Alberto Pasquarelli

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells.

The 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.

A New Diamond Biosensor with Integrated Graphitic Microchannels for Detecting Quantal Exocytic Events from Chromaffin Cells

An MeV ion-microbeam lithographic technique can be successfully employed for the fabrication of an all-carbon miniaturized cellular biosensor based on graphitic microchannels embedded in a single-crystal diamond matrix. The device is 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.

Heterogeneous distribution of exocytotic microdomains in adrenal chromaffin cells resolved by high‐density diamond ultra‐microelectrode arrays

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. 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 ∼5 μm. Using mouse and bovine chromaffin cells, single quantal exocytotic events are recorded from nine microareas of 12–27 μm2. We found an excellent correspondence with recordings from the cell apex using carbon fibre electrodes. 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’. The 9‐Ch NCD‐UMEA brings new insights into the spatial mapping of secretory sites in chromaffin cells.

Development and Characterization of a Diamond-Insulated Graphitic Multi Electrode Array Realized with Ion Beam Lithography

The detection of quantal exocytic events from neurons and neuroendocrine cells is a challenging task in neuroscience. One of the most promising platforms for the development of a new generation of biosensors is diamond, due to its biocompatibility, transparency and chemical inertness. Moreover, the electrical properties of diamond can be turned from a perfect insulator into a conductive material (resistivity ∼mΩ·cm) by exploiting the metastable nature of this allotropic form of carbon. A 16-channels MEA (Multi Electrode Array) suitable for cell culture growing has been fabricated by means of ion implantation. A focused 1.2 MeV He+ beam was scanned on a IIa single-crystal diamond sample (4.5 × 4.5 × 0.5 mm3) to cause highly damaged sub-superficial structures that were defined with micrometric spatial resolution. After implantation, the sample was annealed. This process provides the conversion of the sub-superficial highly damaged regions to a graphitic phase embedded in a highly insulating diamond matrix. Thanks to a three-dimensional masking technique, the endpoints of the sub-superficial channels emerge in contact with the sample surface, therefore being available as sensing electrodes. Cyclic voltammetry and amperometry measurements of solutions with increasing concentrations of adrenaline were performed to characterize the biosensor sensitivity. The reported results demonstrate that this new type of biosensor is suitable for in vitro detection of catecholamine release.

Combined in situ atomic force microscopy and infrared attenuated total reflection spectroelectrochemistry.

A novel analytical platform combining infrared attenuated total reflection (IR-ATR) spectroelectrochemistry (SE) with atomic force microscopy (AFM) using a boron-doped diamond (BDD)-modified ATR crystal is presented. The utility of this combination is demonstrated investigating the electrodeposition of a polymer film via IR spectroscopy, while the surface modification is simultaneously imaged by AFM. The ATR waveguide consists of a single-crystal intrinsic diamond overgrown with a homoepitaxial BDD layer (thickness: ∼100 nm, boron content: ∼5 × 10(20) cm(-3)) to provide electric conductivity. The diamond ATR crystal is shaped in the form of a hemisphere with a beveled top and an octahedronal surface area of approximately 400 μm(2). To demonstrate combined IR-ATR-SE-AFM measurements, the electro-polymerization of 3,4-ethylenedioxothiophene (EDOT) was selected as a model system. Depositions were obtained from aqueous solutions, while changes in IR signature, topography, and electrochemical behavior were recorded in situ and simultaneously during the polymerization process.

Diamond microelectrodes arrays for the detection of secretory cell activity

Diamond applications potential for biosensing devices have been highlighted by several authors, especially concerning the long-term stability of covalent functionalisations on its surface. Additionally, in electrochemistry boron doped diamond electrodes (NA ∼ 1020 cm3) show high corrosion resistance and a large hydrolysis window. These features, recognised and exploited in industrial applications, have up to now found little resonance in the life-sciences. Here we present diamond microelectrode arrays based on (1) nanocrystalline diamond (NCD) thin films and (2) single crystal diamond (SCD). NCD is necessary for large area applications like arrays, but graphitic grain boundaries may influence its behaviour. The ideal case SCD is covered here for comparison. The array design consists of four electrodes whose sensitive area is delimited by means of a patterned photoresist. Two different patterns were used to realise a layout with four independent openings (15 µm diameter) for simultaneous detection on multiple cells and a layout with one single window (25 µm diameter) intersecting all four electrodes to create a quadrupolar detector suitable for mapping the activity of single cells. Early results validated the suitability of both NCD and SCD devices: (1) cyclic-voltammetry measurements confirmed the adrenaline oxidation potential on the presented microelectrodes around 650 mV; (2) alternating applications of 1 mM adrenaline and saline rinsing solutions showed negligible electrode fouling; and (3) interfaced to single adrenal chromaffin cells, the devices clearly detected sustained sequences of quantal events (10–100 pA amplitude, 50–100 ms duration) associated to the vesicular release of adrenaline and noradrenaline during exocytosis induced by cell-depolarisation.

