Simultaneous detection of L-glutamate and nitric oxide from adherently growing cells at known distance using disk shaped dual electrodes |
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| Authors: | Isik Sonnur Castillo Jaime Blöchl Andrea Csöregi Elisabeth Schuhmann Wolfgang |
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| Institution: | 1. Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, BMC B11, 221 84 Lund, Sweden;2. Laboratory of Translational Parkinson''s Disease Research, Center for Neurodegenerative Science, Van Andel Research Institute, 333 Bostwick Ave, N.E., Grand Rapids, MI 49503, USA;3. Developmental and Regenerative Neurobiology, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, BMC A11, 221 84 Lund, Sweden;4. Collège de France, Center for Interdisciplinary Research in Biology (CIRB), Paris, France;5. Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7241, Paris, France;6. Institut National de la Santé et de la Recherche Médicale U1050, Labex Memolife, Paris, France;7. Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA;8. The Udall Center of Excellence in Parkinson''s Disease Research, Michigan State University, Grand Rapids, MI 49503, USA;9. Renovo Neural, Inc., 10000 Cedar Avenue, Cleveland, OH 44106, USA;10. Confocal Microscopy and Quantitative Imaging Core Facility, Van Andel Research Institute, 333 Bostwick Ave, N.E., Grand Rapids, MI 49503, USA;1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China;2. University of Chinese Academy of Sciences, Beijing 100049, China |
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| Abstract: | An ex vivo system for simultaneous detection of nitric oxide (NO) and L-glutamate using integrated dual 250 microm platinum disk electrodes modified individually with suitable sensing chemistries has been developed. One of the sensors was coated with an electrocatalytic layer of Ni tetrasulfonate phthalocyanine tetrasodium salt (Ni-TSPc) covered by second layer of Nafion, which stabilises on the one hand the primary oxidation product NO(+) and prevents interferences from negatively charged compounds such as NO(2)(-). For glutamate determination, the second electrode was modified with a crosslinked redox hydrogel consisting of Os complex modified poly(vinylimidazol), glutamate oxidase and peroxidase. A manual x-y-z micromanipulator on top of an inverted optical microscope was used to position the dual electrode sensor at a defined distance of 5 microm from a cell population under visual control. C6 glioma cells were stimulated simultaneously with bradykinin or VEGF to release NO while KCl was used to invoke glutamate release. For evaluation of the glutamate sensors, in some experiments HN10 cells were used. To investigate the sensitivity and reliability of the system, several drugs were applied to the cells, e.g. Ca(2+)-channel inhibitors for testing Ca(2+)-dependence of the release of NO and glutamate, rotenone for inducing oxidative stress and glutamate antagonists for analysing glutamate release. With these drugs the NO and glutamate release was modulated in a similar way then expected from previously described systems or even in-vivo measurements. We therefore conclude that our system is suitable to analyse stress-induced mechanisms in cell lines. |
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