., Akil, H., Kennedy, R.T., Flagel, S.B. “The In Vivo Neurochemical Profile of Selectively Bred High-Responder and Low-Responder Rats Reveals Baseline, Cocaine-Evoked, and Novelty-Evoked Differences in Monoaminergic Systems,”ACS Chem. Neurosci., 2018, 9(4), 715-724
228) Diefenbach, X.W., Farasat, I., Guetschow, E.D., Welch, C.J., Kennedy, R.T., Sun, S.W., Moore, J.C. “Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry,” ACS Omega, 2018, 3(2), 1498-1508
227) Zestos, A.G., Kennedy, R.T. “Microdialysis Coupled with LC-MS/MS for In Vivo Neurochemical Monitoring,” AAPS, 2017, 19(5), 1284-1293
226) Malec, P.A., Oteri, M., Inferrera, V., Cacciola, F., Mondello, L., Kennedy, R.T. “Determination of Amines and Phenolic Acids in Wine with Benzoyl Chloride Derivitization and Liquid Chromatography-Mass Spectrometry,”, J. Chrom. A, 2017, 1523, 248-256
225) Carpenter, C., Zestos, A.G., Altshuler, R., Sorenson, R.J., Guptaroy, B., Showalter, H.D., Kennedy, R.T., Jutkiewicz, E., Gnegy, M.E. “Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects”, Neurophyschopharmacology, 2017, 42(10), 1940-1949
224) Dawod, M., Arvin, N.E., Kennedy, R.T. “Recent Advances in Protein Analysis by Capillary and Microchip Electrophoresis”, Analyst, 2017, 142(11), 1847-1866
223) Al-Hasani, R., Wong, J-M.T., McGall, J., Mabrouk, O.S., Schmitz, G., Porter-Stransky, K., Bernardi, J.M., Aragona, B., Kennedy, R.T., Bruchas, M.R. “Circuit Dynamics of In Vivo Dynorphin Release in the Nucleus Accumbens”, Alchohol, 2017, 60, 220
222) Longo, F., Mercatelli, D., Novello, S., Arcuri, L., Brugnoli, A., Vincenzi, F., Russo, I., Berti, G., Mabrouk, O. S., Kennedy, R.T., Shimshek, D.R., Varani, K., Bubacco, L., Greggio, E., Morari, M. “Age-dependent dopamine transporter dysfunction and Serine129 phospho-alpha-synuclein overload in G2019S LRRK2 mice”, Acta Neuropathologica Communications, 2017, 5(22), 1–17.
221) Khaksari, M., Mazzoleni, L.R., Ruan, C., Kennedy, R.T., Minerick, A.R. “Determination of water-soluble and fat-soluble vitamins in tears and blood serum of infants and parents by liquid chromatography/mass spectrometry”, Experimental Eye Research, 2017, 155, 54–63.
220) Ouimet, C.M., D’Amico, C.I., Kennedy, R.T. “Advances in capillary electrophoresis and the implications for drug discovery”, Expert Opinion on Drug Discovery, 2017, 12(2), 213-224.
219) Schmudlach, A., Felton, J., Kennedy, R.T., Dovichi, N.J. “Bottom-up proteomics analysis of the secretome of murine islets of Langerhans in elevated glucose levels”, Analyst, 2017, 142, 284–291.
218) Dugan, C.E., Grinias, J.P., Parlee, S.D., El-azzouny, M., Evans, C.R., Kennedy, R.T. “Monitoring cell secretions on microfluidic chips using solid-phase extraction with mass spectrometry”, Anal. Bioanal. Chem. 2017, 409(1), 169–178.
217) Ngernsutivorakul, T., Cipolla, C.M., Dugan, C.E., Jin, S., Morris, M.D., Kennedy, R.T., Esmonde-white, F.W.L. “Design and microfabrication of a miniature fiber optic probe with integrated lenses and mirrors for Raman and fluorescence measurements”, Anal. Bioanal. Chem. 2017, 409(1), 275–285.
216) Makins, C., Ghosh, S., Román-Meléndez, G.D., Malec, P.A., Kennedy, R.T., Marsh, E.N.G. “Does Viperin Function as a Radical S-Adenosyl-L-methionine-dependent Enzyme in Regulating Farnesylpyrophosphate Synthase Expression and Activity?”, JBC, 2016, 291, 26806-26815.
215) Cui, Q., Pitt, J.E., Pamukcu, A., Poulin, J-F., Mabrouk, O.S., Fiske, M.P., Fan, I.B., Augustine, E.C., Young, K.A, Kennedy, R.T., Awatramani, R.C., Chan, S.“Blunted mGluR Activation Disinhibits Striatopallidal Transmission in Parkinsonian Mice”, Cell Reports, 2016, 17(9), 2431-2444.
214) El Azzouny, M., Longacre, M.J., Ansari, I.H., Kennedy, R.T., Burant, C.F., MacDonald, M.J. “Knockdown of ATP citrate lyase in pancreatic beta cells does not inhibit insulin secretion or glucose flux and implicates the acetoacetate pathway in insulin secretion”, Molecular Metabolism, 2016, 5(10), 980-987.
213) Grinias, J.P., Wong, J-M.T., Kennedy, R.T. “Repeatability of gradient ultrahigh pressure liquid chromatography–tandem mass spectrometry methods in instrument-controlled thermal environments”, J. Chrom. A, 2016, 1461, 42-50.
212) Ro, J., Pak, G., Malec, P.A., Lyu, Y., Allison, D.B., Kennedy, R.T., Pletcher, S.D. “Serotonin signaling mediates protein valuation and aging”, Elife, 2016, 5, 1–22.
211) Ouimet, C.M., Shao, H., Rauch, J.N., Dawod, M., Nordhues, B., Dickey, C.A., Gestwicki, J.E., Kennedy, R.T. “Protein Cross-Linking Capillary Electrophoresis for Protein Protein Interaction Analysis”, Anal. Chem., 2016, 88 (16), 8272–8278.
