RESUMO
In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomesâone of the main extracellular vesicles (EVs)âcarrying cell-dependent DNA, mRNA, proteins, etc., play an essential role in cellular communication. Due to experimental limitations, it has been difficult to monitor the real-time release of individual exosomes; this restricts a comprehensive understanding of the basic molecular mechanisms and the functions of exosomes. In this work, we introduce amperometry with microelectrodes to capture the dynamic release of single exosomes from a single living cell, distinguish them from other EVs, and differentiate the molecules inside exosomes and those secreted from LDCVs. We show that, similar to many LDCVs and synaptic vesicles, exosomes released by neuroendocrine cells also contain catecholamine transmitters. This finding reveals a different mode of chemical communication via exosome-encapsulated chemical messengers and a potential interconnection between the two release pathways, changing the canonical view of exocytosis of neuroendocrine cells and possibly neurons. This defines a new mechanism for chemical communication at the fundamental level and opens new avenues in the research of the molecular biology of exosomes in the neuroendocrine and central nervous systems.
Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Comunicação Celular , Membrana Celular/metabolismo , NeurôniosRESUMO
Saline-alkaline stress is one of the major damages that severely affects rice (Oryza sativa L.) growth and grain yield; however, the mechanism of the tolerance remains largely unknown in rice. Herein, we comparatively investigated the transcriptome and metabolome of two contrasting rice subspecies genotypes, Luohui 9 (abbreviation for Chao2R under study, O. sativa ssp. indica, saline-alkaline-sensitive) and RPY geng (O. sativa ssp. japonica, saline-alkaline-tolerant), to identify the main pathways and important factors related to saline-alkaline tolerance. Transcriptome analysis showed that 68 genes involved in fatty acid, amino acid (such as phenylalanine and tryptophan), phenylpropanoid biosynthesis, energy metabolism (such as Glycolysis and TCA cycle), as well as signal transduction (such as hormone and MAPK signaling) were identified to be specifically upregulated in RPY geng under saline-alkaline conditions, implying that a series of cascade changes from these genes promotes saline-alkaline stress tolerance. The transcriptome changes observed in RPY geng were in high accordance with the specifically accumulation of metabolites, consisting mainly of 14 phenolic acids, 8 alkaloids, and 19 lipids based on the combination analysis of transcriptome and metabolome. Moreover, some genes involved in signal transduction as hub genes, such as PR5, FLS2, BRI1, and NAC, may participate in the saline-alkaline stress response of RPY geng by modulating key genes involved in fatty acid, phenylpropanoid biosynthesis, amino acid metabolism, and glycolysis metabolic pathways based on the gene co-expression network analysis. The present research results not only provide important insights for understanding the mechanism underlying of rice saline-alkaline tolerance at the transcriptome and metabolome levels but also provide key candidate target genes for further enhancing rice saline-alkaline stress tolerance.
Assuntos
Oryza , Transcriptoma , Plântula/genética , Oryza/metabolismo , Perfilação da Expressão Gênica/métodos , Metabolômica , Regulação da Expressão Gênica de PlantasRESUMO
Understanding the regulatory mechanisms of exocytosis is essential for uncovering the pathologies of neuronal disorders and developing related pharmaceuticals. In this work intracellular vesicle impact electrochemical cytometry (IVIEC) measurements with different-sized (50-500â nm radius) open carbon nanopipettes (CNPs) were performed to quantify the vesicular content and release kinetics of specific vesicle populations grouped by orifice sizes. Intracellular vesicles with radius below 100â nm were captured and narrowed between 50 and 100â nm. On the basis of this, single vesicular catecholamine concentrations in the intracellular environment were quantified as 0.23-1.1â M. Our results with L-3,4-dihydroxyphenylalanine (L-DOPA)-exposure indicate that L-DOPA regulates exocytosis by increasing the dense core size and vesicular content while catecholamine concentrations did not show obvious alterations. These were all achieved simultaneously and relatively noninvasively with open CNPs.
Assuntos
Carbono/química , Catecolaminas/análise , Levodopa/química , Nanopartículas/químicaRESUMO
We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ion-selective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of -59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h-1), and a linear range of response including physiological and abnormal cell pH levels (6.0-8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells.
Assuntos
Eletrodos Seletivos de Íons , Prótons , Concentração de Íons de Hidrogênio , Potenciometria , Reprodutibilidade dos TestesRESUMO
Stress granules (SGs) are membrane-less organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arsenite-induced SGs, and H2 O2 is the main species. The release kinetics of H2 O2 from single SGs and the number of H2 O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple endpoint. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases (NDs), cancers and viral infections.
