Microelectrode Arrays of Diamond-Insulated Graphitic Channels for Real-Time Detection of Exocytotic Events from Cultured Chromaffin Cells and Slices of Adrenal Glands

Abstract

A microstructured graphitic 4 × 4 multielectrode array was embedded in a single-crystal diamond substrate (4 × 4 μG-SCD MEA) for real-time monitoring of exocytotic events from cultured chromaffin cells and adrenal slices. The current approach relies on the development of a parallel ion beam lithographic technique, which assures the time-effective fabrication of extended arrays with reproducible electrode dimensions. The reported device is suitable for performing amperometric and voltammetric recordings with high sensitivity and temporal resolution, by simultaneously acquiring data from 16 rectangularly shaped microelectrodes (20 × 3.5 μm(2)) separated by 200 μm gaps. Taking advantage of the array geometry we addressed the following specific issues: (i) detect both the spontaneous and KCl-evoked secretion simultaneously from several chromaffin cells directly cultured on the device surface, (ii) resolve the waveform of different subsets of exocytotic events, and (iii) monitoring quantal secretory events from thin slices of the adrenal gland. The frequency of spontaneous release was low (0.12 and 0.3 Hz, respectively, for adrenal slices and cultured cells) and increased up to 0.9 Hz after stimulation with 30 mM KCl in cultured cells. The spike amplitude as well as rise and decay time were comparable with those measured by carbon fiber microelectrodes and allowed to identify three different subsets of secretory events associated with "full fusion" events, "kiss-and-run" and "kiss-and-stay" exocytosis, confirming that the device has adequate sensitivity and time resolution for real-time recordings. The device offers the significant advantage of shortening the time to collect data by allowing simultaneous recordings from cell populations either in primary cell cultures or in intact tissues.

Low pHo boosts burst firing and catecholamine release by blocking TASK‐1 and BK channels while preserving Cav1 channels in mouse chromaffin cells

“…A similar basal release in 2 m m Ca 2+ has been observed, both in isolated bovine chromaffin cell (BCCs) and MCCs of adrenal gland slices (Picollo et al . ). In 10 MCCs, all spontaneously firing, three cells had no amperometric spike activity, whereas the remaining had rare spike events, casually distributed during a recording period of 60 s. Mean spike frequency was 0.02 ± 0.01 Hz (Fig.…”

Quantal Release of Dopamine and Action Potential Firing Detected in Midbrain Neurons by Multifunctional Diamond-Based Microarrays

“…Subsequently, a thermal treatment at 950°C for 2 h in high vacuum (10 -6 mbar) was performed in order to promote the complete graphitization of the modified regions (Hoffman et al, 1996; Hickey et al, 2009; Picollo et al, 2012). A preliminary electrical characterization of the device was then performed as previously described (Picollo et al, 2016a).…”

“…Micro-graphitic single-crystal diamond multielectrode arrays (μG-SCD-MEAs) are a powerful sensor for investigating neurosecretion in living cells (Picollo et al, 2013, 2015b). Previous findings have demonstrated their ability to monitor spontaneous and evoked quantal catecholamine release from cultured mouse and bovine adrenal chromaffin cells (Picollo et al, 2016b) as well as from fresh mouse adrenal slices (Picollo et al, 2016a; Carabelli et al, 2017). Besides providing simultaneous recordings from a variety cells, which have been plated and cultured on the planar array for a number of days, μG-SCD-MEAs possess high-time resolution and sensitivity for the detection of amperometric events with different shape, such as the small amplitude, or previously identified “stand-alone-foot” events (Picollo et al, 2016a).…”

Assessment of acid and thermal oxidation treatments for removing sp2 bonded carbon from the surface of boron doped diamond

“…In this way sp 2 bonded carbon microarray electrodes in diamond have been created for the detection of neurotransmitters. [39,40] Nonetheless, for many applications of diamond (and BDD) removal of any processing-created or growth related forms of sp 2 bonded carbon at the surface of the material is important. In this paper we first examine the effectiveness of two different oxidative treatments at removing surface sp 2 bonded carbon created during the ns-laser micromachining process.…”