210) Jin, S., Furtaw, M.D., Chen, H., Lamb, D.T., Ferguson, S.A., Arvin, N.E., Dawod, M., Kennedy, R.T. “Multiplexed Western Blotting Using Microchip Electrophoresis”, Anal. Chem., 2016, 88 (13), 6703–6710.
209) Grinias, J.P., Whitfield, J.T., Guetschow, E.D., Kennedy, R.T. “An Inexpensive, Open-Source USB Arduino Data Acquisition Device for Chemical Instrumentation” J. Chem. Educ., 2016, 93 (7), 1316–1319.
208) Grinias, J.P., Kennedy, R.T. “Advances in and prospects of microchip liquid chromatography”, Trends Anal. Chem., 2016, 81, 110–117.
207) Zestos, A.G., Mikelman, S.R., Kennedy, R.T., Gnegy, M.E. “PKC beta Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux”, ACS Chem. Neurosci., 2016, 7 (6), 757–766.207)
206) Wong, J-M.T., Malec, P.A., Mabrouk, O.S., Ro, J., Dus, M., Kennedy, R.T. “Benzoyl chloride derivatization with liquid chromatography-mass spectrometry for targeted metabolomics of neurochemicals in biological samples”, J. Chromatogr. A, 2016, 1446, 78–90.
205) Vollbrecht, P.J., Mabrouk, O.S., Nelson, A.D., Kennedy, R.T., Ferrario, C.R. “Pre-Existing Differences and Diet-Induced Alterations in Striatal Dopamine Systems of Obesity-Prone Rats”, Obesity, 2016, 24 (3), 670–677.
204) Glynn, E., Thompson, B., Vadrevu, S., Lu, S., Kennedy, R.T., Ha, J., Sherman, A., Satin, L.S. “Chronic Glucose Exposure Systematically Shifts the Oscillatory Threshold of Mouse Islets: Experimental Evidence for an Early Intrinsic Mechanism of Compensation for Hyperglycemia”, Endocrinology, 2016, 157 (2), 611–623.
203) Lee, W.H., Ngernsutivorakul, T., Mabrouk, O.S., Wong, J-M.T., Dugan, C.E., Pappas, S.S., Yoon, H.J., Kennedy, R.T. “Microfabrication and in Vivo Performance of a Microdialysis Probe with Embedded Membrane”, Anal. Chem., 2016, 88 (2), 1230–1237.
202) Karra, S., Griffith, W.P., Kennedy, R.T., Gorski, W. “Hormone glucagon: electrooxidation and determination at carbon nanotubes”, Analyst, 2016, 141 (8), 2405–2411.
201) Sun, S., Buer, B.C., Marsh, E.N.G., Kennedy, R.T. “A label-free Sirtuin 1 assay based on droplet electrospray ionization mass spectrometry”, Anal. Methods, 2016, 8 (17), 3458–3465.
200) Schmudlach, A., Felton, J., Cipolla, C., Sun, L., Kennedy, R.T., Dovichi, N.J. “Sample preparation protocol for bottom-up proteomic analysis of the secretome of the islets of Langerhans”, Analyst, 2016, 40, 1700–1706.
199) Hamid, A.A., Pettibone, J.R., Mabrouk, O.S., Hetrick, V.L., Schmidt, R., Vander Weele, C.M., Kennedy, R.T., Aragona, B.J., Berke, J.D. “Mesolimbic dopamine signals the value of work“, Nature Neuroscience, 2016, 19 (1), 127-139
198) Guetschow, E.D., Kumar, S., Lombard, D.B., Kennedy, R.T. “Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples”, Anal. Bioanal. Chem., 2016, 408, 721–731
197) Zhou, Y., Wong, J-M.T., Mabrouk, O.S., Kennedy, R.T. “Reducing Adsorption To Improve Recovery and in Vivo Detection of Neuropeptides by Microdialysis with LC-MS”, Anal. Chem., 2015, 87, 9802-9809.
196) El Azzouny, M.A., Evans C.R., Burant, C.F., Kennedy, R.T. “Metabolomics Analysis Reveals that AICAR Affects Glycerolipid, Ceramide and Nucleotide Synthesis Pathways in INS-1 Cells”, PLOS One, 2015, 10(6), e0129029
195) Pappas, S.S., Darr, K., Holley, S.M., Cepeda, C., Mabrouk, O.S., Wong, J-M.T., LeWitt, T.M., Paudel, R., Houlden, H., Kennedy, R.T., Levine, M.S., Dauer, W.T. “Forebrain deletion of the dystonia protein torsinA causes dystonic-like movements and loss of striatal cholinergic neurons”, eLife, 2015, 4, e08352
194) El Azzouny, M.A., Kennedy, R.T., Burant, C.F. “Alterations in Fatty Acid Metabolism Associated with Glucolipotoxicity in beta-Cells” Diabetes, 2015, 64(Supplement 1), A596
193) Patterson, C.M., Wong, J-M.T., Leinninger, G.M., Allison, M.B., Mabrouk, O.S., Kasper, C.L., Gonzalez, I.E., Mackenzie, A., Jones, J.C., Kennedy, R.T., Myers, M.G. “Striatal Dopamine Efflux in Male Mice” Endocrinology, 2015, 156(5), 1692–1700.
192) Li, D.; Mabrouk, O. S.; Liu, T. C.; Tian, F. Y.; Xu, G.; Rengfigo, S.; Choi, S. J.; Mathur, A.; Crooks, C. P.; Kennedy, R. T.; Wang, M. M.; Ghanbari, H.; Borjigin, J. “Asphyxia-Activated Corticocardiac Signaling Accelerates Onset of Cardiac Arrest” PNAS Plus. 2015, E2073–E2082
191) Jin, S.; Kennedy, R. T. “New Developments in Western Blotting” Chinese Chemical Letters, 2015, 4, 416-418.
190) Grinias, J. P.; Kennedy, R. T. “Evaluation of 5 μm Superficially Porous Particles for Capillary and Microfluidic LC Columns.” Chromatography. 2015, 2, 502-514.
