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A New Diamond Biosensor with Integrated Graphitic Microchannels for Detecting Quantal Exocytic Events from Chromaffin Cells
Advanced Materials
Abstract
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.
... The modulation of the ion penetration depth is defined by a degrading stack of metal layers placed directly over the sample surface, essential in order to have the graphitic structure endpoints emerging at the surface. The main limitations ascribable to this technology are the need for a microbeam line on an accelerator facility and the creation of the structures in a serial way [81]. ...
... The carrier is equipped with a perfusion chamber containing the growing medium essential for the cell plating. The device is finally interfaced with the front-end electronic whose performances and characteristics are determined by the kind of measurement performed (amperometry or potentiometry) but are in general similar to those of commercial systems like the amplifier for carbon fibre electrodes or titanium-nitride (TiN) MEAs [32,79,81,109,115]. ...
To understand the working principles of the nervous system is key to figure out its electrical activity and how this activity spreads along the neuronal network. It is therefore crucial to develop advanced techniques aimed to record in real time the electrical activity, from compartments of single neurons to populations of neurons, to understand how higher functions emerge from coordinated activity. To record from single neurons, a technique will be presented to fabricate patch pipettes able to seal on any membrane with a single glass type and whose shanks can be widened as desired. This dramatically reduces access resistance during whole-cell recording allowing fast intracellular and, if required, extracellular perfusion. To simultaneously record from many neurons, biocompatible probes will be described employing multi-electrodes made with novel technologies, based on diamond substrates. These probes also allow to synchronously record exocytosis and neuronal excitability and to stimulate neurons. Finally, to achieve even higher spatial resolution, it will be shown how voltage imaging, employing fast voltage-sensitive dyes and two-photon microscopy, is able to sample voltage oscillations in the brain spatially resolved and voltage changes in dendrites of single neurons at millisecond and micrometre resolution in awake animals.
... In order to overcome this limitation, laser-induced graphitization in diamond can be combined with a preliminary MeV-ion-induced graphitization stage. By taking advantage of the high degree of control on the geometrical properties (depth, thickness) of MeV-ion-induced buried graphitic structures in diamond allowed by the peculiar nuclear energy loss profile of MeV ions [29], this double-step procedure guarantees a better definition in the material micro-structuring [30] and also represents an interesting improvement in the realization of particle detectors [26,31,32], bolometers [33,34], bio-sensors [35][36][37], metallic-dielectric structures [38] and microfluidics [39]. ...
... By allowing a fine tuning of geometrical and structural properties of graphitic layers formed by ion irradiation, laser-induced graphitization offers interesting opportunities for a new level of control in the fabrication of buried graphitic structures in diamond, with appealing applications in different fields in which MeV ion beam lithography and laser graphitization were successfully employed [26,[31][32][33][34][35][36]38,39]. ...
We report on the structural modifications induced by a λ = 532 nm ns-pulsed high-power laser on sub-superficial graphitic layers in single-crystal diamond realized by means of MeV ion implantation. A systematic characterization of the structures obtained under different laser irradiation conditions (power density, number of pulses) and subsequent thermal annealing was performed by different electron microscopy techniques. The main feature observed after laser irradiation is the thickening of the pre-existing graphitic layer. Cross-sectional SEM imaging was performed to directly measure the thickness of the modified layers, and subsequent selective etching of the buried layers was employed to both assess their graphitic nature and enhance the SEM imaging contrast. In particular, it was found that for optimal irradiation parameters the laser processing induces a six-fold increase the thickness of sub-superficial graphitic layers without inducing mechanical failures in the surrounding crystal. TEM microscopy and EELS spectroscopy allowed a detailed analysis of the internal structure of the laser-irradiated layers, highlighting the presence of different nano-graphitic and amorphous layers. The obtained results demonstrate the effectiveness and versatility of high-power laser irradiation for an accurate tuning of the geometrical and structural features of graphitic structures embedded in single-crystal diamond, and open new opportunities in diamond fabrication.
... Our previous studies on the fabrication of conductive graphitic microchannels in single-crystal diamond by DIBL led to the realization of a prototypical single cell biosensor. With such device, the exocytotic activity from single chromaffin cells was measured by amperometry, suggesting the potential of further developing the diamond-based device into a multi cell sensor: the electrochemical performances of the graphitic electrodes embedded into diamond matrix were compared with commercial carbon fibre electrodes demonstrating their compatibility with the state-of-the-art technique [57]. The present work reports our progresses in the realization of multiple conductive micro-structures in single-crystal diamond and on the consequent electrochemical testing of a 16-channel multi electrode array (MEA) biosensor. ...
... This peculiarity is crucial for the detection of the oxidation current of the more common neurotransmitters, which were observed between +650 mV and +850 mV. Moreover, the low leakage currents ensure a limit of detection which allows the recording of spike signals (>20 pA) produced by the exocytic events in neuroendocrine cells [57]. ...
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 ~mOmega·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.
... A relevant number of works has concentrated in the recent years on the application of MeV-ion-induced graphitization to fabricate and functionalize microstructures and devices in single-crystal diamond , including bio-sensors [1], ionizing radiation detectors [2,3] , bo- lometers [4], nano-electromechanical systems (NEMS) [5,6], photonic structures78910 and optical waveguides [11,12] . Laser-induced graphitization has also been employed to fabricate metallo-dielectric struc- tures [13] and ionizing radiation detectors [14] in diamond. ...
... This versatility is due to the fact that both MeV-ion and laser focused beams can locally deliver high power densities in specific regions within the diamond bulk with micrometric spatial resolution in all directions, thus creating confined regions where the diamond lattice structure is critically damaged. In these regions, annealing leads to the graphitization of the damaged structure, whilst the remaining surrounding material is largely restored to pristine diamond, so that well-defined structures can be created by selectively etching the graphitized regions [3,56789101112 or taking advantage of the optical/electrical properties of the graphitized regions [1,2,4,13,14] . At significantly lower damage densities (i.e. ...
