XMEA: A New Hybrid Diamond Multielectrode Array for the In Situ Assessment of the Radiation Dose Enhancement by Nanoparticles.

This work presents a novel multielectrode array (MEA) to quantitatively assess the dose enhancement factor (DEF) produced in a medium by embedded nanoparticles. The MEA has 16 nanocrystalline diamond electrodes (in a cell-culture well), and a single-crystal diamond divided into four quadrants for X-ray dosimetry. DEF was assessed in water solutions with up to a 1000 µg/mL concentration of silver, platinum, and gold nanoparticles. The X-ray detectors showed a linear response to radiation dose (r2 ≥ 0.9999). Overall, platinum and gold nanoparticles produced a dose enhancement in the medium (maximum of 1.9 and 3.1, respectively), while silver nanoparticles produced a shielding effect (maximum of 37%), lowering the dose in the medium. This work shows that the novel MEA can be a useful tool in the quantitative assessment of radiation dose enhancement due to nanoparticles. Together with its suitability for cells' exocytosis studies, it proves to be a highly versatile device for several applications.

Analytical Determination of Serotonin Exocytosis in Human Platelets with BDD-on-Quartz MEA Devices.

Amperometry is arguably the most widely used technique for studying the exocytosis of biological amines. However, the scarcity of human tissues, particularly in the context of neurological diseases, poses a challenge for exocytosis research. Human platelets, which accumulate 90% of blood serotonin, release it through exocytosis. Nevertheless, single-cell amperometry with encapsulated carbon fibers is impractical due to the small size of platelets and the limited number of secretory granules on each platelet. The recent technological improvements in amperometric multi-electrode array (MEA) devices allow simultaneous recordings from several high-performance electrodes. In this paper, we present a comparison of three MEA boron-doped diamond (BDD) devices for studying serotonin exocytosis in human platelets: (i) the BDD-on-glass MEA, (ii) the BDD-on-silicon MEA, and (iii) the BDD on amorphous quartz MEA (BDD-on-quartz MEA). Transparent electrodes offer several advantages for observing living cells, and in the case of platelets, they control activation/aggregation. BDD-on-quartz offers the advantage over previous materials of combining excellent electrochemical properties with transparency for microscopic observation. These devices are opening exciting perspectives for clinical applications.

Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets.

Platelets are probably the most accessible human cells to study exocytosis by amperometry. These cell fragments accumulate biological amines, serotonin in particular, using similar if not the same mechanisms as those employed by sympathetic, serotoninergic, and histaminergic neurons. Thus, platelets have been widely recognized as a model system to study certain neurological and psychiatric diseases. Platelets release serotonin by exocytosis, a process that entails the fusion of a secretory vesicle to the plasma membrane and that can be monitored directly by classic single cell amperometry using carbon fiber electrodes. However, this is a tedious technique because any given platelet releases only 4-8 secretory δ-granules. Here, we introduce and validate a diamond-based multielectrode array (MEA) device for the high-throughput study of exocytosis by human platelets. This is probably the first reported study of human tissue using an MEA, demonstrating that they are very interesting laboratory tools to assess alterations to exocytosis in neuropsychiatric diseases. Moreover, these devices constitute a valuable platform for the rapid testing of novel drugs that act on secretory pathways in human tissues.

Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs.

Diamond-based multiarray sensors are suitable to detect in real-time exocytosis and action potentials from cultured, spontaneously firing chromaffin cells, primary hippocampal neurons, and midbrain dopaminergic neurons. Here, we focus on how amperometric measurements of catecholamine release are performed on micrographitic diamond multiarrays (µG-D-MEAs) with high temporal and spatial resolution by 16 electrodes simultaneously.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

Triggering Neurotransmitters Secretion from Single Cells by X-ray Nanobeam Irradiation.