189) Patterson C. M.; Wong J. M. T.; Leinninger G. M.; Allison M. B.; Mabrouk O. S.; Kasper C. L.; Gonzalez I. E.; Mackenzie A.; Jones J. C.; Kennedy R. T.; Myers M. G. Jr. “Ventral tegmental area neurotensin signaling links the lateral hypothalamus to locomotor activity and striatal dopamine efflux in male mice.” Endocrinology, 2015, 156(5), 1692-1700
188) Cepeda D. E.; Hains L, Li D.; Bull J, Lentz S. I.; Kennedy R. T. “Experimental evaluation and computational modeling of tissue damage from low-flow push-pull perfusion sampling in vivo.” J Neurosci Methods, 2015, 242C, 97-105.
187) Flak JN, Patterson CM, Garfield AS, D’Agostino G, Goforth PB, Sutton AK, Malec PA, Wong JM, Germani M, Jones JC, Rajala M, Satin L, Rhodes CJ, Olson DP, Kennedy RT, Heisler LK, Myers MG Jr. “Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance.” Nat Neurosci. 2014. 17(12):1744-50.
186) Kuo S, Zhou Y, Kim HM, Kato H, Kim RY, Bayar GR, Marcelo CL, Kennedy RT, Feinberg SE. “Biochemical indicators of implantation success of tissue-engineered oral mucosa.“J Dent Res. 2015. 94(1):78-84.
185) Guetschow ED, Steyer DJ, Kennedy RT. “Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.” Anal Chem. 2014, 86(20):10373-9.
184) Vander Weele CM, Porter-Stransky KA, Mabrouk OS, Lovic V, Singer BF, Kennedy RT, Aragona BJ. “Rapid dopamine transmission within the nucleus accumbens: dramatic difference between morphine and oxycodone delivery.” Eur J Neurosci. 2014, 40(7):3041-54.
183) Sun S, Kennedy RT. “Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.” Anal Chem. 2014, 86(18):9309-14
182) Dugan CE, Cawthorn WP, MacDougald OA, Kennedy RT. “Multiplexed microfluidic enzyme assays for simultaneous detection of lipolysis products from adipocytes.” Anal Bioanal Chem. 2014, 406(20):4851-9
181) El-Azzouny M, Evans CR, Treutelaar MK, Kennedy RT, Burant CF. “Increased Glucose Metabolism and Glycerolipid Formation by Fatty Acids and GPR40 Receptor Signaling Underlies the Fatty Acid Potentiation of Insulin Secretion.” JOURNAL OF BIOLOGICAL CHEMISTRY. 2014, 289(19):13575-13588
180) Bosse KE, Jutkiewicz EM, Schultz-Kuszak KN, Mabrouk OS, Kennedy RT, Gnegy ME, Traynor JR. “Synergistic activity between the delta-opioid agonist SNC80 and amphetamine occurs via a glutamatergic NMDA-receptor dependent mechanism.” NEUROPHARMACOLOGY. 2014, 77:19-27
179) Dugan CE, Kennedy RT. “ Measurement of Lipolysis Products Secreted by 3T3-L1 Adipocytes Using Microfluidics.” METHODS OF ADIPOSE TISSUE BIOLOGY, Book Series: Methods in Enzymology. 2014, 538:195-209
178) Mabrouk OS, Semaan DZ, Mikelman S, Gnegy ME, Kennedy RT. “Amphetamine stimulates movement through thalamocortical glutamate release.” JOURNAL OF NEUROCHEMISTRY. 2014, 128(1):152-161
177) Nie J, Kennedy RT. “Capillary liquid chromatography fraction collection and postcolumn reaction using segmented flow microfluidics.” JOURNAL OF SEPARATION SCIENCE. 2013, 36(21-22):3471-3477
176) Rauch JN，Nie J，Buchholz TJ，Gestwicki JE，Kennedy RT. “Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions.” Analytical Chemistry. 2013, 85(20):9824-9831
175) Kennedy RT. “Emerging trends in in vivo neurochemical monitoring by microdialysis.” CURRENT OPINION IN CHEMICAL BIOLOGY. 2013, 17(5):860-7
174) Davda D, El Azzouny MA, Hernandez JL, Majmudar JD, Kennedy RT, Martin BR. “Profiling Targets of the Irreversible Palmitoylation Inhibitor 2-Bromopalmitate.” ACS CHEMICAL BIOLOGY. 2013, 8(9):1912-7
173) Jin S, Anderson GJ, Kennedy RT. “Western blotting using microchip electrophoresis interfaced to a protein capture membrane.” Analytical Chemistry. 2013, 85(12):6073-9.
172) Zhou Y, Mabrouk OS, Kennedy RT. “Rapid Preconcentration for Liquid Chromatography-Mass Spectrometry Assay of Trace Level Neuropeptides.” Journal of American Society Mass Spectrometry 2013 Apr 17. [Epub ahead of print]
171) Lee WH, Slaney TR, Hower RW, Kennedy RT. “Microfabricated sampling probes for in vivo monitoring of neurotransmitters.” Analytical Chemistry 2013, 85(8):3828-31.
170) Akiyama M, Liew CW, Lu S, Hu J, Martinez R, Hambro B, Kennedy RT, Kulkarni RN. “X-Box Binding Protein 1 Is Essential for Insulin Regulation of Pancreatic α-Cell Function.” Diabetes. 2013, 62(7):2439-49.
169) Lorenz MA, El Azzouny MA, Kennedy RT, Burant CF. “Metabolome response to glucose in the β-cell line INS-1 832/13.” Journal of Biological Chemistry 2013, 288(15):10923-35.
168) Slaney TR, Mabrouk OS, Porter-Stransky KA, Aragona BJ, Kennedy RT. “Chemical gradients within brain extracellular space measured using low flow push-pull perfusion sampling in vivo.” ACS Chemical Neuroscience. 2013, 4(2):321-9.
167) Hershey ND, Kennedy RT. “In Vivo Calibration of Microdialysis Using Infusion of Stable-Isotope Labeled Neurotransmitters.” ACS Chemical Neuroscience. 2013, in press.