... Furthermore, CFEs have a short lifetime [4]. To address these limitations, over the past decade, we have developed prototypes of multi-electrode arrays known as MicroGraphited-Diamond-Multi Electrode Arrays (µG-D-MEAs) [5][6][7]. These prototypes showcase the capability to simultaneously capture quantal exocytotic events in real-time from both neurosecretory cells and midbrain neurons cultured directly on µG-D-MEAs. ...
MicroGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As μG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.
... The MEA device ( Fig. 1) is composed by an artificial diamond in which graphitic tracks have been implanted using the MeV ion beam lithography technique [26]. ...
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.Key wordsDiamond microelectrode devicesChromaffin cellsExocytosisIon beam lithography
... Diamond is widely applied in biosensing [1], cutting/grinding processing [2,3], optics [4][5][6], and many other fields for its excellent properties, such as the ultrahigh hardness, the superior thermal conductivity, and the excellent chemical stability [7,8]. The pulsed laser has been widely employed in the precision micromachining of the diamond for its high energy density, good controllability, and high processing accuracy [9][10][11][12][13][14]. Due to the complex transient heating process occurred during the pulsed laser ablation of the diamond [15,16], in the laser heat-affected zone (HAZ), both the physical and chemical properties are subjected to large variations, owing to the phase transition and the microstructure change [17][18][19][20]. ...
In this paper, a comprehensive finite element (FE) simulation model of the heat transfer for laser ablation of a single crystalline diamond (SCD) with a scanning laser beam in Gaussian shape is developed. The model takes into account the material properties, the geometric structure, and the thermal boundary conditions. It is employed to study the dependence of the temperature distribution under varying laser machining parameters. The distribution characteristics of the temperature field, the temperature evolution, and the heat conduction on the diamond surface are obtained, analyzed, and discussed. The law describing the influence of the laser parameters on the temperature field on the diamond surface is established. After comparing the numerically estimated thermal penetration depth of the pulsed laser in the diamond with the experimentally ablated groove characterized by a scanning electron microscope (SEM), a white light interferometer (WLI) and the Raman spectroscopy analysis of the deposited metamorphic layer, it is found that the developed model shows an excellent predictive capability and provides a promising tool for the future optimization of the machining parameters.
... [9 12] -In addition, it is noteworthy that diamond is also an attractive candidate for fabricating cellular biosensors due to its inherent biocompatibility. [13] In fact, single crystalline diamond (SCD) intrinsically holds numerous excellent properties for optical, photonic, and quantum-optical applications. Due to its wide light transmission spectrum and comparatively high refractive index, it is appealing to use SCD to manufacture light-guiding structures operating at wavelengths in the range from ultraviolet (UV) to midinfrared ray. ...
Comprehending the implantation related characteristic quantities of multi‐high‐energy proton irradiation of diamond is of significant technological interest for sensing applications, fabrication of three‐dimensional photonic crystals, and color centers (e.g. NV⁻ center) based quantum computing. In this paper, proton projected ranges and defect distributions in the (010) plane (Y‐normal plane) of microwave plasma chemical vapour deposition (MPCVD) grown diamond irradiated with 0.5‐2.0 MeV proton (H⁺ or molecular H2+ ions) in a non‐channeling (near [001], Z‐axis) direction, were experimentally measured via cross sectional optical microscopy and high resolution confocal Raman spectroscopy mapping. It was found that the projected ranges of the experimental findings and the simulation results indicate good consistencies, while there is considerable disagreement between them in regards to longitudinal straggling and lateral straggling. Based on the comparisons and analyses, we obtained a clear view of protons implanted in single crystalline diamond in the high‐energy range theoretically and experimentally.
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... Its suitability to the fabrication of integrated cellular sensors for in vitro measurements has already been demonstrated in a series of previous works. 18 −20 As shown in Figure 1, each fabricated subsuperficial conductive microchannel is characterized by two emerging end-points, one in correspondence of the biological sample under investigation (i.e., the cells plated in the central region of the device) and the other at the input of the acquisition electronic chain (i.e., the readout contacts at the peripheral region). ...
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 nano-beam 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 multi-electrode array. We have demonstrated that, in specific conditions, X-rays can alter cell activity by promoting dopamine exocytosis: such effect is potentially very attractive for a more effective treatment of tumours.
... Several strategies have been developed for the fabrication of multielectrode arrays with different techniques and different materials. To list a few: platinum ultramicroelectrodes [6], platinum electrodes covered by iridium films [7], sensors based on CNT ITO modified electrodes [8], nitrogen-doped diamond-like-carbon [9], boron-doped diamond [10,11] and Micro Graphitic -Diamond -Multi-Electrode Arrays (μG-D-MEAs) [12][13][14][15][16]. ...
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 ± 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.
... In spite of this, no data concerning the detection of quantal release and electrical activity from the same cultured neurons using the same multiarray prototypes have been reported, to the best of our knowledge. Micro-graphitic single-crystal diamond multielectrode arrays (µG-SCD-MEAs) are a powerful sensor for investigating neurosecretion in living cells (Picollo et al., 2013(Picollo et al., , 2015b. Previous findings have demonstrated their ability to monitor spontaneous and evoked quantal catecholamine release from cultured mouse and bovine adrenal chromaffin cells (Picollo et al., 2016b) as well as from fresh mouse adrenal slices (Picollo et al., 2016a;Carabelli et al., 2017). ...
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.
... Among the aforementioned materials, ITO and diamond are advantageous alternatives of CFEs because they show quite similar characteristics such as high biocompatibility, outstanding electrochemical responsiveness as well as chemical inertness [57,58]. More importantly, their optical transparency endows them the inherent superiority to couple amperometry with fluorescence microscopy. ...
Vesicular exocytosis is a ubiquitous bio-process involved in plenty of normal and pathologic events in living cells and has received increasing research attention. After decades of progress, amperometry has been demonstrated to be one of the most powerful techniques to monitor the exocytotic release in real time because of its remarkable advantages such as facility to implement, high sensitivity and sub-millisecond temporal resolution. In this review, we provide an overview of recent advances in electrochemical courses related to exocytotic secretions. The principle for amperometric characterization of quantal exocytosis is briefly introduced. Development of mircrofabricated architectures for amperometric detection and their contributions are summarized. An intimate coupling of amperometry and fluorescence imaging for in-situ exocytosis tracking are highlighted.
