RESUMEN
Single cell amperometry has proven to be a powerful and well-established method for characterizing single vesicular exocytotic events elicited at the level of excitable cells under various experimental conditions. Nevertheless, most of the reported characteristics are descriptive, being mostly concerned with the morphological characteristics of the recorded current spikes (maximum current intensities, released charge, rise and fall times, etc.) which are certainly important but do not provide sufficient kinetic information on exocytotic mechanisms due to lack of quantitative models. Here, continuing our previous efforts to provide rigorous models rationalizing the kinetic structures of frequently encountered spike types (spikes with unique exponential decay tails and kiss-and-run events), we describe a new theoretical approach enabling a quantitative kinetic modeling of all types of exocytotic events giving rise to current spikes exhibiting exponential decay tails. This model follows directly from the fact that the condensation of long intravesicular polyelectrolytic strands by high concentrations of monocationic neurotransmitter molecules leads to a matrix structure involving two compartments in constant kinetic exchanges during release. This kinetic model has been validated theoretically (direct and inverse problems) and its experimental interest established by the analysis of the amperometric spikes relative to chromaffin and PC12 cells previously published by some of us.
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Exposure to widely used inert fibrous nanomaterials (for example, glass fibres or carbon nanotubes) may result in asbestos-like lung pathologies, becoming an important environmental and health concern. However, the origin of the pathogenesis of such fibres has not yet been clearly established. Here we report an electrochemical nanosensor that is used to monitor and quantitatively characterize the flux and dynamics of reactive species release during the frustrated phagocytosis of glass nanofibres by single macrophages. We show the existence of an intense prolonged release of reactive oxygen and nitrogen species by single macrophages near their phagocytic cups. This continued massive leakage of reactive oxygen and nitrogen species damages peripheral cells and eventually translates into chronic inflammation and lung injury, as seen during in vitro co-culture and in vivo experiments.
Asunto(s)
Nanofibras , Nanotubos de Carbono , Oxígeno , Nanotubos de Carbono/química , Fagocitosis , Macrófagos , Especies Reactivas de OxígenoRESUMEN
Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by ß-amyloid (Aß) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aß42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aß42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aß amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aß42-induced neuronal hyperactivity and neuronal degeneration.
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Enfermedad de Alzheimer , Ácido Glutámico , Ratones , Animales , Ácido Glutámico/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Encéfalo/metabolismo , Fragmentos de Péptidos/metabolismoRESUMEN
Reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NADH) are important intracellular redox-active molecules involved in various pathological processes including inflammation, neurodegenerative diseases, and cancer. However, the fast dynamic changes and mutual regulatory kinetic relationship between intracellular ROS and NADH in these biological processes are still hard to simultaneously investigate. A dual-channel nanowire electrode (DC-NWE) integrating two conductive nanowires, one functionalized with platinum nanoparticles and the other with conductive polymer, was nanofabricated for the selective and simultaneous real-time monitoring of intracellular ROS and NADH release by mitochondria in single living MCF-7 tumoral cells stimulated by resveratrol. The production of ROS was observed to occur tenths of a second before the release of NADH, a significant new piece of information suggesting a mechanism of action of resveratrol. Beyond the importance of the specific data gathered in this study, this work established the feasibility of simultaneously monitoring multiple species and analyzing their kinetics relationships over sub-second time scales thanks to dual-channel nanowire electrodes. It is believed that this concept and its associated nanoelectrochemical tools might benefit to a deeper understanding of mutual regulatory relationship between intracellular crucial molecular markers during physiological and pathological processes as well as for evaluating medical treatments.
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Técnicas Biosensibles , Nanopartículas del Metal , NAD/química , Especies Reactivas de Oxígeno , Cinética , Resveratrol , Platino (Metal) , Oxidación-ReducciónRESUMEN
Reactive oxygen and nitrogen species (ROS/RNS) are generated by macrophages inside their phagolysosomes. This production is essential for phagocytosis of damaged cells and pathogens, i.e., protecting the organism and maintaining immune homeostasis. The ability to quantitatively and individually monitor the four primary ROS/RNS (ONOO-, H2O2, NO, and NO2-) with submillisecond resolution is clearly warranted to elucidate the still unclear mechanisms of their rapid generation and to track their concentration variations over time inside phagolysosomes, in particular, to document the origin of ROS/RNS homeostasis during phagocytosis. A novel nanowire electrode has been specifically developed for this purpose. It consisted of wrapping a SiC nanowire with a mat of 3 nm platinum nanoparticles whose high electrocatalytic performances allow the characterization and individual measurements of each of the four primary ROS/RNS. This allowed, for the first time, a quantitative, selective, and statistically robust determination of the individual amounts of ROS/RNS present in single dormant phagolysosomes. Additionally, the submillisecond resolution of the nanosensor allowed confirmation and measurement of the rapid ability of phagolysosomes to differentially mobilize their enzyme pools of NADPH oxidases and inducible nitric oxide synthases to finely regulate their homeostasis. This reveals an essential key to immune responses and immunotherapies and rationalizes its biomolecular origin.