Assuntos
Técnicas Eletroquímicas , Neoplasias/diagnóstico , Espécies Reativas de Oxigênio/análise , Humanos , Neoplasias/metabolismo , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Viroses/diagnóstico , Viroses/metabolismoRESUMO
Resistive-pulse sensing is a technique widely used to detect single nanoscopic entities such as nanoparticles and large molecules that can block the ion current flow through a nanopore or a nanopipette. Although the species of interest, e.g., antibodies, DNA, and biological vesicles, are typically produced by living cells, so far, they have only been detected in the bulk solution since no localized resistive-pulse sensing in biological systems has yet been reported. In this report, we used a nanopipette as a scanning ion conductance microscopy (SICM) tip to carry out resistive-pulse experiments both inside immobilized living cells and near their surfaces. The characteristic changes in the ion current that occur when the pipet punctures the cell membrane are used to monitor its insertion into the cell cytoplasm. Following the penetration, cellular vesicles (phagosomes, lysosomes, and/or phagolysosomes) were detected inside a RAW 264.7 macrophage. Much smaller pipettes were used to selectively detect 10 nm Au nanoparticles in the macrophage cytoplasm. The in situ resistive-pulse detection of extracellular vesicles released by metastatic human breast cells (MDA-MB-231) is also demonstrated. Electrochemical resistive-pulse experiments were carried out by inserting a conductive carbon nanopipette into a macrophage cell to sample single vesicles and measure reactive oxygen and nitrogen species (ROS/RNS) contained inside them.
Assuntos
Lisossomos/química , Nanopartículas Metálicas/análise , Fagossomos/química , Espécies Reativas de Nitrogênio/análise , Espécies Reativas de Oxigênio/análise , Animais , Linhagem Celular Tumoral , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Ouro/química , Humanos , Nanopartículas Metálicas/química , Camundongos , Microscopia/instrumentação , Microscopia/métodos , Células RAW 264.7RESUMO
In this work, open carbon nanopipettes (CNPs) with radius between 50 and 600 nm were used to control translocation of different-sized vesicles through the pipette orifice followed by nanoelectrochemical analysis. Vesicle impact electrochemical cytometry (VIEC) was used to determine the number of catecholamine molecules expelled from single vesicles onto an inner-wall carbon surface, where the duration of transmitter release was quantified and correlated to the vesicle size all in the same nanotip. This in turn allowed us to both size and count molecules for vesicles in a living cell. Here, small and sharp open CNPs were employed to carry out intracellular VIEC with minimal invasion and high sensitivity. Our findings with VIEC reveal that the vesicular content increases with vesicle size. The release kinetics of vesicular transmitters and dense core size have the same relation with the vesicle size, implying that the vesicular dense core size determines the speed of each release event. This direct correlation unravels one of the complexities of exocytosis.
Assuntos
Carbono/química , Catecolaminas/química , Nanocápsulas/química , Permeabilidade da Membrana Celular , Células Cromafins , Liberação Controlada de Fármacos , Técnicas Eletroquímicas , Humanos , Cinética , Tamanho da Partícula , Vesículas Secretórias/metabolismo , Imagem Individual de MoléculaRESUMO
Carbon nanotube yarn microelectrodes (CNTYMEs) are an alternative to carbon-fiber microelectrodes (CFMEs) with interesting electrochemical properties because analyte is momentarily trapped in cavities between the CNTs. Here, we compare fast-scan cyclic voltammetry (FSCV) detection of catecholamines, including dopamine, norepinephrine, and epinephrine, at CNTYMEs, CFMEs, as well as cavity carbon nanopipette electrodes (CNPEs). At CFMEs, current decreases dramatically at high FSCV repetition frequencies. At CNTYMEs, current is almost independent of FSCV repetition frequency because the analytes are trapped in the crevices between CNTs, and thus the electrode acts like a thin-layer cell. At CFMEs, small cyclization product peaks are observed due to an intramolecular cyclization reaction to form leucocatecholamine, which is electroactive, and these peaks are largest for the secondary amine epinephrine. At CNTYMEs, more of the leucocatecholamine cyclization product is detected for all catecholamines because of the enhanced trapping effects, particularly at higher repetition rates where the reaction occurs more frequently and more product is accumulated. For epinephrine, the secondary peaks have larger currents than the primary oxidation peaks at 100 Hz, and similar trends are observed with faster scan rates and 500 Hz repetition frequencies. Finally, we examined CNPEs, which also momentarily trap neurotransmitters. Similar to CNTYMEs, at CNPEs, catecholamines have robust cyclization peaks, particularly at high repetition rates. Thus, CNTYMEs and CNPEs have thin layer cell behavior that facilitates high temporal resolution measurements, but catecholamines CVs are complicated by cyclization reactions. However, those additional peaks could be useful in discriminating the analytes, particularly epinephrine and norepinephrine.