... Intrinsic SCD cantilevers fabricated via ion beam assisted lift-off (IAL) [24][25][26] (Figure S1, Supporting information) were adopted to demonstrate the on-chip SCD NEMS/MEMS based on SEA transduction. The single crystal nature of the SCD cantilevers was confirmed by Raman and high-resolution transmission microscopy, which can be found elsewhere. ...
All‐electrical nano‐electromechanical or micro‐electromechanical systems (NEMS/MEMS) with integrated sensing and actuation are essential to fulfill the functionalities of single‐crystal diamond (SCD) NEMS/MEMS by integrated circuits for sensors or quantum systems. However, the implementation of all‐electrical on‐chip SCD NEMS/MEMS transducers has encountered bottlenecks due to the lack of shallow dopants in diamond and the difficulty of integrating active materials on diamond. In the present work, an on‐chip SCD NEMS/MEMS with unprecedented figure‐of‐merits is demonstrated by proposing a self‐sensing enhancing actuation scheme to circumvent the bottlenecks in diamond. The on‐chip SCD NEMS/MEMS show high self‐sensitivity, low actuation voltage, little energy dissipation, and high‐frequency and high‐temperature operation (873 K). The present work provides a platform for the development of ultrahigh‐performance and robust SCD NEMS/MEMS in sensors and quantum science fields, superior to other semiconductors.
... We are currently extending the device to 1000 electrodes, a number that cannot be achieved using external connections for each electrode. Various materials have been tested as electrodes for amperometry, including diamond-like carbon [18,27], indium tin oxide [27,45], boron-doped diamond [8,42], and graphitic electrodes embedded in a diamond substrate [8,[36][37][38][39]. Each of these materials has their unique property for measuring quantal release events. ...
Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.
... T h e co nd uc t i v e p oly m er po l y( 3, 4ethylenedioxythiophene) (PEDOT) doped with poly(styrene sulfonate) has high transparency, flexibility, and biocompatibility [60,71] and has been shown to be suitable for amperometric detection of release [49], including quantal exocytosis [93]. Microstructured graphitic multielectrode arrays embedded in a single-crystal diamond were realized as sub-superficial conductive micropaths by means of a deep ion beam lithography technique: by focusing ion beam writing through variablethickness masks, the approach allows to regulate the depth at which the channels are formed and their emergence at specific locations of the sample surface [65,66,68]. Micrographitic arrays can detect quantal exocytosis from isolated cells and adrenal slices, and are sensitive enough to distinguish full fusion from stand-alone foot signals [45,67]. ...
Carbon-fiber electrodes (CFEs) are the gold standard for quantifying the release of oxidizable neurotransmitters from single vesicles and single cells. Over the last 15 years, microfabricated devices have emerged as alternatives to CFEs that offer the possibility of higher throughput, subcellular spatial resolution of exocytosis, and integration with other techniques for probing exocytosis including microfluidic cell handling and solution exchange, optical imaging and stimulation, and electrophysiological recording and stimulation. Here we review progress in developing electrochemical electrode devices capable of resolving quantal exocytosis that are fabricated using photolithography.
... Multisite detection of exocytosis from cultured chromaffin cells and adrenal slices has been also assessed by a new DBMs generation. In these devices, the sensitive elements are sub-superficial graphitic micro-channels in a single-crystal diamond (µG-SCD) in different geometries 83,84,42,43,85 . Bovine chromaffin cells can be cultured on these devices without protein coating, with a density of 150.000 cells per chip, to ensure that each of the 16 (or 64) microelectrodes contact one cell. ...
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.
... In this work, the electrical control of the charge state in NV ensembles is investigated in a singlecrystal diamond substrate structured with sub-superficial graphitic micro-electrodes fabricated by means of MeV ion beam lithography [22]. The employed fabrication technique allows to define arbitrary electrode geometries with micrometric resolution in the diamond bulk (i.e. up to several micrometers below the sample surface) by exploiting the radiation-induced graphitization of the material occurring at the end of the MeV ion penetration range, and has already been successfully adopted to realize different integrated devices in diamond, such as bolometers [23,24], particle detectors [25], cellular biosensors [26,27] and IR emitters [28]. More pertinently to this work, sub-superficial graphitic electrodes were previously employed to stimulate electroluminescence from diamond color centers, both in multi-photon [29] and single-photon [30] emission regimes. ...
The control of the charge state of nitrogen-vacancy (NV) centers in diamond is of primary importance for the stabilization of their quantum-optical properties, in applications ranging from quantum sensing to quantum computing. To this purpose, in this work current-injecting micro-electrodes were fabricated in bulk diamond for NV charge state control. Buried (i.e. 3 {\mu}m in depth) graphitic micro-electrodes with spacing of 9 {\mu}m were created in single-crystal diamond substrates by means of a 6 MeV C scanning micro-beam. The high breakdown field of diamond was exploited to electrically control the variation in the relative population of the negative (NV-) and neutral (NV0) charge states of sub-superficial NV centers located in the inter- electrode gap regions, without incurring into current discharges. Photoluminescence spectra acquired from the biased electrodes exhibited an electrically induced increase up to 40% in the NV- population at the expense of the NV0 charge state. The variation in the relative charge state populations showed a linear dependence from the injected current at applied biases smaller than 250 V, and was interpreted as the result of electron trapping at NV sites, consistently with the Space Charge Limited Current interpretation of the abrupt current increase observed at 300 V bias voltage. In correspondence of such trap-filling-induced transition to a high-current regime, a strong electroluminescent emission from the NV0 centers was observed. In the high-current-injection regime, a decrease in the NV- population was observed, in contrast with the results obtained at lower bias voltages. These results disclose new possibilities in the electrical control of the charge state of NV centers located in the diamond bulk, which are characterized by longer spin coherence times.