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Nanopartículas del Metal , Oxígeno , Homeostasis , Peróxido de Hidrógeno , Nitrógeno , Fagosomas , Platino (Metal) , Especies de Nitrógeno Reactivo/química , Especies Reactivas de Oxígeno/químicaRESUMEN
If hydrogen can be stored and carried safely at a high density, hydrogen-fuel cells offer effective solutions for vehicles. The stable chemisorption of atomic hydrogen on single layer graphene (SLG) seems a perfect solution in this regard, with a theoretical maximum storage capacity of 7.7 wt %. However, generating hydrogenated graphene from H2 requires extreme temperatures and pressures. Alternatively, hydrogen adatoms can easily be produced under mild conditions by the electroreduction of protons in solid/liquid systems. Graphene is electrochemically inert for this reaction, but H-chemisorption on SLG can be carried out under mild conditions via a novel Pt-electrocatalyzed "spillover-surface diffusion-chemisorption" mechanism, as we demonstrate using dynamic electrochemistry and isotopic Raman spectroscopy. The apparent surface diffusion coefficient (â¼10-5 cm2 s-1), capacity (â¼6.6 wt %, â¼85.7% surface coverage), and stability of hydrogen adatoms on SLG at room temperature and atmospheric pressure are significant, and they are perfectly suited for applications involving stored hydrogen atoms on graphene.
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Quantitative measurements of intravesicular glutamate (Glu) and of transient exocytotic release contents directly from individual living neurons are highly desired for understanding the mechanisms (full or sub-quantal release?) of synaptic transmission and plasticity. However, this could not be achieved so far due to the lack of adequate experimental strategies relying on selective and sensitive Glu nanosensors. Herein, we introduce a novel electrochemical Glu nanobiosensor based on a single SiC nanowire that can selectively measure in real-time Glu fluxes released via exocytosis by large Glu vesicles (ca. 125â nm diameter) present in single hippocampal axonal varicosities as well as their intravesicular content before exocytosis. These measurements revealed a sub-quantal release mode in living hippocampal neurons, viz., only ca. one third to one half of intravesicular Glu molecules are released by individual vesicles during exocytotic events. Importantly, this fraction remained practically the same when hippocampal neurons were pretreated with L-Glu-precursor L-glutamine, while it significantly increased after zinc treatment, although in both cases the intravesicular contents were drastically affected.
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Ácido Glutámico/metabolismo , Nanotecnología , Neuronas/citología , Animales , Supervivencia Celular , Células Cultivadas , Electroquímica , Nanocables/química , Vesículas Sinápticas/metabolismoRESUMEN
Since the early work of Bernard Katz, the process of cellular chemical communication through exocytosis, quantal release, has been considered to be all or none. Recent evidence has shown exocytosis to be partial or "subquantal" at single-cell model systems, but there is a need to understand this at communicating nerve cells. Partial release allows nerve cells to control the signal at the site of release during individual events, for which the smaller the fraction released, the greater the range of regulation. Herein, we show that the fraction of the vesicular octopamine content released from a living Drosophila larval neuromuscular neuron is very small. The percentage of released molecules was found to be only 4.5 % for simple events and 10.7 % for complex (i.e., oscillating or flickering) events. This large content, combined with partial release controlled by fluctuations of the fusion pore, offers presynaptic plasticity that can be widely regulated.
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Exocitosis , Espacio Intracelular/química , Nanotecnología , Neuronas/citología , Animales , Supervivencia Celular , Electroquímica , Neuronas/metabolismo , Análisis de la Célula IndividualRESUMEN
Zinc, a suspected potentiator of learning and memory, is shown to affect exocytotic release and storage in neurotransmitter-containing vesicles. Structural and size analysis of the vesicular dense core and halo using transmission electron microscopy was combined with single-cell amperometry to study the vesicle size changes induced after zinc treatment and to compare these changes to theoretical predictions based on the concept of partial release as opposed to full quantal release. This powerful combined analytical approach establishes the existence of an unsuspected strong link between vesicle structure and exocytotic dynamics, which can be used to explain the mechanism of regulation of synaptic plasticity by Zn2+ through modulation of neurotransmitter release.
