181 research outputs found
Microelectrode arrays of diamond-insulated graphitic channels for real time detection of exocytotic events from cultured chromaffin cells and slices of adrenal glands
A microstructured graphitic 4x4 multielectrode array was embedded in a single
crystal diamond substrate (4x4 {uG-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 (20x3.5 um^2) separated by 200 um 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, iii) monitoring quantal
secretory events from thin slices of the adrenal gland. The frequency of
spontaneous release was low (0.12 Hz 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 to "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
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.Comment: 24 pages, 5 figure
Nanodiamonds-induced effects on neuronal firing of mouse hippocampal microcircuits
Fluorescent nanodiamonds (FND) are carbon-based nanomaterials that can
efficiently incorporate optically active photoluminescent centers such as the
nitrogen-vacancy complex, thus making them promising candidates as optical
biolabels and drug-delivery agents. FNDs exhibit bright fluorescence without
photobleaching combined with high uptake rate and low cytotoxicity. Focusing on
FNDs interference with neuronal function, here we examined their effect on
cultured hippocampal neurons, monitoring the whole network development as well
as the electrophysiological properties of single neurons. We observed that FNDs
drastically decreased the frequency of inhibitory (from 1.81 Hz to 0.86 Hz) and
excitatory (from 1.61 Hz to 0.68 Hz) miniature postsynaptic currents, and
consistently reduced action potential (AP) firing frequency (by 36%), as
measured by microelectrode arrays. On the contrary, bursts synchronization was
preserved, as well as the amplitude of spontaneous inhibitory and excitatory
events. Current-clamp recordings revealed that the ratio of neurons responding
with AP trains of high-frequency (fast-spiking) versus neurons responding with
trains of low-frequency (slow-spiking) was unaltered, suggesting that FNDs
exerted a comparable action on neuronal subpopulations. At the single cell
level, rapid onset of the somatic AP ("kink") was drastically reduced in
FND-treated neurons, suggesting a reduced contribution of axonal and dendritic
components while preserving neuronal excitability.Comment: 34 pages, 9 figure
Nanodiamond quantum sensors reveal temperature variation associated to hippocampal neurons firing
Temperature is one of the most relevant parameters for the regulation of
intracellular processes. Measuring localized subcellular temperature gradients
is fundamental for a deeper understanding of cell function, such as the genesis
of action potentials, and cell metabolism. Here, we detect for the first time
temperature variations (1{\deg}C) associated with potentiation and depletion of
neuronal firing, exploiting a nanoscale thermometer based on optically detected
magnetic resonance in nanodiamonds. Our results provide a tool for assessing
neuronal spiking activity under physiological and pathological conditions and,
conjugated with the high sensitivity of this technique (in perspective
sensitive to < 0.1{\deg}C variations), pave the way to a systematic study of
the generation of localized temperature gradients. Furthermore, they prompt
further studies explaining in detail the physiological mechanism originating
this effect.Comment: 27 pages, 5 figures, 3 table
A facility for the test of large area muon chambers at high rates
Operation of large area muon detectors at the future Large Hadron Collider (LHC) will be characterized by large sustained hit rates over the whole area, reaching the range of kHz/\scm. We describe a dedicated test zone built at CERN to test the performance and the aging of the muon chambers currently under development. A radioactive source delivers photons causing the sustained rate of random hits, while a narrow beam of high energy muons is used to directly calibrate the detector performance. A system of remotely controlled lead filters serves to vary the rate of photons over four orders of magnitude, to allow the study of performance as a function of rate
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