... The high Raman coefficient is also useful for implementing solid state quantum memories, a vital component for any quantum optical technology enabling the storage and retrieval of quantum information 10 . In addition, diamond serves as an ideal platform for biophotonics due to a high level of biocompatibility 11 . The efficiency of many of these technologies would be improved by the ability to confine and route light using a network of optical waveguides 12 . ...
Three dimensional waveguides within the bulk of diamond are manufactured using ultrafast laser fabrication. High intensities within the focal volume of the laser cause breakdown of the diamond into a graphitic phase leading to a stress induced refractive index change in neighboring regions. Type II waveguiding is thus enabled between two adjacent graphitic tracks, but supporting just a single polarization state. We show that adaptive aberration correction during the laser processing allows the controlled fabrication of more complex structures beneath the surface of the diamond which can be used for 3D waveguide splitters and Type III waveguides which support both polarizations.
... Device microfabrication. The SCD-MEAs were realized using optical-grade single-crystal artificial diamond substrates by means of an advanced MeV ion beam lithography technique, whose effectiveness was previously demonstrated by the proof-of-concept single-electrode device reported in 17 . ...
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.
... Ion-beam-induced damage on diamond crystals has been observed and studied in the past only either in the low ion energy range (i.e., b10 MeV) [10][11][12][13] or for light ion beams [14][15][16][17][18]. In both cases nuclear stopping power is far larger than its electronic counterpart. ...
... Thus, micro-beam writing can be employed in the fabrication of conductive channels or pads under the surface of diamond, while pulsed laser graphitization is suitable for fabrication of conductive columns, perpendicular to the surface, or of conductive channels, at the surface level. In this way, electrodes inside diamond can be implemented in three-dimensional diamond detectors, or in micro-electrodes arrays employed in studies of biological tissues [12,13], or in Stark-effect tuned optical micro-cavities [14], just to mention some of the possible applications. Moreover, the optical modification of the material induced by ion implantation can be used to implement light guides in microoptical devices. ...
... Our findings also suggest that quantal events recorded from the apex (with CFEs) or from the bottom of a chromaffin cell (with the 9-Ch NCD-UMEA) are comparable. This is in good agreement with recordings from cell bottom using planar arrays of different materials and geometry, and confirms the convenience of using planar microchips for detecting secretion (Chen et al. 1994;Hafez et al. 2005;Gao et al. 2010;Picollo et al. 2013). However, these observations are at variance with previous findings on BCCs (Amatore et al. 2007), reporting different secretion efficiency between the apex and the bottom of the cell using ITO devices. ...
Key points
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 μ m .
Using mouse and bovine chromaffin cells, single quantal exocytotic events are recorded from nine microareas of 12–27 μm ² . 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.
Abstract
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 μ m . 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 μm ² . 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.
... Ion implantation has been widely applied to the fabrication and functionalization of single-crystal diamond, with application in diverse fields such as optics and photonics [1][2][3][4][5][6][7][8], bio-sensors [9], particle detectors [10,11] and micro-electromechanical systems (MEMS) [12][13][14]. Several fabrication schemes can be implemented by exploiting light ions in the 10 2 -10 3 keV energy range, whose strongly non-uniform damage depth profile allows the creation of heavily damaged buried layers which graphitize after thermal annealing, whilst the structure of the surrounding material is largely restored [15][16][17]. ...
We present a phenomenological model and finite element simulations to describe the depth variation of mass density and strain of ion-implanted single-crystal diamond. Several experiments are employed to validate the approach: firstly, samples implanted with 180 keV B ions at relatively low fluences are characterized using high-resolution x-ray diffraction; secondly, the mass density variation of a sample implanted with 500 keV He ions, well above its amorphization threshold, is characterized with electron energy loss spectroscopy. At high damage densities, the experimental depth profiles of strain and density display a saturation effect with increasing damage and a shift of the damage density peak towards greater depth values with respect to those predicted by TRIM simulations, which are well accounted for in the model presented here. The model is then further validated by comparing transmission electron microscopy-measured and simulated thickness values of a buried amorphous carbon layer formed at different depths by implantation of 500 keV He ions through a variable-thickness mask to simulate the simultaneous implantation of ions at different energies.
The investigation of secondary effects induced by ionizing radiation represents a new and ever-growing research field in radiobiology. This new paradigm cannot be investigated only using standard instrumentation and methodologies, but rather requires novel technologies to achieve significant progress. In this framework, we developed diamond-based sensors that allow simultaneous real-time measurements with a high spatial resolution of the secretory activity of a network of cells cultured on the device, as well as of the dose at which they are exposed during irradiation experiments. The devices were functionally characterized by testing both the above-mentioned detection schemes, namely: amperometric measurements of neurotransmitter release from excitable cells (such as dopamine or adrenaline) and dosimetric evaluation using different ionizing particles (alpha particle and X-ray photons). Finally, the sensors were employed to investigate the effects induced by X-rays on the exocytotic activity of PC12 neuroendocrine cells by monitoring the modulation of the dopamine release in real-time.
In the present work, we report on the fabrication of a diamond-based device targeted to the detection of quantal neurotransmitter release. We have developed Multi-electrode Arrays with 16 independent graphitic channels fabricated by means of Deep Ion Beam Lithography (DIBL). These devices are capable of detecting the in vitro exocytotic event from neurosecretory cells, while overcoming several critical limitations of standard amperometric techniques.
Synaptic transmission is based on quantal release of neurotransmitters. Alterations of the molecular mechanisms and components governing exocytosis are at the basis of several neurological and neurodegenerative diseases. The aim of this chapter is to provide an overview on the most recent advances of boron-doped diamond (BDD) and graphitic multiarrays in monitoring quantal release of oxidizable neurotransmitters with submillisecond time resolution.