The employment of ionizing radiation is a powerful tool in cancer therapy, but beyond targeted effects, many studies have highlighted the relevance of its off-target consequences. An exhaustive understanding of the mechanisms underlying these effects is still missing, and no real-time data about signals released by cells during irradiation are presently available. We employed a synchrotron X-ray nanobeam to perform the first real-time simultaneous measurement of both X-ray irradiation and in vitro neurotransmitter release from individual adrenal phaeochromocytoma (PC12) cells plated over a diamond-based multielectrode array. We have demonstrated that, in specific conditions, X-rays can alter cell activity by promoting dopamine exocytosis, and such an effect is potentially very attractive for a more effective treatment of tumors.

In situ self-assembling Au-DNA complexes for targeted cancer bioimaging and inhibition.

Cancer remains one of the most challenging diseases to treat. For accurate cancer diagnosis and targeted therapy, it is important to assess the localization of the affected area of cancers. The general approaches for cancer diagnostics include pathological assessments and imaging. However, these methods only generally assess the tumor area. In this study, by taking advantage of the unique microenvironment of cancers, we effectively utilize in situ self-assembled biosynthetic fluorescent gold nanocluster-DNA (GNC-DNA) complexes to facilitate safe and targeted cancer theranostics. In in vitro and in vivo tumor models, our self-assembling biosynthetic approach allowed for precise bioimaging and inhibited cancer growth after one injection of DNA and gold precursors. These results demonstrate that in situ bioresponsive self-assembling GNC-PTEN (phosphatase and tensin homolog) complexes could be an effective noninvasive technique for accurate cancer bioimaging and treatment, thus providing a safe and promising cancer theranostics platform for cancer therapy.

Simultaneous multisite detection of quantal release from PC12 cells using micro graphitic-diamond multi electrode arrays.

Micro graphitic - diamond - multi electrode arrays (µG-D-MEAs) are suitable for measuring multisite quantal dopamine (DA) release from PC12 cells. Following cell stimulation with high extracellular KCl and electrode polarization at +650 mV, amperometric spikes are detected with a mean frequency of 0.60 ±â€¯0.16 Hz. In each recording, simultaneous detection of secretory events is occurred in approximately 50% of the electrodes. Kinetic spike parameters and background noise are preserved among the different electrodes. Comparing the amperometric spikes recorder under control conditions with those recorders from PC12 cells previously incubated for 30 min with the dopamine precursor Levodopa (L-DOPA, 20 µM) it appears that the quantal size of amperometric spikes is increased by 250% and the half-time width (t1/2) by over 120%. On the contrary, L-DOPA has no effect on the frequency of secretory events. Overall, these data demonstrate that the µG-D-MEAs represent a reliable bio-sensor to simultaneously monitor quantal exocytotic events from different cells and in perspective can be exploited as a drug-screening tool.

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

Micro-Graphitic Single Crystal Diamond Multi Electrode Arrays (µG-SCD-MEAs) have so far been used as amperometric sensors to detect catecholamines from chromaffin cells and adrenal gland slices. Besides having time resolution and sensitivity that are comparable with carbon fiber electrodes, that represent the gold standard for amperometry, µG-SCD-MEAs also have the advantages of simultaneous multisite detection, high biocompatibility and implementation of amperometric/potentiometric protocols, aimed at monitoring exocytotic events and neuronal excitability. In order to adapt diamond technology to record neuronal activity, the µG-SCD-MEAs in this work have been interfaced with cultured midbrain neurons to detect electrical activity as well as quantal release of dopamine (DA). µG-SCD-MEAs are based on graphitic sensing electrodes that are embedded into the diamond matrix and are fabricated using MeV ion beam lithography. Two geometries have been adopted, with 4 × 4 and 8 × 8 microelectrodes (20 µm × 3.5 µm exposed area, 200 µm spacing). In the amperometric configuration, the 4 × 4 µG-SCD-MEAs resolved quantal exocytosis from midbrain dopaminergic neurons. KCl-stimulated DA release occurred as amperometric spikes of 15 pA amplitude and 0.5 ms half-width, at a mean frequency of 0.4 Hz. When used as potentiometric multiarrays, the 8 × 8 µG-SCD-MEAs detected the spontaneous firing activity of midbrain neurons. Extracellularly recorded action potentials (APs) had mean amplitude of ∼-50 µV and occurred at a mean firing frequency of 0.7 Hz in 67% of neurons, while the remaining fired at 6.8 Hz. Comparable findings were observed using conventional MEAs (0.9 and 6.4 Hz, respectively). To test the reliability of potentiometric recordings with µG-SCD-MEAs, the D2-autoreceptor modulation of firing was investigated by applying levodopa (L-DOPA, 20 µM), and comparing µG-SCD-MEAs, conventional MEAs and current-clamp recordings. In all cases, L-DOPA reduced the spontaneous spiking activity in most neurons by 70%, while the D2-antagonist sulpiride reversed this effect. Cell firing inhibition was generally associated with increased APs amplitude. A minority of neurons was either insensitive to, or potentiated by L-DOPA, suggesting that AP recordings originate from different midbrain neuronal subpopulations and reveal different modulatory pathways. Our data demonstrate, for the first time, that µG-SCD-MEAs are multi-functional biosensors suitable to resolve real-time DA release and AP firing in in vitro neuronal networks.

