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

Awareness, treatment, and control of major cardiovascular risk factors in a small-scale Italian community : results of a screening campaign

Introduction: Hypertension, hypercholesterolemia, and diabetes are the main causes of cardiovascular diseases in developed countries. However, these conditions are still poorly recognized and treated. Objective: This study aimed at estimating the prevalence, awareness, treatment, and control rates of major cardiovascular risk factors in an unselected sample of individuals of a small community located in northern Italy. Methods: We screened 344 sequential subjects in this study. Data collection included family and clinical history, anthropometric data, blood pressure, blood glucose, and serum cholesterol values. Individual cardiovascular risk profiles were assessed by risk charts of the Progetto Cuore. Results: Based on personal history and/or measured values, 78.2% of subjects had hypercholesterolemia (total cholesterol levels > 190 mg/dL), 61.0% had central obesity (waist circumference 65 94 cm for men and 6580 cm for women), 51.2% had arterial hypertension (blood pressure 65 140/90 mmHg), 8.1% had diabetes (blood glucose 65 126 mg/dL), 22.7% had impaired fasting glucose (blood glucose 100\u2013125 mg/dL), and 35.5% were overweight (body mass index 25\u201329 kg/m2). Alcohol drinkers and smokers accounted for 46.2% and 22.4% of subjects, respectively. Awareness of hypertension, hypercholesterolemia, and diabetes was poor, and control of these risk factors, except for diabetes, was even worse. Prevalence of high blood pressure, high serum cholesterol, overweight, and obesity significantly increased with aging. Hypercholesterolemia and obesity were significantly more common in women, while overweight and diabetes in men. In 15.4% of participants, the risk of a major cardiovascular event in the next 10 years was either high or very high. Conclusion: In a small community in a wealthy region of Italy, the prevalence of major cardiovascular risk factors is high, while awareness, treatment, and control are poor. Such a result highlights the importance of screening campaigns as a strategy to improve early diagnosis and access to treatment, and thus effective prevention of cardiovascular diseases in the general population

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

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