166) Mabrouk OS, Falk T, Sherman SJ, Kennedy RT, Polt R. “CNS penetration of the opioid glycopeptide MMP-2200: a microdialysis study.” Neuroscience Letters 2012, 531(2):99-103.
165) DiFeliceantonio AG, Mabrouk OS, Kennedy RT, Berridge KC. “Enkephalin surges in dorsal neostriatum as a signal to eat.”Current Biology 2012, 22(20):1918-24.
164) Mabrouk OS, Kennedy RT. “Simultaneous oxytocin and arg-vasopressin measurements in microdialysates using capillary liquid chromatography-mass spectrometry.” Journal of Neuroscience Methods 2012, 209(1):127-33.
163) Sun S, Slaney TR, Kennedy RT. “Label free screening of enzyme inhibitors at femtomole scale using segmented flow electrospray ionization mass spectrometry.” Analytical Chemsitry 2012, 84(13):5794-800.
162) Song P, Hershey ND, Mabrouk OS, Slaney TR, Kennedy RT.”Mass spectrometry “sensor” for in vivo acetylcholine monitoring.”Analytical Chemistry 2012, 84(11):4659-64.
161) Morioka T, Dishinger JF, Reid KR, Liew CW, Zhang T, Inaba M, Kennedy RT, Kulkarni RN. “Enhanced GLP-1- and sulfonylurea-induced insulin secretion in islets lacking leptin signaling.” Molecular Endocrinology 2012, 26(6):967-76.
160) Payeur AL, Lorenz MA, Kennedy RT. “Analysis of fatty acid composition in insulin secreting cells by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry.”J Chromatogr B Analyt Technol Biomed Life Sci. 2012, 893-894:187-92
159) Shackman JG, Reid KR, Dugan CE, Kennedy RT. “Dynamic monitoring of glucagon secretion from living cells on a microfluidic chip.” Analytical and Bioanalytical Chemistry 2012 Mar;402(9):2797-803.
158) Hesdorffer C.S., E. Malchinkhuu, A. Biragyn, Mabrouk OS, Kennedy RT, Madara K, Taub DD, Longo DL, Schwartz JB, Ferrucci L,& Goetzl EJ. “Distinctive immunoregulatory effects of adenosine on T cells of older humans.” FASEB Journal, (2012) 26, 1301-10.
157) Peng Song, Omar S. Mabrouk, Neil D. Hershey, and Robert T. Kennedy. “In Vivo Neurochemical Monitoring Using Benzoyl Chloride Derivatization and Liquid Chromatography–Mass Spectrometry“. Analytical Chemistry, 2012, 84(1), 412–419.
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.
118) Dishinger JF, Kennedy RT. “Serial immunoassays in parallel on a microfluidic chip for monitoring hormone secretion from living cells”, ANALYTICAL CHEMISTRY 79 (3): 947-954 FEB 1 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
Abstracts
120) High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-B beta, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-B beta and Fyn complex barely affected. IC50 of both selective and nonselective inhibitors were determined and compared for different proteins. The IC50 of the nonselective inhibitor was 49 +/- 9, 323 +/- 42, and 228 +/- 19 mu M (n = 3) for Src, SH2-B beta, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions.
119) Capillary liquid chromatography-mass spectrometry (cLC-MS) was coupled on-line to microdialysis sampling to monitor endogenous acetylcholine (ACh) from the rodent brain. In vivo microdialysate sampled at 0.6 mu L/min from the striatum of ketamine or chloral hydrate anesthetized rats was loaded onto a sample loop and then injected onto a similar to 5 cm long strong cation exchange (SCX) capillary column. A step gradient was used to separate the analyte from ionization suppressing salts contained in dialysate in 2.4 min. Sampling coupled on-line with cLC-MS allowed for high temporal resolution (data points at 2.4 min intervals), good reproducibility (10-15% relative standard deviation, R.S.D.), and sensitive limits of detection (0.04 nM or 8 amol injected). The method successfully monitored basal and stimulated levels (induced by increased K+ concentrations) of ACh from the anesthetized rat without necessitating perfusion of an acetyleholinesterase (AChE) inhibitor. Absolute and percent basal levels of ACh from rats receiving different anesthetics were also compared.
118) A microfluidic chip that allows for the continuous monitoring of cellular secretions from multiple independent living samples was developed. Performance of the device was characterized through the analysis of insulin secretion from islets of Langerhans. The chip contained four individual channel networks, each capable of performing electrophoresis-based immunoassays of the perfusate from islets. In the networks, islets were housed in a chamber that was continuously perfused with pressure-driven biological media at 0.6 mu L min(-1). Electroosmosis was used to pull perfusate containing secreted insulin into 4-cm-long reaction channels where it mixed with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for 60 s. The reaction streams were sampled at 6.25-s intervals and analyzed in parallel using an on-chip capillary electrophoresis separation with laser-induced fluorescence detection by a scanning confocal microscope. The limit of detection for insulin was 10 nM. The device was used to complete over 1450 immunoassays of biological samples in less than 40 min, allowing the parallel monitoring of insulin release from four islets every 6.25 s.
117) We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha(2A) adrenoreceptor-G(alpha o1) wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha(2a) adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 mu M. The EC50 increased to 9.4 +/- 5 mu M with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.
116) Microdialysis sampling was coupled on-line to micellar electrokinetic chromatography (MEKC) to monitor extracellular dopamine concentration in the brains of rats. Microdialysis probes were perfused at 0.3 mu L/min and the dialysate mixed on-line with 6 mM naphthalene-2,3-dicarboxaldehye and 10 mM potassium cyanide pumped at 0.12 mu L/min each into a reaction capillary. The reaction mixture was delivered into a flow-gated interface and separated at 90-s intervals. The MEKC separation buffer consisted of 30 mM phosphate, 6.5 mM SDS, and 2 mM HP-beta-CD at pH 7.4, and the electric field was 850 V/cm applied across a 14-cm separation distance. Analytes were detected by laser-induced fluorescence excited using the 413-nm line of a 14-mW diode-pumped laser. The detection limit for dopamine was 2 nM when sampling by dialysis. The basal dopamine concentration in dialysates collected from the striatum of anesthetized rats was 18 +/- 3 nM (n = 12). The identity of the putative dopamine peak was confirmed by showing that dopamine uptake inhibitors increased the peak and dopamine synthesis inhibitors eliminated the peak. The utility of this method for behavioral studies was demonstrated by correlating dopamine concentrations in vivo and with psychomotor behavior in freely moving rats following the intravenous administration of cocaine. Over 60 additional peaks were detected in the electropherograms, suggesting the potential for monitoring many other substances in vivo by this method.