Correction to: Monitoring dynamic release of intracellular hydrogen peroxide through a microelectrode based enzymatic biosensor.

The authors would like to call the reader's attention to the fact that unfortunately Alberto Pasquarelli's and Kay-Eberhard Gottschalk's affiliations were wrong in the original publication.

Tailoring spin defects in diamond by lattice charging.

Atomic-size spin defects in solids are unique quantum systems. Most applications require nanometre positioning accuracy, which is typically achieved by low-energy ion implantation. A drawback of this technique is the significant residual lattice damage, which degrades the performance of spins in quantum applications. Here we show that the charge state of implantation-induced defects drastically influences the formation of lattice defects during thermal annealing. Charging of vacancies at, for example, nitrogen implantation sites suppresses the formation of vacancy complexes, resulting in tenfold-improved spin coherence times and twofold-improved formation yield of nitrogen-vacancy centres in diamond. This is achieved by confining implantation defects into the space-charge layer of free carriers generated by a boron-doped diamond structure. By combining these results with numerical calculations, we arrive at a quantitative understanding of the formation and dynamics of the implanted spin defects. These results could improve engineering of quantum devices using solid-state systems.

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.

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 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.

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.

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

Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose-response calibrations, set the lowest detectable concentration of adrenaline to ∼5 µm. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12-27 µm2. In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The 'non-active' ('silent') zones covered 24% of the total and persisted for 6-8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution.

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 16­channels 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.

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.

Tooth tissue engineering: the influence of hydrophilic surface on nanocrystalline diamond films for human dental stem cells.

New techniques for tissue engineering (TE) are rapidly emerging. The basic concept of autologous TE is to isolate cells from small biopsy specimens, and to expand these cells in culture for subsequent seeding onto biodegradable scaffolds. Nanocrystalline diamond films have attracted the attention of researchers from a variety of different areas in recent years, due to their unique and exceptional properties. In this approach, human dental stem cells (hDSCs) were characterized by flow cytometry and grown on diamond films with hydrogen (H)-terminated and oxygen (O)-terminated surfaces for 28 days, and then removed by lysis and washing with distilled water. Energy dispersive spectroscopy analysis was performed, showing that the regions with O-terminated surfaces contained much higher levels of deposited calcium, oxygen, and phosphorus. These results suggest that the extracellular matrix was considerably more developed in the O-terminated regions, as compared with the H-terminated regions. In addition, optical microscopy of hDSCs cultured on the diamond substrate with H- and O-terminated surfaces, before washing with distilled water, showed preferential directions of the cells arrangement, where orthogonal lines suggest that the cells appeared to be following the O-terminated regions or hydrophilic surface. These findings suggest that O-terminated diamond surfaces prepared on biodegradable scaffolds can be useful for mineralized dental tissue formation.

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.