Laser writing with ultrashort pulses provides a potential route for the manufacture of three-dimensional wires, waveguides and defects within diamond. We present a transmission electron microscopy (TEM) study of the intrinsic structure of the laser modifications and reveal a complex distribution of defects. Electron energy loss spectroscopy (EELS) indicates that the majority of the irradiated region remains as $sp^3$ bonded diamond. Electrically-conductive paths are attributed to the formation of multiple nano-scale, $sp^2$-bonded graphitic wires and a network of strain-relieving micro-cracks.
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 μm2) 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, 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.
Graphitic wires embedded beneath the surface of single crystal diamond are promising for a variety of applications. Through a combination of ultra short (femtosecond) pulsed fabrication, high numerical aperture focusing and adaptive optics, graphitic wires can be written along any 3D path. Here, we demonstrate a non-reciprocal directional dependence to the graphitization process: the features are distinct when the fabrication direction is reversed. The non-reciprocal effects are significantly determined by the laser power, the fabrication speed, the light polarization and pulse front tilt. The influences of these factors are studied.
Focused MeV ion beams with micrometric resolution are suitable tools for the direct writing of conductive graphitic channels buried in an insulating diamond bulk, as already demonstrated for different device applications. In this work we apply this fabrication method to the electrical excitation of color centers in diamond, demonstrating the potential of electrical stimulation in diamond-based single-photon sources. Differently from optically-stimulated light emission from color centers in diamond, electroluminescence (EL) requires a high current flowing in the diamond subgap states between the electrodes. With this purpose, buried graphitic electrode pairs, 10 1/4m spaced, were fabricated in the bulk of a single-crystal diamond sample using a 6MeV C microbeam. The electrical characterization of the structure showed a significant current injection above an effective voltage threshold of 150 V, which enabled the stimulation of a stable EL emission. The EL imaging allowed to identify the electroluminescent regions and the residual vacancy distribution associated with the fabrication technique. Measurements evidenced isolated electroluminescent spots where non-classical light emission in the 560-700nm spectral range was observed. The spectral and auto-correlation features of the EL emission were investigated to qualify the non-classical properties of the color centers.
Graphitic wires embedded beneath the surface of single crystal diamond are demonstrated. Through a combination of ultrashort (femtosecond) pulsed fabrication, high numerical aperture focusing and adaptive optics, wires are created with sub micrometre dimensions that can follow any three dimensional path within the diamond. The increased level of focal control available through the use of adaptive optics appears particularly important in the generation of high quality wires, with measured conductivities over an order of magnitude greater than previous laser-induced graphitic wires in diamond. Applications for the embedded wires are discussed.
Diamond multi electrode arrays (MEAs) and ultra-micro electrode array (uMEA) fabricated from boron-doped nanocrystalline (BNCD) thin-films, show excellent performances when detecting cell activity in both amperometric and potentiometric applications. Furthermore they are suitable for delivering electrical stimulation to elicit bioelectric events. Results obtained with our previous 9-channels uMEA for single cells at high spatial resolution, and 16-channels MEA for multicellular samples, validated the signal quality of diamond MEAs in comparison with competing technologies. The latest progresses are reported in this paper were we describe a new generation of devices with enhanced performances: a 12-channel uMEA for recordings from a single cell at high spatial resolution, and a 64-channel MEA to accommodate larger multicellular samples. The technology is based on a 200 μm thin high-temperature glass with the same thermal expansion coefficient of silicon and a spin-on seeding method. Preliminary experiments show excellent electrodes activity and a strongly improved transparency.
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.
In the present work we report about a parallel-processing ion beam
fabrication technique whereby high-density sub-superficial graphitic
microstructures can be created in diamond. Ion beam implantation is an
effective tool for the structural modification of diamond: in particular
ion-damaged diamond can be converted into graphite, therefore obtaining an
electrically conductive phase embedded in an optically transparent and highly
insulating matrix. The proposed fabrication process consists in the combination
of Deep Ion Beam Lithography (DIBL) and Focused Ion Beam (FIB) milling. FIB
micromachining is employed to define micro-apertures in the contact masks
consisting of thin (<10 um) deposited metal layers through which ions are
implanted in the sample. A prototypical single-cell biosensor was realized with
the above described technique. The biosensor has 16 independent electrodes
converging inside a circular area of 20 um diameter (typical neuroendocrine
cells size) for the simultaneous recording of amperometric signals.
Focused MeV ion microbeams are suitable tools for the direct writing of
conductive graphitic channels buried in an insulating diamond bulk, as
demonstrated in previous works with the fabrication of multi-electrode ionizing
radiation detectors and cellular biosensors. In this work we investigate the
suitability of the fabrication method for the electrical excitation of colour
centres in diamond. Differently from photoluminescence, electroluminescence
requires an electrical current flowing through the diamond sub-gap states for
the excitation of the colour centres. With this purpose, buried graphitic
electrodes with a spacing of 10 micrometers were fabricated in the bulk of a
detector-grade CVD single-crystal diamond sample using a scanning 1.8 MeV He
micro-beam. The current flowing in the gap region between the electrodes upon
the application of a 250 V bias voltage was exploited as the excitation pump
for the electroluminescence of different types of colour centres localized in
the above-mentioned gap. The bright light emission was spatially mapped using a
confocal optical microscopy setup. The spectral analysis of electroluminescence
revealed the emission from neutrally-charged nitrogen-vacancy centres ($NV^0$,
$\lambda_{ZPL}$ = 575 nm), as well as from cluster crystal dislocations
(A-band, {\lambda} = 400-500 nm). Moreover, an electroluminescence signal with
appealing spectral features (sharp emission at room temperature, low phonon
sidebands) from He-related defects was detected ($\lambda_{ZPL}$ = 536.3 nm,
$\lambda_{ZPL}$ = 560.5 nm); a low and broad peak around {\lambda} = 740 nm was
also observed and tentatively ascribed to Si-V or GR1 centres. These results
pose interesting future perspectives for the fabrication of
electrically-stimulated single-photon emitters in diamond for applications in
quantum optics and quantum cryptography.