115) A chip fabricated by multilayer soft lithography of poly(dimethylsiloxane) was created for separations-based sensing of neurotransmitters in vivo. The chip incorporated a pneumatically actuated peristaltic pump and valving system to combine low-flow push -pull perfusion sampling, on-line derivatization, and flow-gated injection onto an embedded fused-silica capillary for high speed separation of amine neurotransmitters from the brain of living animals. Six 160 mm wide by 10 mm high control channels, actuated with an overlapping 60 degrees pulse sequence, simultaneously drove sample and buffers through fluidic channels of the same dimensions. Tunable sampling flow rates of 40 to 130 nL min(-1) and separation buffer flow rates of 380 to 850 nL min(-1) were achieved with actuation frequencies between 3 and 10 Hz. On-line sampling of amine neurotransmitters with separation efficiencies in excess of 250 000 plates, detection limits of similar to 40 nM, and relative standard deviations of 4% for glutamate and aspartate were achieved in vitro. Electropherograms with resolution of gamma-aminobutyric acid, glutamine, taurine, serine, glycine, o-phosphorylethanolamine, glutamate, and aspartate could be collected every 30 s for over 4 h in vivo. It was also shown that pharmacological agents could be delivered and subsequent changes in neurotransmitter profile could be measured when delivering either 70 mM K+ artificial cerebrospinal fluid or 200 mu M L-trans-pyrrolidine-2,4-dicarboxilic acid with the chip. These results demonstrate the ability of this chip to sample and monitor chemicals in the complex environment of the central nervous system with high selectivity and sensitivity over extended periods.
114) Capillary liquid chromatography (CLC) coupled off-line with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/ TOF-MS were explored for identification and quantification of neuropeptides in microwave-fixed rat brain tissue. Sample was separated by gradient elution on 50-mu m-inner diameter reversed-phase columns at 180 nL/min. Effluent was mixed with matrix solution and transferred to a MALDI target plate by pulsed electric field deposition, yielding sample spots with 200-300-mu m diameter. Mass detection limits as low as 2 amol, corresponding to 1 pM concentration, were achieved for neuropeptides. CLC-MALDI-TOF- MS analysis of microwave-fixed rat striatum tissue yielded detection of over 400 distinctive peaks. CLC-MALDI-TOF/TOF-MS allowed identification of 10 peptides including 3 novel peptides. Quantification was evaluated using substance P as analyte and N-15(3)-labeled substance P as an internal standard. Quantification of substance P revealed similar to 6.8-fold higher levels than previously reported in the rat striatum. This increase is attributed to use of microwave fixation, which prevented degradation of the peptide, aggressive extraction procedures, and accounting for oxidation of substance P in the analysis. These results demonstrate that CLC-MALDI-TOF-MS is a versatile tool for neuropeptide analysis in brain tissue by allowing for detection, identification, and quantification.
113) A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CELIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or G alpha(s)-GTP gamma S as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected EC50 values for forskolin and G alpha(s)-GTP gamma S of 27 +/- 6 mu M and 317 +/- 56 nM, respectively. These EC50 values compared well to those previously reported using [alpha-P-32]ATP as substrate. When AC was concurrently activated with 2.5 mu M forskolin and 25 nM G alpha(s)-GTP gamma S, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2′-(or-3′)-O-(N-methylanthraniloyl) guanosine 5′-triphosphate with an IC50 of 9 +/- 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.
112) Capillary electrophoresis (CE) was coupled to negative mode electrospray ionisation-mass spectrometry (MS) for separation and detection of phosphorylated and acidic metabolites in extracts of prokaryotes. Unlike previous CE-MS systems for metabolite analysis, a sheathless interface was used to improve sensitivity. To accomplish this, the separation capillary was modified by creating a porous junction near the outlet where the electrospray voltage and cathodic voltage for CE were applied. The outlet of the capillary was pulled to a 5 microm inner diameter to form an electrospray emitter and had a frit fabricated near the exit to prevent clogging. During analysis pressure was applied at the inlet of the separation column to create sufficient flow towards the detector. Limits of detection for 19 metabolites in full scan mode ranged from 20 nM for ADP ribose to 2.5 microM for alpha-ketoglutarate for 40 nL injections. Extracts of Escherichia coli, strain DH5-alpha, were analyzed using this system. In full scan mode, 118 different metabolites were detected. Tandem mass spectrometry was also employed to attempt identification. Reproducible fragmentation of 19 parent peaks was found and 10 of these produced spectra that were consistent with identification obtained from matching to compounds in the MetaCyc database. These results demonstrate the utility of a sensitive CE-MS system for large scale metabolite detection in biological samples.
111) Microfluidic electrophoresis devices were coupled on-line to microdialysis for in vivo monitoring of primary amine neurotransmitters in rat brain. The devices contained a sample introduction channel for dialysate, a precolumn reactor for derivatization with o-phthaldialdehyde, a flow-gated injector, and a separation channel. Detection was performed using confocal laser-induced fluorescence. In vitro testing revealed that the initial device design had detection limits for amino acids of ~200 nM, relative standard deviation of peak heights of 2%, and separations within 95 s with up to 30 200 theoretical plates when applying an electric field of 370 V/cm. A second device design that allowed electric fields of 1320 V/cm to be applied while preserving the reaction time allowed separations within 20 s with up to 156 000 theoretical plates. Flow splitting into the electrokinetic network from hydrodynamic flow in the sample introduction channel was made negligible for sampling flow rates from 0.3 to 1.2 L/min by placing a 360-m-diameter fluidic access hole that had flow resistance (0.15-7.2) × 108-fold lower than that of the electrokinetic network at the junction of the sample introduction channel and the electrokinetic network. Using serial injections, the device allowed the dialysate stream to be analyzed at 130-s intervals. In vivo monitoring was demonstrated by using the microdialysis/microfluidic device to record glutamate concentrations in the striatum of an anesthetized rat during infusion of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid. These results prove the feasibility of using a microfabricated fluidic system coupled to sampling probes for chemical monitoring of complex media such as mammalian brain.
110) Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 m wide by 15 m high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100 000 theoretical plates were acquired at ~20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were ~60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10% in vivo. Basal concentrations of glutamate were 1.9 ± 0.6 M (n = 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K+ through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of ~45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.
109) A sample preparation method that combines a modified target plate with a nanoscale reversed-phase column (nanocolumn) was developed for detection of neuropeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A gold-coated MALDI plate was modified with an octadecanethiol (ODT) self-assembled monolayer to create a hydrophobic surface that could concentrate peptide samples into a 200-500-µm diameter spot. The spot sizes generated were comparable to those obtained for a substrate patterned with 200-µm hydrophilic spots on a hydrophobic substrate. The sample spots on the ODT-coated plate were 100-fold smaller than those formed on an unmodified gold plate with a 1-µl sample and generated 10 to 50 times higher mass sensitivity for peptide standards by MALDI-TOF MS. When the sample was deposited on an ODT-modified plate from a nanocolumn, the detection limit for peptides was as low as 20 pM for 5-µl samples corresponding to 80 amol deposited. This technique was used to analyze extracts of microwave-fixed tissue from rat brain striatum. Ninety-eight putative peptides were detected including several that had masses matching neuropeptides expected in this brain region such as substance P, rimorphin, and neurotensin. Twenty-three peptides had masses that matched peaks detected by capillary liquid chromatography with electrospray ionization MS.
108) The effect of sample extraction and preparation on neuropeptidomic analysis of brain tissue by capillary liquid chromatography with tandem mass spectrometry was investigated. In agreement with previous reports, analysis of peptide extracts of brain tissue from animals sacrificed by microwave irradiation, which fixes tissue, allows identification of neuronally derived peptides whereas similar analysis of tissue from animals sacrificed without fixation does not. A comparison of a physical method for cell lysis (sonication) to physical combined with chemical cell lysis (sonication with detergent treatment) revealed that the latter method increased the number of neuronally derived peptides positively identified by ~3-fold, from 16 to 44, for analysis of microwave-fixed rat striatum. Use of synaptosome preparations also allowed detection of neuronally derived peptides (23 positively identified) without a requirement of microwave fixation, suggesting that this method may be a useful alternative for sample preparation. Although numerous peptides were identified in these experiments, several known neuropeptides were not detected including neuropeptide Y and neurotensin. Chemical properties such as hydrophobicity and atypical gas-phase fragmentation were found to account for the inability to detect these peptides. These results suggest that further improvement in sample preparation and automated spectral interpretation are needed to provide better coverage of neuropeptides in mammalian tissues. A total of 39 novel neuronally dervived peptides, including some originating from proenkephalin and phosphatidylethanolamine binding protein, were identified in striatum and synaptosome.
107) Metabolites in islets of Langerhans and Escherichia coli strain DH5- were analyzed using negative-mode, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For analysis of anionic metabolites by MALDI, 9-aminoacridine as the matrix yielded a far superior signal in comparison to -cyano-4-hydroxycinnamic acid, 2,5-dihydrobenzoic acid, 2,4,6,-trihydroxyacetophenone, and 3-hydroxypicolinic acid. Limits of detection for metabolite standards were as low as 15 nM for GDP, GTP, ADP, and ATP and as high as 1 M for succinate in 1-L samples. Analysis of islet extracts allowed detection of 44 metabolites, 29 of which were tentatively identified by matching molecular weight to compounds in METLIN and KEGG databases. Relative quantification was demonstrated by comparing the ratio of selected di- and triphosphorylated nucleotides for islets incubated with different concentrations of glucose. For islets at 3 mM glucose, concentration ratios of ATP/ADP, GTP/GDP, and UTP/UDP were 1.9 ± 1.39, 1.12 ± 0.50, and 0.79 ± 0.35 respectively, and at 20 mM glucose stimulation, the ratios increased to 4.13 ± 1.89, 5.62 ± 4.48, and 4.30 ± 4.07 (n = 3). Analysis was also performed by placing individual, intact islets on a MALDI target plate with matrix and impinging the laser directly on the dried islet. Direct analysis of single islets allowed detection of 43 metabolites, 28 of which were database identifiable. A total of 43% of detected metabolites from direct islet analysis were different from those detected in islet extracts. The method was extended to prokaryotic cells by analysis of extracts from E. coli. Sixty metabolites were detected, 39 of which matched compounds in the MetaCyc database. A total of 27% of the metabolites detected from prokaryotes overlapped those found in islets. These results show that MALDI can be used for detection of metabolites in complex biological samples.
106) Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-B (SH2-B (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the Kd = 82 ± 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-B (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing Kd (101 ± 12 nM in good agreement with FA measurements) and dissociation rate (koff = 0.95 ± 0.02 s-1 corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining Kd or koff; however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.
105) Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 ‘-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max cat) steady-state and K(m) values were determined from assays for each substrate-protein pair. When BGTP was the substrate, maximum turnover numbers for Galpha and Galpha(i1) were 8.3 +/- 1 x 10(-3) and 3.0 +/- 0.2 x 10(-2) s(-1), respectively, and K(m) values were 120 +/- 60 and 940 +/- 160 nm. Assays with BGTPgammaS yielded maximum turnover numbers of 1.6 +/- 0.1 x 10(-4) and 5.5 +/- 0.3 x 10(-4) s(-1) for Galpha and Galpha(i1); K(m) values were 14 (o)(+/-)8 and 87 +/- 22 nm. Acceleration of Galpha GTPase activity by regulators of G protein signaling (RGS) was demonstrated in both steady-state and pseudo-single-turnover assay formats with BGTP. Nanomolar RGS increased the rate of enzyme product formation (BODIPY(R) FL GDP (BGDP)) by 117-213% under steady-state conditions and accelerated the rate of G protein-BGTP complex decay by 199 -778% in pseudo-single-turnover assays. Stimulation of GTPase activity by RGS proteins was inhibited 38-81% by 40 mum YJ34, a previously reported peptide RGS inhibitor. Taken together, these results illustrate that Galpha subunits utilize BGTP as a substrate similarly to GTP, making BGTP a useful fluorescent indicator of G protein activity. The unexpected levels of BGTPgammaS hydrolysis detected suggest that caution should be used when interpreting data from fluorescence assays with this probe.
104) In vivo microdialysis sampling was coupled to capillary liquid chromatography (LC)/electrospray ionization quadrupole ion trap mass spectrometry (MS) to monitor [Met]enkephalin and [Leu]enkephalin in the striatum of anesthetized and freely-moving rats. The LC system utilized a high-pressure pump to load 2.5 µl samples and desalt the 25 µm i.d. by 2 cm long column in 12 min. Samples were eluted with a separate pump at ~100 nl/min. A rapid gradient effectively separated the endogenous neuropeptides in 4 min. A comparison was made for operating the mass spectrometer in the MS² and MS³ modes for detection of the peptides. In standard solutions, the detection limits were similar at 1-2 pM (2-4 amol injected); however, the reproducibility was improved with MS³ as the relative standard deviation was
103) A microfluidic device that incorporates continuous perfusion and an on-line electrophoresis immunoassay was developed, characterized, and applied to monitoring insulin secretion from single islets of Langerhans. In the device, a cell chamber was perfused with cell culture media or a balanced salt solution at 0.6 to 1.5 uL/min. The flow was driven by gas pressure applied off-chip. Perfusate was continuously sampled at 2 nL/min by electroosmosis through a separate channel on the chip. The perfusate was mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and monoclonal anti-insulin antibody and allowed to react for 60 s as the mixture traveled down a 4 cm long reaction channel. The cell chamber and reaction channel were maintained at 37 °C. The reaction mixture was injected onto a 1.5 cm separation channel as rapidly as every 6 s, and the free FITC-insulin and the FITC-insulin-antibody complex were separated under an electric field of 500 to 600 V/cm. The immunoassay had a detection limit of 0.8 nM and a relative standard deviation of 6% during 2 h of continuous operation with standard solutions. Individual islets were monitored for up to 1 h while perfusing with different concentrations of glucose. The immunoassay allowed quantitative monitoring of classical biphasic and oscillatory insulin secretion with 6 s sampling frequency following step changes in glucose from 3 to 11 mM. The 2.5 cm x 7.6 cm microfluidic system allowed for monitoring islets in a highly automated fashion. The technique should be amenable to studies involving other tissues or cells that release chemicals.
102) A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at P-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+, secretion coupling and any application requiring rapid assays of secretion.
101) A novel approach to detecting affinity interactions that combines fluorescence anisotropy with capillary electrophoresis (FACE) was developed. In the method, sample is injected into a capillary filled with buffer that contains a fluorescent probe that possesses low fluorescence anisotropy. If proteins or other large molecules in the sample bind the fluorescent probe, their migration through the capillary can be detected as a positive anisotropy shift. Thus, the method provides both separation and confirmation of binding to the probe. Calculations based on combining the Perrin equation and dissociation constant were used to predict the effect of conditions on aniostropy detection. These calculations predict that low probe concentrations yield the best sensitivity while higher concentrations increase the dynamic range for detection of binding partner. The assay was applied to detection of G proteins using BODIPY FL GTPgammaS as the fluorescent probe. Experimental measurements exhibited trends in anisotropy with varying probe and protein concentrations that were consistent with the calculations. The limit of detection for G(alphai1) was 3 nM when the electrophoresis buffer contained 250 nM BODIPY FL GTPgammaS. FACE affinity assay is envisioned as a method that can quantify selected binding partners and screen complex samples for compounds that possess affinity for a particular small molecule that is used as a probe.
100) Endogenous peptides from brain extracellular fluid of live rats were analyzed using capillary liquid chromatography (LC)-tandem mass spectrometry (MS²). A 4-mm-long microdialysis probe perfused at 0.6 uL/min implanted into the striatum of anesthetized male rats was used to collect 3.6 uL dialysate fractions that were injected online into the capillary LC-MS² system for analysis. A total of 3349 MS² spectra were collected from 13 different animals under basal conditions and during localized depolarization evoked by infusion of a high-K+ solution through the microdialysis probe. Subtractive analysis revealed a total of 859 MS² spectra that were observed only during depolarization. From these spectra, 29 peptide sequences (25 were peptides not previously observed) from 6 different protein precursors were identified using database searching software. Proteins identified include precursors to neuropeptides, synaptic proteins, blood proteins, and transporters. The identified peptides represent candidates for neurotransmitters, neuromodulators, and markers of synaptic activity or brain tissue damage. A screen for neuroactivity of novel proenkephalin fragments that were found was performed by infusing the peptides into the brain while monitoring amino acid neurotransmitters by microdialysis sampling combined with capillary electrophoresis. Three of the six tested peptides evoked significant increases in various neuroactive amino acids. These results demonstrate that this combination of methods can identify novel neurotransmitter candidates and screen for potential neuroactivity.
