Anal. Chem. 2005, 77, 681-686
Nanofabricated Carbon-Based Detector Vladimir Parpura*
Department of Cell Biology and Neuroscience and Center for Nanoscale Science and Engineering, University of California, Riverside, California 92521
The procedure for the fabrication of a detector termed NACAD, nanofabricated carbon-based detector, is described. I characterized the mechanical properties of this detector using atomic force and scanning electron microscopy techniques. This detector exhibits a smooth surface (rms 2-4 nm) and easy access for atomic force microscopy tips, necessary features for biological studies of secretory granules and vesicles, which store transmitters and release them via exocytosis. The NACAD does not impair the elastic properties of granular matrixes deposited onto a detector, as they show their typical Young’s moduli and ion exchanger properties; divalent histamine shrinks them, while monovalent sodium causes their swelling. Additionally, the NACADs’ electrochemical properties allowed amperometric measurements of serotonin released from intact secretory granules isolated from mast cells, after removal of their granular membranes using a mild detergent treatment. Thus, this detector will aid future studies of single secretory granules and vesicles and their insoluble matrixes. Secretory granules store secretory products, which are released from their intragranular lumen into the extracellular space via exocytosis. Uvna¨s and Åborg have proposed that secretory granules contain a matrix that binds secretory products and acts as an ion exchanger that governs the release of these products by exchanging them with counterions, while keeping fixed charges in the matrix electroneutral.1 Experiments using giant granules, which have diameters up to several micrometers, isolated from mutant beige mouse mast cells and light microscopy addressed the role of a matrix in this ion exchange mechanism.2-4 When the membranes of secretory granules were removed, the negatively charged intragranular matrixes were exposed to the bathing solution containing different cations. Such matrixes were condensed in the presence of divalent cations, while they swelled in the presence of monovalent cations. Although those data have been informative, most secretory granules are smaller than 1 µm in diameter; synaptic vesicles measure ∼50 nm, while mast cell granules are ∼600 nm in diameter.5,6 Thus, these submicrometer* Phone and fax: (951) 827-2074. E-mail:
[email protected]. (1) Uvna¨s, B.; Åborg, C. H. Acta Physiol. Scand. 1983, 119, 225-234. (2) Brodwick, M. S.; Curran, M.; Edwards, C. J. Membr. Biol. 1992, 126, 159169. (3) Curran, M. J.; Brodwick, M. S. J. Gen. Physiol. 1991, 98, 771-790. (4) Fernandez, J. M.; Villalon, M.; Verdugo, P. Biophys. J. 1991, 59, 10221027. (5) Chock, S. P.; Schmauder-Chock, E. A. J. Biol. Chem. 1989, 264, 28622868. 10.1021/ac0487404 CCC: $30.25 Published on Web 12/14/2004
© 2005 American Chemical Society
sized granules have to be studied with alternative instrumentation aside from light microscopy. Electron microscopy (EM) offers high-resolution, yet static, images of synaptic vesicles and secretory granules.5,7 However, because EM studies require fixation and dehydration of the sample, the functional properties of any matrix are destroyed, hampering detailed examination of the luminal structures of vesicles and granules, and their possible role as ion exchangers. Relatively recently, atomic force microscopy (AFM) has emerged as a technique that can investigate samples under physiological conditions while offering high temporal (∼1 ms) and x-y (