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.

Coronary MR Imaging : Navigator Echo Biofeedback Increases Navigator Efficiency-Initial Experience

RATIONALE AND OBJECTIVES The aim of this study was to investigate whether a respiratory biofeedback system could increase navigator efficiency and maintain image quality compared to conventional respiratory-gated magnetic resonance coronary angiography (MRCA). MATERIALS AND METHODS Eighteen healthy volunteers underwent MRCA using three different respiratory-gating protocols. A conventional expiratory free-breathing (FB) sequence was compared to two approaches using navigator echo biofeedback (NEB), a midinspiratory approach (NEBin) and an expiratory approach (NEBex). Navigator data reflecting the position of the diaphragm relative to a 3-mm gating window were made available to the subject using a video projector in combination with a Plexiglas screen and mirror goggles. Image quality was graded by two radiologists in consensus using a visual score ranging from 1 (not visible) to 4 (excellent vessel depiction). RESULTS The NEB approaches improved navigator efficiency (71.1% with NEBex and 68.0% with NEBin vs 42.2% with FB), thus reducing total imaging time. This difference was statistically significant (P(NEBin)=.007; P(NEBex)=.001). Image quality in the NEBex group was comparable to that in the FB group (median score, 2.44 vs 2.52), but it proved to be significantly lower (median score, 1.94 vs 2.52) for the right coronary artery and the left anterior descending coronary artery in the NEBin group. CONCLUSION NEB maintains image quality and significantly increases navigator efficiency, thereby decreasing total imaging time by about 40% compared to a conventional FB acquisition strategy.

Planar Diamond-Based Multiarrays to Monitor Neurotransmitter Release and Action Potential Firing: New Perspectives in Cellular Neuroscience

High biocompatibility, outstanding electrochemical responsiveness, inertness, and transparency make diamond-based multiarrays (DBMs) first-rate biosensors for in vitro detection of electrochemical and electrical signals from excitable cells together, with potential for in vivo applications as neural interfaces and prostheses. Here, we will review the electrochemical and physical properties of various DBMs and how these devices have been employed for recording released neurotransmitter molecules and all-or-none action potentials from living cells. Specifically, we will overview how DBMs can resolve localized exocytotic events from subcellular compartments using high-density microelectrode arrays (MEAs), or monitoring oxidizable neurotransmitter release from populations of cells in culture and tissue slices using low-density MEAs. Interfacing DBMs with excitable cells is currently leading to the promising opportunity of recording electrical signals as well as creating neuronal interfaces through the same device. Given the recent increasingly growing development of newly available DBMs of various geometries to monitor electrical activity and neurotransmitter release in a variety of excitable and neuronal tissues, the discussion will be limited to planar DBMs.

Technical reportCoronary MR Imaging: Navigator Echo Biofeedback Increases Navigator Efficiency—Initial Experience

RATIONALE AND OBJECTIVES The aim of this study was to investigate whether a respiratory biofeedback system could increase navigator efficiency and maintain image quality compared to conventional respiratory-gated magnetic resonance coronary angiography (MRCA). MATERIALS AND METHODS Eighteen healthy volunteers underwent MRCA using three different respiratory-gating protocols. A conventional expiratory free-breathing (FB) sequence was compared to two approaches using navigator echo biofeedback (NEB), a midinspiratory approach (NEBin) and an expiratory approach (NEBex). Navigator data reflecting the position of the diaphragm relative to a 3-mm gating window were made available to the subject using a video projector in combination with a Plexiglas screen and mirror goggles. Image quality was graded by two radiologists in consensus using a visual score ranging from 1 (not visible) to 4 (excellent vessel depiction). RESULTS The NEB approaches improved navigator efficiency (71.1% with NEBex and 68.0% with NEBin vs 42.2% with FB), thus reducing total imaging time. This difference was statistically significant (P(NEBin)=.007; P(NEBex)=.001). Image quality in the NEBex group was comparable to that in the FB group (median score, 2.44 vs 2.52), but it proved to be significantly lower (median score, 1.94 vs 2.52) for the right coronary artery and the left anterior descending coronary artery in the NEBin group. CONCLUSION NEB maintains image quality and significantly increases navigator efficiency, thereby decreasing total imaging time by about 40% compared to a conventional FB acquisition strategy.