Amperometric detection of quantal catecholamine secretion from individual cells by an ion beam microfabricated single crystalline diamond biosensor

It is shown that buried graphitic channels fabricated in monocrystalline diamond by selective damage induced by focused MeV ions, can be considered an effective alternative to the commonly used carbon-fibers to detect the catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis. Quantal secretory responses have been measured from stimulated chromaffin cells, which were positioned on the graphitic microelectrode, polarized to +800 mV. Sequences of amperometric spikes started after cell stimulation with the KCl solution, with amplitudes well above the background noise within the range of 8–180 pA and comparable with signals obtained by conventional carbon fiber electrodes

Kelvin probe characterization of buried graphitic microchannels in single-crystal diamond

In this work, we present an investigation by Kelvin Probe Microscopy (KPM) of buried graphitic microchannels fabricated in single-crystal diamond by direct MeV ion microbeam writing. Metal deposition of variable-thickness masks was adopted to implant channels with emerging endpoints and high temperature annealing was performed in order to induce the graphitization of the highly-damaged buried region. When an electrical current was flowing through the biased buried channel, the structure was clearly evidenced by KPM maps of the electrical potential of the surface region overlying the channel at increasing distances from the grounded electrode. The KPM profiling shows regions of opposite contrast located at different distances from the endpoints of the channel. This effect is attributed to the different electrical conduction properties of the surface and of the buried graphitic layer. The model adopted to interpret these KPM maps and profiles proved to be suitable for the electronic characterization of buried conductive channels, providing a non-invasive method to measure the local resistivity with a micrometer resolution. The results demonstrate the potential of the technique as a powerful diagnostic tool to monitor the functionality of all-carbon graphite/diamond devices to be fabricated by MeV ion beam lithography.Comment: 21 pages, 5 figure

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

Direct experimental observation of nonclassicality in ensembles of single photon emitters

In this work we experimentally demonstrate for the first time a recently proposed criterion adressed to detect nonclassical behavior in the fluorescence emission of ensembles of single-photon emitters. In particular, we apply the method to study clusters of NV centres in diamond observed via single-photon-sensitive confocal microscopy. Theoretical considerations on the behavior of the parameter at any arbitrary order in presence of poissonian noise are presented and, finally, the opportunity of detecting manifold coincidences is discussed

Creation of pure non-crystalline diamond nanostructures via room-temperature ion irradiation and subsequent thermal annealing

Carbon exhibits a remarkable range of structural forms, due to the availability of sp3, sp2 and sp1 chemical bonds. Contrarily to other group IV elements such as silicon and germanium, the formation of an amorphous phase based exclusively on sp3 bonds is extremely challenging due to the strongly favored formation of graphitic-like structures at room 19 temperature and pressure. As such, the formation of a fully sp3-bonded carbon phase requires 20 an extremely careful (and largely unexplored) definition of the pressure and temperature across the phase diagram. Here, we report on the possibility of creating full-sp3 amorphous nanostructures within the bulk crystal of diamond with room-temperature ion-beam irradiation, followed by an annealing process that does not involve the application of any external mechanical pressure. As confirmed by numerical simulations, the (previously unreported) radiation-damage-induced formation of an amorphous sp2-free phase in diamond is determined by the buildup of extremely high internal stresses from the surrounding lattice, which (in the case of nanometer-scale regions) fully prevent the graphitization process. Besides the relevance of understanding the formation of exotic carbon phases, the use of focused/collimated ion beams discloses appealing perspectives for the direct fabrication of such nanostructures in complex three-dimensional geometries

Estimates and multivariable risk assessment of mid-buccal gingival recessions in an Italian adult population according to the 2018 World Workshop Classification System

Objectives: The aim of this cross-sectional study was to provide estimate of mid-buccal gingival recession (GR) according to the 2018 World Workshop Classification System and to explore GR risk indicators in a representative urban population in North-West of Italy. Material and methods: This is a secondary analysis using data collected in an epidemiological study enrolling a representative sample of 736 adults, living in Turin. GR prevalence was defined as the presence of at least one mid-buccal GR ≥ 1 mm. GRs were categorized according to the 2018 classification system (RT1, RT2, RT3) and to different severity cutoffs. Logistic regression analysis was performed to identify RT GR risk indicators. Results: Mid-buccal GR ≥ 1 mm affected 57.20% of subjects and 14.56% of teeth. When considering RT1 GRs, their prevalence was 40.90% and 6.29% at the patient and tooth level. RT2 and RT3 GRs affected 25.82% and 36.68% of the study population, respectively. RT1 GRs occurred mostly on maxillary and mandibular premolars and maxillary canines, while RT2 and RT3 GRs on maxillary molars and mandibular incisors. Older age, high education, and full-mouth plaque score (FMPS) 60% were significant contributors to RT2 and RT3 GRs. Conclusions: RT1 and RT3 are fairly common findings in this Italian population and are significantly associated to different contributing factors and tooth type distribution pattern. Clinical relevance: Prevention strategies should target different socio-demographic, behavioral, and clinical risk indicators based on the RT classes