Microelectrode arrays offer the potential to electrochemically monitor concentrations of molecules at high spatial resolution. However, current systems are limited in the number of sensor sites, signal resolution, and throughput. Here, we present a fully integrated complementary metal oxide semiconductor (CMOS) system with an array of 32 × 32 working electrodes to perform electrochemical measurements like amperometry and voltammetry. The array consists of platinum electrodes with a center-to-center distance of 100 μm and electrode diameters of 5 to 50 μm. Currents in the range from 10 μA down to pA can be measured. The current is digitized by sigma-delta converters at a maximum resolution of 13.3 bits. The integrated noise is 220 fA for a bandwidth of 100 Hz, allowing for detection of pA currents. Currents can be continuously acquired at up to 1 kHz bandwidth, or the whole array can be read out rapidly at a frame rate of up to 90 Hz. The results of the electrical characterization meet the requirements of a wide range of electrochemical methods including cyclic voltammograms and amperometric images of high spatial and temporal resolution.
We present experimental results and numerical finite element analysis to describe surface swelling due to the creation of buried graphite-like inclusions in diamond substrates subjected to MeV ion implantation. Numerical predictions are compared to experimental data for MeV proton and helium implantations, performed with scanning ion microbeams. Swelling values are measured with white-light interferometric profilometry in both cases. Simulations are based on a model which accounts for the through-the-thickness variation of mechanical parameters in the material, as a function of ion type, fluence and energy. Surface deformation profiles and internal stress distributions are analyzed and numerical results are seen to adequately fit experimental data. Results allow us to draw conclusions on structural damage mechanisms in diamond for different MeV ion implantations. (c) 2010 Elsevier B.V. All rights reserved.
We report on the systematic characterization of conductive micro-channels
fabricated in single-crystal diamond with direct ion microbeam writing. Focused
high-energy (~MeV) helium ions are employed to selectively convert diamond with
micrometric spatial accuracy to a stable graphitic phase upon thermal
annealing, due to the induced structural damage occurring at the end-of-range.
A variable-thickness mask allows the accurate modulation of the depth at which
the microchannels are formed, from several {\mu}m deep up to the very surface
of the sample. By means of cross-sectional transmission electron microscopy
(TEM) we demonstrate that the technique allows the direct writing of amorphous
(and graphitic, upon suitable thermal annealing) microstructures extending
within the insulating diamond matrix in the three spatial directions, and in
particular that buried channels embedded in a highly insulating matrix emerge
and electrically connect to the sample surface at specific locations. Moreover,
by means of electrical characterization both at room temperature and variable
temperature, we investigate the conductivity and the charge-transport
mechanisms of microchannels obtained by implantation at different ion fluences
and after subsequent thermal processes, demonstrating that upon
high-temperature annealing, the channels implanted above a critical damage
density convert to a stable graphitic phase. These structures have significant
impact for different applications, such as compact ionizing radiation
detectors, dosimeters, bio-sensors and more generally diamond-based devices
with buried three-dimensional all-carbon electrodes.
In this paper we review and provide an overview to the understanding of the chemical vapor deposition (CVD) of diamond materials with a particular focus on the commonly used microwave plasma-activated chemical vapor deposition (MPCVD). The major topics covered are experimental measurements in situ to diamond CVD reactors, and MPCVD in particular, coupled with models of the gas phase chemical and plasma kinetics to provide insight into the distribution of critical chemical species throughout the reactor, followed by a discussion of the surface chemical process involved in diamond growth.
We studied wild-type (WT) and Cav1.3(-/-) mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca(2+) channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca(2+) buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivating BK currents sustained mainly the afterhyperpolarization of the short APs (2-3 ms) and only partially the pacemaker current during the long interspike (300-500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca(2+) current and the corresponding Ca(2+)-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.
A microchip was applied to electrically depolarize rat pheochromocytoma (PC12) cells and to simultaneously detect exocytotic catecholamine release amperometrically. Results demonstrate exocytosis elicited by flowing cells through an electric field generated by a potentiostat circuit in a microchannel, as well as exocytosis triggered by application of an extracellular voltage pulse across. Electrical finite element model (FEM) analysis illustrated that larger cells experienced greater depolarizing excitation from the extracellular electric fields due to the smaller shunt path and higher resistance to current flow in the channel around the cell. Consistent with these simulations, data recorded from cell clusters and large cells exhibited increased release rates relative to data from the smaller cells. Overall, the system was capable of resolving single vesicle quantal release, in the zeptomole range, as well as the kinetics associated with the vesicle fusion process. Analysis of spike population statistics suggested detection of catecholamines from multiple release sites around the cells. The potential for such a device to be used in flow cytometry to evoke and detect exocytosis was demonstrated.
Neurons and endocrine cells secrete neurotransmitter and hormones in discrete packets in a process called quantal exocytosis. Electrochemical microelectrodes can detect spikes in current resulting from the oxidation of individual quanta of transmitter only if the electrodes are small and directly adjacent to release sites on the cell. Here we report development of a microchip device that uses microfluidic traps to automatically target individual or small groups of cells to small electrochemical electrodes. Microfluidic channels and traps were fabricated by multi-step wet etch of a silicon wafer whereas Pt electrodes were patterned in register with the trap sites. We demonstrate high-resolution amperometric measurement of quantal exocytosis of catecholamines from chromaffin cells on the device. This reusable device is a step towards developing high-throughput lab-on-a-chip instruments for recording quantal exocytosis to increase the pace of basic neuroscience research and to enable screening of drugs that target exocytosis.
