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Improved Surface-Patterned Platinum Microelectrodes for the Study of Exocytotic Events Khajak Berberian,*,† Kassandra Kisler,‡ Qinghua Fang,‡ and Manfred Lindau†,‡ Department of Biomedical Engineering, and School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853 Surface-patterned platinum microelectrodes insulated with 300 nm thick fused silica were fabricated using contact photolithography. These electrodes exhibit low noise and were used for monitoring single vesicle exocytosis from chromaffin cells by constant potential amperometry as well as fast-scan cyclic voltammetry. Amperometric spike parameters were consistent with those obtained with conventional carbon fiber electrodes. Catecholamine voltammograms acquired with platinum electrodes exhibited redox peaks with full width at halfmaximum of ∼45 mV, much sharper than those of carbon fiber electrode recordings. The time course of voltammetrically measured release events was similar for platinum and carbon fiber electrodes. The fused-silicainsulated platinum electrodes could be cleaned and reused repetitively and allowed incorporation of micrometer precision surface-patterned poly-D-lysine. Poly-D-lysinefunctionalized devices were applied to stimulate mast cells and record single release events without serotonin preloading. Microfabricated platinum electrodes are thus able to record single exocytotic events with high resolution and should be suitable for highly parallel electrode arrays allowing simultaneous measurements of single events from multiple cells. Exocytosis is a fundamental cellular mechanism by which cells extrude the contents of membrane bound vesicles into the extracellular space. Many techniques have been employed to investigate single exocytotic events including whole-cell patchclamp capacitance measurements,1-4 cell-attached capacitance * To whom correspondence should be addressed. Fax: 001-607-255-7658. E-mail:
[email protected]. † Department of Biomedical Engineering. ‡ School of Applied and Engineering Physics. (1) Breckenridge, L. J.; Almers, W. Nature 1987, 328, 814–817. (2) Lindau, M. Q. Rev. Biophys. 1991, 24, 75–101. (3) Zimmerberg, J.; Curran, M.; Cohen, F. S.; Brodwick, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1585–1589. (4) Fernandez, J. M.; Neher, E.; Gomperts, B. D. Nature 1984, 312, 453–455. (5) Lollike, K.; Borregaard, N.; Lindau, M. J. Cell Biol. 1995, 129, 99–104. (6) He, L.; Wu, X. S.; Mohan, R.; Wu, L. G. Nature 2006, 444, 102–105. (7) Han, X.; Wang, C. T.; Bai, J.; Chapman, E. R.; Jackson, M. B. Science 2004, 304, 289–292. (8) Debus, K.; Lindau, M. Biophys. J. 2000, 78, 2983–2997. (9) Chow, R. H.; Ru ¨ den, L. v.; Neher, E. Nature 1992, 356, 60–63. (10) Wightman, R. M. Science 2006, 311, 1570–1574. (11) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, D. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J., Jr.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754–10758.
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measurements,5-8 carbon fiber amperometry,9-11 patch amperometry,12-14 and total internal reflection fluorescence microscopy.15-19 The probably most widely used method for the study of single exocytotic events utilizes carbon fiber electrodes (CFEs),20 which record the release of oxidizable compounds, such as catecholamine release from chromaffin cells9-11 or even dopamine release from dopaminergic neurons21-24 at extraordinary resolution. Low noise and high temporal resolution has been achieved, due to the CFE’s small size and fast response time. There are, however, two limitations to the CFE technique: (i) in order to obtain statistical significance, experiments need to be performed on a large number of cells,25,26 which is timeconsuming for single-cell experiments under a microscope, and (ii) the simultaneous electrochemical detection of release and fluorescence imaging of the same vesicle is not readily possible. Fluorescence imaging can often provide important complementary information on vesicle motion17 or molecular events associated with vesicle exocytosis.27 To overcome this limitation, surface-patterned electrochemical microelectrodes have recently been fabricated using platinum (Pt)28,29 or indium-tin oxide (ITO)30,31 as the working electrode (12) Dernick, G.; Alvarez De Toledo, G.; Lindau, M. In Electrochemical Methods for Neuroscience; Michael, A. C., Borland, L. M., Eds.; CRC Press: Boca Raton, FL, 2006; pp 315-336. (13) Dernick, G.; Gong, L. W.; Tabares, L.; Alvarez de Toledo, G.; Lindau, M. Nat. Methods 2005, 2, 699–708. (14) Albillos, A.; Dernick, G.; Horstmann, H.; Almers, W.; Alvarez de Toledo, G.; Lindau, M. Nature 1997, 389, 509–512. (15) Steyer, J. A.; Horstmann, H.; Almers, W. Nature 1997, 388, 474–478. (16) Axelrod, D. J. Biomed. Opt. 2001, 6, 6–13. (17) Allersma, M. W.; Bittner, M. A.; Axelrod, D.; Holz, R. W. Mol. Biol. Cell 2006, 17, 2424–2438. (18) Wang, M. D.; Axelrod, D. Dev. Dyn. 1994, 201, 29–40. (19) Axelrod, D.; Thompson, N. L.; Burghardt, T. P. J. Microsc. 