99) An analytical method was developed to monitor amino acids collected by in vivo microdialysis. Microdialysate was continuously derivatized on-line by mixing 6 mM naphthalene-2,3-diearboxaldehyde (NDA) and 10 mM potassium cyanide with the dialysate stream in a fused ailien capillary to form fluorescent products. Reaction time, determined by the flow rate and volume of reaction capillary, was 3 min. Derivatized amino acids were continuously delivered into a flow-gated interface and periodically injected onto a capillary electrophoresis unit equipped with a laser-induced fluorescence detection based on a commercial microscope. Separation was performed in the micellar electrokinetic chromatography mode using 30 mM sodium dodecyl sulfate in 15 mM phosphate buffer at pH 8.0 as the separation media. An electric field of 1.3 kV/cm was applied across a 10 cm long, 10 um internal diameter separation capillary. These conditions allowed 17 amino acid derivatives to be resolved in less than 30 s. On-line injections could be performed at 30 s intervals for in vivo samples. Detection limits were from 10 to 30 nM for the amino acids. The method was applied to monitor the acute ethanol-induced amino acid level changes in freely moving rats. The results demonstrate the utility of the method to reveal dynamics of amino acid concentration in vivo.
98) Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY® FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in similar to 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.
97) The mechanism by which heat shock disrupts development of the two-cell bovine embryo was examined. The reduction in the proportion of embryos that became blastocysts caused by heat shock was not exacerbated when embryos were cultured in air (20.95% O-2) as compared with 5% O-2. In addition, heat shock did not reduce embryonic content of glutathione, cause a significant alteration in oxygen consumption, or change embryonic ATP content. When embryos were heat-shocked at the two-cell stage and allowed to continue development until 72 h post insemination, heat-shocked embryos had fewer total nuclei and a higher percentage of them were condensed. Moreover, embryos became blocked in development at the eight-cell stage. The lack of effect of the oxygen environment on the survival of embryos exposed to heat shock, as well as the unchanged content of glutathione, suggest that free radical production is not a major cause for the inhibition in development caused by heat shock at the two-cell stage. In addition, heat shock appears to have no immediate effect on oxidative phosphorylation since no differences in ATP content were observed. Finally, the finding that heat shock causes a block to development at the eight-cell stage implies that previously reported mitochondrial damage caused by heat shock or other heat shock-induced alterations in cellular physiology render the embryo unable to proceed past the eight-cell stage.
96) Ethanol increases taurine efflux in the nucleus accumbens or ventral striatum (VS), a dopaminergic terminal region involved in positive reinforcement. However, this has been found only at ethanol doses above 1 g/kg intraperitoneally, which is higher than what most rats will self-administer. We used a sensitive on-line assay of microdialysate content to test whether lower doses of ethanol selectively increase taurine efflux in VS as opposed to other dopaminergic regions not involved in reinforcement (e.g., dorsal striatum; DS). Adult male rats with microdialysis probes in VS or DS were injected with ethanol (0, 0.5, 1, and 2 g/kg intraperitoneally), and the amino acid content of the dialysate was measured every 11 sec using capillary electrophoresis and laser-induced fluorescence detection. In VS, 0.5 g/kg ethanol significantly increased taurine levels by 20% for 10 min. A similar increase was seen after 1 g/kg ethanol, which lasted for about 20 min after injection. A two-phased taurine efflux was observed with the 2.0 g/kg dose, where taurine was increased by 2-fold after 5 min but it remained elevated by 30% for at least 60 min. In contrast, DS exhibited much smaller dose-related increases in taurine. Glycine, glutamate, serine, and gamma-aminobutyric acid were not systematically affected by lower doses of ethanol; however, 2 g/kg slowly decreased these amino acids in both brain regions during the hour after injection. These data implicate a possible role of taurine in the mechanism of action of ethanol in the VS. The high sensitivity and time resolution afforded by capillary electrophoresis and laser-induced fluorescence detection will be useful for detecting subtle changes of neuronally active amino acids levels due to low doses of ethanol.
95) The development of an efficient method for high-throughput analysis of multiple electropherograms or chromatograms collected in series is presented. The method, encoded in a computer program designated “Cutter”, utilizes batch processing for determining chromatographic figures of merit (CFOM) including peak centroid times, heights, areas, signal-to-noise ratios (S/N), variance (sigma(2)), skew, excess, and plate number (N) across a set of separations collected serially. The software was validated using simulated data with varying S/N, skew, and excess. The accuracy of the analysis was comparable to or improved over commercial software with area calculation relative errors (RE) below 5% for simulated data with S/N = 5. File sets containing 1300 electropherograms were analyzed in 5 min, representing a nearly 200-fold reduction in analysis time from other methods. Incorporated within the program is a novel method for automated peak deconvolution using an Empirically Transformed Gaussian function. Area measurements of deconvoluted peaks were within 3% of the true value of a simulated data set with S/N = 5 and resolution (R-s) = 1 for equivalent peaks, and within 10% when the ratio of the overlapped peak heights was 10:1.
94) Mice with deletion of insulin receptor substrate (IRS)-1 (IRS-1 knockout [KO] mice) show mild insulin resistance and defective glucose-stimulated insulin secretion and reduced insulin synthesis. To further define the role of IRS-1 in islet function, we examined the insulin secretory defect in the knockouts using freshly isolated islets and primary ß-cells. IRS-1 KO ß-cells exhibited a significantly shorter increase in intracellular free Ca2+ concentration ([Ca2+]i) than controls when briefly stimulated with glucose or glyceraldehyde and when L-arginine was used to potentiate the stimulatory effect of glucose. These changes were paralleled by a lower number of exocytotic events in the KO ß-cells in response to the same secretagogues, indicating reduced insulin secretion. Furthermore, the normal oscillations in intracellular Ca2+ and O2 consumption after glucose stimulation were dampened in freshly isolated KO islets. Semiquantitative RT-PCR showed a dramatically reduced islet expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2b and -3 in the mutants. These data provide evidence that IRS-1 modulation of insulin secretion is associated with Ca2+ signaling and expression of SERCA-2b and -3 genes in pancreatic islets and provides a direct link between insulin resistance and defective insulin secretion.
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 (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
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. While70% 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) Not Available
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) Not Available
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) Not Available
20) Not Available
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) Not Available
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.
11) Not Available
10) Not Available
9) 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.
8.) Not Available
7) Not Available
6) 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.
5) Not Available
4) Not Available
3) Not Available
2) Not Available
1) Not Available
You must be logged in to post a comment.