a b s t r a c tThe quantal release of oxidizable molecules can be successfully monitored by means of polarized carbon fiber microelectrodes (CFEs) positioned in close proximity to the cell membrane. To partially overcome certain CFE limitations, mainly related to their low spatial resolution and lack of optical transparency, we developed a planar boron-doped nanocrystalline diamond (NCD) prototype, grown on a transparent sapphire wafer. Responsiveness to applied catecholamines as well as the electrochemical and optical properties of the NCD-based device were first characterized by cyclic voltammetry and optical transmittance measurements. By stimulating chromaffin cells positioned on the device with external KCl, well-resolved quantal exocytotic events could be detected either from one NCD microelectrode, or simultaneously from an array of four microelectrodes, indicating that the chip is able to monitor secretory events (amperometric spikes) from a number of isolated chromaffin cells. Spikes detected by the planar NCD device had comparable amplitudes, kinetics and vesicle diameter distributions as those measured by conventional CFEs from the same chromaffin cell.

Key pointsr A planar nanocrystalline diamond array with nine ultra-microelectrodes (9-Ch NCD-UMEA) has been designed for high spatial resolution of amperometric recordings in single chromaffin cells.r The 9-Ch NCD-UMEA operates in voltammetric and amperometric mode to reveal low doses of adrenaline, dopamine and serotonin. The lowest detectable concentration of adrenaline is ß5 μM.r Using mouse and bovine chromaffin cells, single quantal exocytotic events are recorded from nine microareas of 12-27 μm 2 . We found an excellent correspondence with recordings from the cell apex using carbon fibre electrodes. r In the bovine, secretion is heterogeneous. There are areas of high and medium activity covering 54% of the cell surface and areas of low and no activity covering the remainder. The 'non-active zones' (silent) cover 24% of the cell surface and persist for minutes as the 'active zones' . r The 9-Ch NCD-UMEA brings new insights into the spatial mapping of secretory sites in chromaffin cells.Abstract Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose-response calibrations, set the lowest detectable concentration of adrenaline to ß5 μM. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12-27 μm 2 . In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The 'non-active' ('silent') zones covered 24% of the total and persisted for 6-8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution.

a b s t r a c tSemiconductor nanocrystal quantum dots (QDs) possess an enormous potential of applications in nanomedicine, drug delivery and bioimaging which derives from their unique photoemission and photostability characteristics. In spite of this, however, their interactions with biological systems and impact on human health are still largely unknown. Here we used neurosecretory mouse chromaffin cells of the adrenal gland for testing the effects of CdSeeZnS coreeshell quantum dots (5e36 nM) on Ca 2þ channels functionality and Ca 2þ -dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSeeZnS QDs (!16 nM) showed that the semiconductor nanocrystal is effectively internalized into the cells without affecting cell integrity (no changes of membrane resistance and cell capacitance). QDs reduced the size of Ca 2þ currents by w28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at þ10 mV, where Ca 2þ currents are maximal, was reduced by 29%. CdSeeZnS QDs reduced the size of the readily releasable pool (RRP) of secretory vesicles by 32%, the frequency of release by 33% and the overall quantity of released catecholamines by 61%, as measured by carbon fibers amperometry. In addition, the Ca 2þ -dependence of exocytosis was reduced, whereas the catecholamine content of single granules, as well as the kinetics of release, remained unaltered. These data suggest that exposure to CdSeeZnS QDs impairs Ca 2þ influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.

The quantal release of catecholamines from neuroendocrine cells is a key mechanism which has been investigated with a broad range of materials and devices, among which carbon-based materials such as carbon fibers, diamond-like carbon, carbon nanotubes and nanocrystalline diamond. In the present work we demonstrate that a MeV-ion-microbeam lithographic technique can be successfully employed for the fabrication of an all-carbon miniaturized cellular bio-sensor based on graphitic micro-channels embedded in a single-crystal diamond matrix. The device was functionally characterized for the in vitro recording of quantal exocytic events from single chromaffin cells, with high sensitivity and signal-to-noise ratio, opening promising perspectives for the realization of monolithic all-carbon cellular biosensors.Submitted to 2

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