1983, 129, 19– 28. (20) Gonon, F.; Cespuglio, R.; Ponchon, J. L.; Buda, M.; Jouvet, M.; Adams, R. N.; Pujol, J. F. C. R. Hebd. Seances Acad. Sci. D 1978, 286, 1203–1206. (21) Jaffe, E. H.; Marty, A.; Schulte, A.; Chow, R. H. J. Neurosci. 1998, 18, 3548– 3553. (22) Pothos, E. N.; Davila, V.; Sulzer, D. J. Neurosci. 1998, 18, 4106–4118. (23) Staal, R. G.; Mosharov, E. V.; Sulzer, D. Nat. Neurosci. 2004, 7, 341–346. (24) Pothos, E.; Desmond, M.; Sulzer, D. J. Neurochem. 1996, 66, 629–636. (25) Mosharov, E. V.; Sulzer, D. Nat. Methods 2005, 2, 651–658. (26) Colliver, T. L.; Hess, E. J.; Pothos, E. N.; Sulzer, D.; Ewing, A. G. J. Neurochem. 2000, 74, 1086–1097. (27) An, S. J.; Almers, W. Science 2004, 306, 1042–1046. (28) Dias, A. F.; Dernick, G.; Valero, V.; Yong, M. G.; James, C. D.; Craighead, H. G.; Lindau, M. Nanotechnology 2002, 13, 285–289. (29) Hafez, I.; Kisler, K.; Berberian, K.; Dernick, G.; Valero, V.; Yong, M. G.; Craighead, H. G.; Lindau, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13879–13884. 10.1021/ac900674g CCC: $40.75 2009 American Chemical Society Published on Web 09/25/2009
material. Here, we describe an improved design of reusable surface-patterned, glass-insulated platinum microelectrodes (PtEs) and characterize their properties in amperometric as well as fastscan cyclic voltammetric modes for their use in the study of single exocytotic events. MATERIALS AND METHODS Cell Preparation, Reagents, and Solutions. Bovine chromaffin cells were cultured on 8 mm glass coverslips as described.32 Mast cells were isolated from the peritoneum of adult SpragueDawley rats as described.33 The solution used for all electrochemical recordings contained (in mM) 140 NaCl, 5 KCl, 5 CaCl2, 1 MgCl2, 10 HEPES/NaOH, 20 glucose (pH 7.3). Dopamine hydrochloride, (±)-epinephrine hydrochloride, DL-norepinephrine hydrochloride, and poly-D-lysine (PDL) were all purchased from Sigma (Milwaukee, WI) and used without further purification. Experiments were performed at room temperature, at day 1 after isolation for chromaffin cells and at the day of isolation for mast cells. CFE Fabrication. Carbon fiber electrodes were fabricated as described.11 Briefly, a single carbon fiber (5 µm diameter) was inserted in a borosilicate glass capillary (1.8 mm o.d., Hilgenberg GmbH, Germany). The capillary was then pulled using a pipet puller (model P-97, Sutter instrument, U.S.A.) producing two CFEs, which were separated with scissors. CFE tips were dipped in melting wax (Sticky Wax, Kerr Corporation, U.S.A.) for 2 min and subsequently cut using a blade (no. 10, Feather Safety Razor Co., Japan). Prior to experiments, CFEs were backfilled with 3 M KCl solution. PtE Microfabrication. Single or triple PtEs were surfacepatterned on 4 in. diameter borosilicate glass wafers of 160-190 µm thickness (ThermoFisher Scientific, Portsmouth, NH) using contact photolithography techniques and metal liftoff. Briefly, the fabrication procedure was as follows. The glass wafers were cleaned with acetone and baked for ∼3 min on a hot plate at 115 °C. The wafers were then vapor-primed with hexamethyldisilazane (HMDS) in a YES LP-III (Yield Engineering Systems, Inc., Livermore, CA) oven. Following vapor priming, the wafers were spin-coated (4000 rpm for 30 s) with Shipley S1813 photoresist and baked on a hot plate at 115 °C for 1 min to remove excess solvent. A mask containing the electrode design was used to selectively expose the wafers to UV light using a contact aligner (EV620, EV Group, Scharding, Austria). Wafers were then baked for 2.5 min on a hot plate at 115 °C (post-exposure bake) to harden the resist. Subsequently, wafers were ammonia-baked in an image reversal oven (YES oven) and exposed to UV light for 60 s using the contact aligner (flood expose). Following UV exposure, the photoresist was developed in a positive developer (AZ 300MIF, which contains 2.38% by weight tetramethyl ammonium hydroxide) for 1 min. Residual photoresist (