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Neurotransmisores/genética , Células PC12/metabolismo , Transmisión Sináptica/genética , Zinc/química , Animales , Transporte Biológico , RatasRESUMEN
The existence of a homeostatic mechanism regulating reactive oxygen/nitrogen species (ROS/RNS) amounts inside phagolysosomes has been invoked to account for the efficiency of this process but could not be unambiguously documented. Now, intracellular electrochemical analysis with platinized nanowire electrodes (Pt-NWEs) allowed monitoring ROS/RNS effluxes with sub-millisecond resolution from individual phagolysosomes impacting onto the electrode inserted inside a living macrophage. This shows for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results establish the existence of the long-postulated ROS/RNS homeostasis and allows its kinetics and efficiency to be quantified. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.
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Técnicas Electroquímicas/métodos , Electrodos , Homeostasis , Macrófagos/metabolismo , Fagosomas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Macrófagos/citología , Ratones , Oxidación-ReducciónRESUMEN
The release of reactive oxygen and nitrogen species (ROS/RNS) by macrophages undergoing phagocytosis is crucial for the efficiency of the immune system. In this work, platinized carbon nanoelectrodes were used to detect, characterize, and quantify for the first time the intracellular production rates of the four primary ROS/RNS (i.e., H2O2, ONOO-, NOâ¢, and NO2-) inside single phagolysosomes of living RAW 264.7 murine macrophages stimulated by interferon-γ and lipopolysaccharide (IFN-γ/LPS) to mimic an in vivo inflammatory activation. The time-dependent concentrations of the four primary ROS/RNS in individual phagolysosomes monitored using a four-step chronoamperometric method evidenced a high variability of their production rates. This intrinsic variability unravels the complexity of phagocytosis.
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Macrófagos/citología , Fagosomas/metabolismo , Especies de Nitrógeno Reactivo/química , Especies Reactivas de Oxígeno/química , Animales , Supervivencia Celular , Electroquímica , Ratones , Células RAW 264.7 , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In the last decade, following fluorescent dyes and protein tags, pH sensitive false fluorescent neurotransmitters (FFN) were introduced and were valuable for labeling secretory vesicles and monitoring exocytosis at living cells. In particular, the synthetic analog of neurotransmitters FFN102 was shown to be an electroactive probe. Here, we show that FFN102 is suitable to be used as a bioanalytic probe at the widely used PC12 cell model. FFN102 was uptaken in the secretory vesicles of PC12 cells, partially replacing the endogenous dopamine stored in these vesicles. The different oxidation potentials of dopamine and FFN102 allowed to determine that ca. 12% of dopamine was replaced by FFN102. Moreover, the FFN102 was found to be over released through the initial fusion pore suggesting that it was mostly uptaken in fast diffusion compartment of the vesicles.
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Dopamina/metabolismo , Colorantes Fluorescentes/metabolismo , Neurotransmisores/metabolismo , Vesículas Secretoras/metabolismo , Animales , Compartimento Celular , Técnicas Electroquímicas/métodos , Electrodos , Exocitosis , Células PC12 , RatasRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DAergic) neurons and low level of dopamine (DA) in the midbrain. Recent studies suggested that some natural products can protect neurons against injury, but their role on neurotransmitter release and the underlying mechanisms remained unknown. In this work, nanoelectrode electrochemistry was used for the first time to quantify DA release inside single DAergic synapses. Our results unambiguously demonstrated that harpagide, a natural product, effectively enhances synaptic DA release and restores DA release at normal levels from injured neurons in PD model. These important protective and curative effects are shown to result from the fact that harpagide efficiently inhibits the phosphorylation and aggregation of α-synuclein by alleviating the intracellular reactive oxygen level, being beneficial for vesicle loading and recycling. This establishes that harpagide offers promising avenues for preventive or therapeutic interventions against PD and other neurodegenerative disorders.
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Rotating ring-disk electrode (RRDE) is a generator-collector electrochemical system widely used as an electroanalytical and kinetic device. However, RRDEs are costly and difficult to fabricate, particularly when the electrode material is fragile, small, and scarce. Taking advantage of readily available 3D printing technology an alternative generator-collector system was developed: rotating acentric binary-disk electrode (RABDE). RABDE consists of two close acentric disk electrodes arranged in a cylindrical matrix so that the line connecting their centers is perpendicular to radius of the device passing through the center of generator electrode. In contrast to RRDE that is based on radial flow velocity for mass transfer between the generator and the collector, RABDE mostly takes advantage of the larger tangential flow velocity. RABDE thus exhibits higher current densities than RRDE for a same rotation rate and evidences much better electroanalytical performances. These increased performances were tested and quantified using typical analytes: potassium ferricyanide system, copper ion system, and oxygen reduction reaction in alkaline solution.