Photorelease of caged Ca(2+) is a uniquely powerful tool to study the dynamics of Ca(2+)-triggered exocytosis from individual cells. Using photolithography and other microfabrication techniques, we have developed transparent microchip devices to enable photorelease of caged Ca(2+), together with electrochemical detection of quantal catecholamine secretion from individual cells or cell arrays as a step towards developing high-throughput experimental devices. A 100 nm thick transparent indium-tin-oxide (ITO) film was sputter-deposited onto glass coverslips, which were then patterned into 24 cell-sized working electrodes (approximately 20 microm by 20 microm). We loaded bovine chromaffin cells with acetoxymethyl (AM) ester derivatives of the Ca(2+) cage NP-EGTA and Ca(2+) indicator dye fura-4F, then transferred these cells onto the working ITO electrodes for amperometric recordings. Upon flash photorelease of caged Ca(2+), a uniform rise of [Ca(2+)](i) within the target cell leads to quantal release of oxidizable catecholamines measured amperometrically by the underlying ITO electrode. We observed a burst of amperometric spikes upon rapid elevation of [Ca(2+)](i) and a "priming" effect of sub-stimulatory [Ca(2+)](i) on the response of cells to subsequent [Ca(2+)](i) elevation, similar to previous reports using different techniques. We conclude that UV photolysis of caged Ca(2+) is a suitable stimulation technique for higher-throughput studies of Ca(2+)-dependent exocytosis on transparent electrochemical microelectrode arrays.
As demonstrated in previous works, implantation with a MeV ion microbeam through masks with graded thickness allows the formation of conductive micro-channels in diamond which are embedded in the insulating matrix at controllable depths [P. Olivero et al., Diamond Relat. Mater. 18 (5–8), 870–876 (2009)]. In the present work we report about the systematic electrical characterization of such micro-channels as a function of several implantation conditions, namely: ion species and energy, implantation fluence. The current–voltage (IV) characteristics of the buried channels were measured at room temperature with a two point probe station. Significant parameters such as the sheet resistance and the characteristic exponent (α) of the IV power-law trend were expressed as a function of damage density, with satisfactory compatibility between the results obtained in different implantation conditions.
The LNL proton microprobe, operational since spring 1993 in the Laboratori Nazionali di Legnaro, has been used extensively in a wide ranging experimental programme. This paper overviews the setup, outlines the original solutions adopted and specifies the performance. Finally, the changes being carried out in view of single event experiments are briefly reviewed.
This paper presents a review of state-of-the-art reports of homoepitaxial diamond growth. Crystal growth and characterization methods that are commonly used for homoepitaxial diamond studies are first described briefly. Then, effects of each process parameter on the growth mode of homoepitaxial diamond are discussed. Methane concentration and substrate temperature were two dominant parameters that determine the growth rate and crystalline quality of diamond films, while conditions of diamond substrates including crystal orientation and pretreatments should be optimized for high-quality diamond deposition. Attempts to increase growth rate of homoepitaxial diamond films while keeping their crystalline quality are discussed in the later section. Increase of microwave power density for plasma-assisted chemical vapor deposition was found to be effective for the high-rate growth of diamond. Free-exciton recombination emission was intensively observed from homoepitaxial films grown at higher growth rate of 3.5 μm/h under the high power density condition. In addition, boron-doped samples grown under the high-power conditions showed high Hall mobility of 1620 cm2 V s−1 at room temperature. The plasma power density is thus one of the important parameters of diamond deposition from the viewpoint of electronic device application. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
SRIM is a software package concerning the Stopping and Range of Ions in Matter. Since its introduction in 1985, major upgrades are made about every six years. Currently, more than 700 scientific citations are made to SRIM every year. For SRIM-2010, the following major improvements have been made: (1) About 2800 new experimental stopping powers were added to the database, increasing it to over 28,000 stopping values. (2) Improved corrections were made for the stopping of ions in compounds. (3) New heavy ion stopping calculations have led to significant improvements on SRIM stopping accuracy. (4) A self-contained SRIM module has been included to allow SRIM stopping and range values to be controlled and read by other software applications. (5) Individual interatomic potentials have been included for all ion/atom collisions, and these potentials are now included in the SRIM package. A full catalog of stopping power plots can be downloaded at www.SRIM.org. Over 500 plots show the accuracy of the stopping and ranges produced by SRIM along with 27,000 experimental data points. References to the citations which reported the experimental data are included.
We report on a novel method for the fabrication of three-dimensional buried graphitic micropaths in single crystal diamond with the employment of focused MeV ions. The use of implantation masks with graded thickness at the sub-micrometer scale allows the formation of conductive channels which are embedded in the insulating matrix at controllable depths. In particular, the modulation of the channels depth at their endpoints allows the surface contacting of the channel terminations with no need of further fabrication stages.In the present work we describe the sample masking, which includes the deposition of semi-spherical gold contacts on the sample surface, followed by MeV ion implantation. Because of the significant difference between the densities of pristine and amorphous or graphitized diamond, the formation of buried channels has a relevant mechanical effect on the diamond structure, causing localized surface swelling, which has been measured both with interferometric profilometry and atomic force microscopy. The electrical properties of the buried channels are then measured with a two point probe station: clear evidence is given that only the terminal points of the channels are electrically connected with the surface, while the rest of the channels extends below the surface. IV measurements are employed also to qualitatively investigate the electrical properties of the channels as a function of implantation fluence and annealing.
Voltage gated Ca(2+) channels are effective voltage sensors of plasma membrane which convert cell depolarizations into Ca(2+) signaling. The chromaffin cells of the adrenal medulla utilize a large number of Ca(2+) channel types to drive the Ca(2+)-dependent release of catecholamines into blood circulation, during normal or stress-induced conditions. Some of the Ca(2+) channels expressed in chromaffin cells (L, N, P/Q, R and T), however, do not control only vesicle fusion and catecholamine release. They also subserve a variety of key activities which are vital for the physiological and pathological functioning of the cell, like: (i) shaping the action potentials of electrical oscillations driven either spontaneously or by ACh stimulation, (ii) controlling the action potential frequency of tonic or bursts firing, (iii) regulating the compensatory and excess endocytosis following robust exocytosis and (iv) driving the remodeling of Ca(2+) signaling which occurs during stressors stimulation. Here, we will briefly review the well-established properties of voltage-gated Ca(2+) channels accumulated over the past three decades focusing on the most recent discoveries on the role that L- (Cav1.2, Cav1.3) and T-type (Cav3.2) channels play in the control of excitability, exocytosis and endocytosis of chromaffin cells in normal and stress-mimicking conditions.
