Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on SubstrateIntegrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00129
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Simultaneous Fluorescent and Amperometric Detection Of Catecholamine Release From Neuroendocrine Cells With Transparent Diamond MEAs Alberto Pasquarelli1*, Andrea Marcantoni2, Daniela Gavello2, Alfio Battiato3, Federico Picollo3, Paolo Olivero3, Emilio Carbone4 and Valentina Carabelli4 University of Ulm, Institute of Electron Devices and Circuits, Germany University of Turin, Dept. Drug Science and Technology, Italy 3 University of Torino, Physics Department, Italy 4 University of Torino, Dept. Drug Science and Technology, Italy 1 2
Motivation Among all materials suitable for the fabrication of inert electrodes (Type-0) for amperometry, diamond is the one that shows the best pros/cons ratio. It is in fact very sensitive, chemically inert, biocompatible and transparent. This latter property was used so far only for cell inspection and not for optical measurements. In this work, we demonstrate for the first time the suitability of diamond MEAs for simultaneously detecting amperometric and fluorescence signals associated with calcium transients and catecholamine release in in vitro chromaffin cells. This is now possible thanks to the recently improved technology of the diamond MEAs, which allows to reach transmittance of ~ 50% in the whole optical range of interest, while keeping excellent amperometric sensitivity. Material and Methods New 16Ch-MEAs were fabricated on a 200 µm thick hightemperature glass carrier (AF32eco, Schott, Mainz, Germany), having the same thermal expansion coefficient of silicon. This material property leads to a minimal compressive stress of the diamond film once cooled-down after growth and therefore the risk of delamination is very low even without any adhesion promoter, like amorphous silicon, which can spoil the optical properties. The growth of the nanocrystalline diamond (NCD) film was done in two microwave plasma CVD reactors, one dedicated to the growth of intrinsic material and the other to the growth of doped material. The whole growth procedure comprised three main steps. The first one consisted in seeding the thoroughly cleaned glass substrate with a nanodiamond suspension, needed to provide a startinglayer. The second step was the deposition of a ~1 µm thick intrinsic NCD-film, which was then overgrown in the following third step with a ~ 200 nm thick boron-doped NCD-layer (BDD). This double-layer technique yields better electrical properties of the conducting structures, i.e. conductivity and background noise, than the single deposition of just a BDD-layer. The 16 channel MEA-pattern was achieved by means of standard microfabrication processes, here including optical lithography, hard and soft masks, dry and wet etching, physical and chemical vapor deposition (PVD and CVD) of materials, following the same procedure described in [1]. Thus the final device
presented 16 electrodes with a circular active area of 20 µm in diameter, arranged on a 4x4 grid with 200 µm pitch. For the measurements, the chips were mounted onto our home-designed readout electronics, which fits the table of an inverted microscope, and the signals were acquired by means of a National Instruments USB-6216 unit under a LabView software application [1,2]. Bovine chromaffin cells were prepared following the standard protocol described in [2], plated onto the diamond MEA and kept in water-saturate 5% CO2 atmosphere, at 37 °C for 24 hours, to allow full expression of voltage-gated calcium channels prior recordings. Catecholamine secretion was triggered by applying a depolarizing external solution containing 30 mM KCl. Calcium imaging Chromaffin cells were loaded with the fluorescent calcium indicator Fura2-AM dye (3 µM) (Invitrogen, Molecular Probes, Oregon, USA) in the extracellular solution for 1 h. The fluorescent dye was then washed-out. An inverted microscope (Leica DMI3000B, Wetzlar, Germany) equipped with a short-arc xenon gas discharge lamp (Ushio, Cypress, California, USA) and a monochromator (Till Photonics, Grafelfing, Germany) was used for measuring Fura2-AM fluorescence. Images were projected onto a EMCCD camera (QuantEM:512SC, Photometrics, Tucson, Arizona, USA) and collected every 200 ms. Calcium transients were recorded and stored using Metafluor software (Molecular Devices, California, USA). Results In the diamond areas, the microfabricated chips exhibited an integral transmittance of ~50% in a wide wavelength range (from ~300 nm to near infrared). The electrical recording setup was working in the amperometric mode, with a transimpedance gain factor of 100 MOhm over a bandwidth from DC to 1 kHz. Using these operating parameters, the background noise was below 1 pA rms. Simultaneous amperometric and fluorescence recordings of calcium-dependent secretory bursts were obtained using an extracellular solution containing 30 mM KCl and 10 mM CaCl2. This caused an increased F340/F380 ratio (from 0.5 to 4.5) and the simultaneous appearance of amperometric spikes, revealing the calciumdependence of catecholamine release in bovine chromaffin cells. After a rapid solution exchange (wash with standard saline solution), the release of catecholamines stopped and the fluorescence signal completely decayed. KCl stimulation and wash-out could be repeated, proving the reproducibility of secretory activity (Fig.1). Discussion and Conclusion This work brings new impelling evidence that diamond MEAs are suitable for concurrent amperometric and calciumimaging recordings. Simultaneous detection of amperometric spikes and intracellular calcium loadings in cell populations or networks using low-density diamond MEAs represents a key improvement targeted to provide multi-parametric analysis of calcium-regulated processes. In this preliminary study, the fluorescence image information is integral on the entire cell and has a coarse time resolution. Under these recording conditions, it is not possible to optically resolve the sites of individual secretory events. This issue will be addressed in future works of our group. References 1. Granado et al., Physica Status Solidi (a) 2015, 212: 2445-2453 2. Picollo et al., Scientific Reports 2016, 6: 20682 Figure Legend Figure 1: Top, raw fluorescence images showing the FURA2 emission under low/high intracellular calcium concentration corresponding to rest and calcium-dependent catecholamine release induced by KCl stimulation. Center, temporal profile of the specific FURA2 emission related to intracellular calcium. Time bars show the duration of KCl perfusion and wash. Bottom, amperometric spike bursts detected in coincidence with the FURA2 fluorescence changes.
Figure 1
Keywords: Exocytosis, Fura-2, multimodality, nanocrystalline diamond Conference: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016. Presentation Type: Poster Presentation Topic: MEA Meeting 2016 Citation: Pasquarelli A, Marcantoni A, Gavello D, Battiato A, Picollo F, Olivero P, Carbone E and Carabelli V (2016). Simultaneous Fluorescent and Amperometric Detection Of Catecholamine Release From Neuroendocrine Cells With Transparent Diamond MEAs. Front. Neurosci. Conference Abstract: MEA Meeting 2016 | 10th International Meeting on Substrate-Integrated Electrode Arrays. doi: 10.3389/conf.fnins.2016.93.00129 Received: 22 Jun 2016; Published Online: 24 Jun 2016. * Correspondence: Dr. Alberto Pasquarelli, University of Ulm, Institute of Electron Devices and Circuits, Ulm, Germany,
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