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Applications of conducting carbon materials for highly efficient electrochemical energy devices require a greater fundamental understanding of heterogeneous electron-transfer (ET) mechanisms. This task, however, is highly challenging experimentally, because an adsorbing carbon surface may easily conceal its intrinsic reactivity through adventitious contamination. Herein, we employ nanoscale scanning electrochemical microscopy (SECM) and cyclic voltammetry to gain new insights into the interplay between heterogeneous ET and adsorption of a Co(III)/Co(II)-complex redox couple at the contamination-free surface of electron-beam-deposited carbon (eC). Specifically, we investigate the redox couple of tris(1,10-phenanthroline)cobalt(II), Co(phen)32+, as a promising mediator for dye-sensitized solar cells and redox flow batteries. A pristine eC surface overlaid with KCl is prepared in vacuum, protected from contamination in air, and exposed to an ultrapure aqueous solution of Co(phen)32+ by the dissolution of the protective KCl layer. We employ SECM-based nanogap voltammetry to quantitatively demonstrate that Co(phen)32+ is adsorbed on the pristine eC surface to electrostatically self-inhibit outer-sphere ET of nonadsorbed Co(phen)33+ and Co(phen)32+. Strong electrostatic repulsion among Co(phen)32+ adsorbates is also demonstrated by SECM-based nanogap voltammetry and cyclic voltammetry. Quantitatively, self-inhibitory ET is characterized by a linear decrease in the standard rate constant of Co(phen)32+ oxidation with a higher surface concentration of Co(phen)32+ at the formal potential. This unique relationship is consistent not with the Frumkin model of double layer effects, but with the Amatore model of partially blocked electrodes as extended for self-inhibitory ET. Significantly, the complicated coupling of electron transfer and surface adsorption is resolved by combining nanoscale and macroscale voltammetric methods.
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An innovative microfluidic platform was designed to monitor electrochemically four primary reactive oxygen (ROS) and reactive nitrogen species (RNS) released by aerobic cells. Taking advantage of the space confinement and electrode performances under flow conditions, only a few experiments were sufficient to directly provide significant statistical data relative to the average behavior of cells during oxidative-stress bursts. The microfluidic platform comprised an upstream microchamber for cell culture and four parallel microchannels located downstream for separately detecting H2O2, ONOO-, NO·, and NO2-. Amperometric measurements were performed at highly sensitive Pt-black electrodes implemented in the microchannels. RAW 264.7 macrophage secretions triggered by a calcium ionophore were used as a way to assess the performance, sensitivity, and specificity of the integrated microfluidic device. In comparison with some previous evaluations achieved from single-cell measurements, reproducible and relevant determinations validated the proof of concept of this microfluidic platform for analyzing statistically significant oxidative-stress responses of various cell types.
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The production of reactive oxygen and nitrogen species (ROS and RNS) in human cells is implicated in various diseases, including cancer. Micrometer-sized electrodes coated with Pt black and platinized Pt nanoelectrodes have previously been used for the detection of primary ROS and RNS in biological systems. In this Article, we report the development of platinized carbon nanoelectrodes with well-characterized geometry and use them as scanning electrochemical microscopy (SECM) tips to measure ROS and RNS inside noncancerous and metastatic human breast cells. By performing time-dependent quantitative amperometric measurements at different potentials, the relative concentrations of four key ROS/RNS in the cell cytoplasm and their dynamics were determined and used to elucidate the chemical origins and production rates of ROS/RNS in nontransformed and metastatic human breast cells.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Técnicas Electroquímicas , Metástasis de la Neoplasia , Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Carbono/química , Electrodos , Humanos , Tamaño de la Partícula , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living-cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC-core-shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.
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Nanocables/química , Especies de Nitrógeno Reactivo/análisis , Especies Reactivas de Oxígeno/análisis , Animales , Compuestos Inorgánicos de Carbono/química , Técnicas Electroquímicas , Electrodos , Células HeLa , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Microscopía Fluorescente , Fagosomas/metabolismo , Células RAW 264.7 , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Silicona/químicaRESUMEN
Existing methods offer little direct and real-time information about stretch-triggered biochemical responses during cell mechanotransduction. A novel stretchable electrochemical sensor is reported that takes advantage of a hierarchical percolation network of carbon nanotubes and gold nanotubes (CNT-AuNT). This hybrid nanostructure provides the sensor with excellent time-reproducible mechanical and electrochemical performances while granting very good cellular compatibility, making it perfectly apt to induce and monitor simultaneously transient biochemical signals. This is validated by monitoring stretch-induced transient release of small signaling molecules by both endothelial and epithelial cells cultured on this sensor and submitted to stretching strains of different intensities. This work demonstrates that the hybrid CNT-AuNT platform offers a versatile and highly sensitive way to characterize and quantify short-time mechanotransduction responses.