The study of single cell dynamics has been greatly adapted in biological and medical research and applications. In this work a novel microfluidic electrochemical sensor with carbon nanotubes (CNTs) modified indium tin oxide (ITO) microelectrode was developed for single cells release monitoring. The sensitivity of the electrochemical sensor after CNTs surface modification was improved by 2.5-3 orders of magnitude. The developed CNTs modified ITO sensor was successfully employed to monitor the dopamine release from single living rat pheochromocytoma (PC 12) cells. Its ultrahigh sensitivity, transparency and need for fewer agents enable this smart electrochemical sensor to become a powerful tool in recording dynamic release from various living tissues and organs optically and electrically.
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.
In the present work we report about the investigation of the conduction mechanism of sp2 carbon micro-channels in single crystal diamond. The structures are fabricated with a technique which employs a MeV focused
ion-beam to damage diamond in conjunction with variable thickness masks. This process changes significantly the structural
properties of the target material, because the ion nuclear energy loss induces carbon conversion from sp3 to sp2 state mainly at the end of range of the ions (few micrometers). Furthermore, placing a mask with increasing thickness on
the sample it is possible to modulate the channels depth at their endpoints, allowing their electrical connection with the
surface. A single-crystal HPHT diamond sample was implanted with 1.8 MeV He+ ions at room temperature, the implantation fluence was set in the range 2.1×1016-6.3×1017 ions cm-2, determining the formation of micro-channels with a graded level of damage extending down to a depth of about 3 μm. After
deposition of metallic contacts at the channels’ endpoints, the electrical characterization was performed measuring the I-V
curves at variable temperatures in the 80-690 K range. The Variable Range Hopping model was used to fit the experimental data
in the ohmic regime, allowing the estimation of characteristic parameters such as the density of localized states at the Fermi
level. A value of 5.5×1017 states cm-3 eV-1 was obtained, in satisfactory agreement with values previously reported in literature. The power-law dependence between current
and voltage is consistent with the space charge limited mechanism at
moderate electric fields.
In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.
Biophysical events involved in late stages of exocytosis occur at highly localized areas of cells on millisecond and submillisecond time scales. Thus, methodologies with high spatio-temporal resolution are required to achieve measurements at individual secretory cells. Much has been learned about the mechanisms and kinetics of vesicular release through analysis with the carbon fiber microelectrode techniques amperometry and cyclic voltammetry. Coupling of these techniques with other methods such as patch-clamp continues to reveal details of the secretion process. It is now clear that extrusion of the vesicular contents is a more complex process than previously believed. Vesicle-cell fusion, revealed by cell capacitance measurements, is temporally dissociated from secretion measured amperometrically. The stability imparted by interaction and association of vesicle contents at rest results in a rate-limiting extrusion process after full fusion. Furthermore, the presence of partial fusion events and the occurrence of nonquantized release have been revealed with electrochemical tools.
We have modeled single-atom radiation damage events in diamond by a molecular-dynamics simulation, using an empirical interatomic potential to describe the interaction between the atoms in diamond. We find that the damage threshold energy needed to displace a single atom is well above simple estimates based on the diamond cohesive energy. The threshold derived from our simulations is approximately 50 eV, and is relatively insensitive to the direction of initial motion of the displaced atom. The high threshold energy is due to a rapid dissipation of kinetic energy from the bombarded atom into incoherent vibrational energy of its neighboring atoms before the displaced atom can overcome the structural energy barrier to defect formation. This rapid dissipation can be understood qualitatively by noting that when the kinetic energy of a carbon atom is comparable to the damage threshold energy, its velocity is comparable to the speed of sound in diamond.
Over the past 20 years, the technological impediments to fabricating electrodes of micrometer dimensions have been largely
overcome. These small electrodes can be readily applied to probe chemical events at the surface of tissues or individual biological
cells; they can even be used to monitor concentration changes within intact animals. These measurements can be made on rapid
time scales and with minimal perturbation of the system under study. Several recent applications have provided important insights
into chemical processes at cells and in tissues. Examples include molecular flux measurements at the surface of single cells
and through skin—which can offer insights into oxidative stress, exocytosis, and drug delivery—and real-time brain neurotransmitter
monitoring in living rats, which reveals correlations between behavior and molecular events in the brain. Such findings can
promote interdisciplinary collaborations and may lead to a broader understanding of the chemical aspects of biology.
alpha(1H) T-type channels recruited by beta(1)-adrenergic stimulation in rat chromaffin cells (RCCs) are coupled to fast exocytosis with the same Ca(2+) dependence of high-threshold Ca(2+) channels. Here we show that RCCs exposed to chronic hypoxia (CH) for 12-18 h in 3% O(2) express comparable densities of functional T-type channels that depolarize the resting cells and contribute to low-voltage exocytosis. Following chronic hypoxia, most RCCs exhibited T-type Ca(2+) channels already available at -50 mV with the same gating, pharmacological and molecular features as the alpha(1H) isoform. Chronic hypoxia had no effects on cell size and high-threshold Ca(2+) current density and was mimicked by overnight incubation with the iron-chelating agent desferrioxamine (DFX), suggesting the involvement of hypoxia-inducible factors (HIFs). T-type channel recruitment occurred independently of PKA activation and the presence of extracellular Ca(2+). Hypoxia-recruited T-type channels were partially open at rest (T-type 'window-current') and contributed to raising the resting potential to more positive values. Their block by 50 microm Ni(2+) caused a 5-8 mV hyperpolarization. The secretory response associated with T-type channels could be detected following mild cell depolarizations, either by capacitance increases induced by step depolarizations or by amperometric current spikes induced by increased [KCl]. In the latter case, exocytotic bursts could be evoked even with 2-4 mm KCl and spike frequency was drastically reduced by 50 microm Ni(2+). Chronic hypoxia did not alter the shape of spikes, suggesting that hypoxia-recruited T-type channels increase the number of secreted vesicles at low voltages, without altering the mechanism of catecholamine release and the quantal content of released molecules.
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