RESUMO
The reaction of formaldehyde (HCHO) with the solution containing Au nanoparticles and [Ag(NH3)2]+ through Tollens' reaction is described to form Au@Ag nanoparticles (Au@AgNPs), which are used as a SERS enhancement material. A minimized hydrodistillation device with air condense is designed to obtain a clean aqueous background for SERS analysis and simplified pretreatment process. A good linear relationship is found between the SERS signals and the concentration of HCHO in the range 0.3 to 55 mg L-1 when p-aminothiophenol (p-ATP) is selected as the Raman signal molecule, and the detection limit is 0.08 mg L-1. Stable SERS signals could be achieved due to both homogeneous formation of Au@AgNPs and clean aqueous background. The relative standard deviation (RSD) of 15 batches samples is found to be 7.1%. The proposed approach has successfully been applied to the determination of HCHO residues in beer samples with low interferences. The recoveries of spiked samples are in the range 96 to 117% with RSDs lower than 5.9%. Graphical Abstract.
RESUMO
Resistive-pulse sensing with biological or solid-state nanopores and nanopipettes has been widely employed in detecting single molecules and nanoparticles. The analytical signal in such experiments is the change in ionic current caused by the molecule/particle translocation through the pipet orifice. This paper describes a new version of the resistive-pulse technique based on the use of carbon nanopipettes (CNP). The measured current is produced by electrochemical oxidation/reduction of redox molecules at the carbon surface and responds to the particle translocation. In addition to counting single entities, this technique enables qualitative and quantitative analysis of the electroactive material they contain. Using liposomes as a model system, we demonstrate the capacity of CNPs for (1) conventional resistive-pulse sensing of single liposomes, (2) electrochemical resistive-pulse sensing, and (3) electrochemical identification and quantitation of redox species (e.g., ferrocyanide, dopamine, and nitrite) contained in a single liposome. The small physical size of a CNP suggests the possibility of single-entity measurements in biological systems.
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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.
Assuntos
Macrófagos/citologia , Fagossomos/metabolismo , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Oxigênio/química , Animais , Sobrevivência Celular , Eletroquímica , Camundongos , Células RAW 264.7 , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Ion transport controlled by electrostatic interactions is an important phenomenon in biological and artificial membranes, channels, and nanopores. Here, we employ carbon-coated nanopipets (CNPs) for studying permselective electrochemistry in a conductive nanopore. A significant accumulation (up to 2000-fold) of cationic redox species and anion depletion inside a CNP by diffuse-layer and surface-charge effects in a solution of low ionic strength were observed as well as the shift of the voltammetric midpeak potential. Finite-element simulations of electrostatic effects on CNP voltammograms show permselective ion transport in a single conducting nanopore and semiquantitatively explain our experimental data. The reported results are potentially useful for improving sensitivity and selectivity of CNP sensors for ionic analytes.
RESUMO
Microelectrodes are typically used for neurotransmitter detection, but nanoelectrodes are not because there is a trade-off between spatial resolution and sensitivity that is dependent on surface area. Cavity carbon-nanopipette electrodes (CNPEs), with tip diameters of a few hundred nanometers, have been developed for nanoscale electrochemistry. Here, we characterize the electrochemical performance of CNPEs with fast-scan cyclic voltammetry (FSCV) for the first time. Dopamine detection using cavity CNPEs, with a depth equivalent to a few radii, is compared with that using open-tube CNPEs, an essentially infinite geometry. Open-tube CNPEs have very slow temporal responses that change over time as the liquid rises in the CNPE. However, a cavity CNPE has a fast temporal response to a bolus of dopamine that is not different from that of a traditional carbon-fiber microelectrode. Cavity CNPEs, with tip diameters of 200-400 nm, have high currents because the small cavity traps and increases the local dopamine concentration. The trapping also leads to an FSCV frequency-independent response and the appearance of cyclization peaks that are normally observed only with large concentrations of dopamine. CNPEs have high dopamine selectivity over ascorbic acid (AA) because of the repulsion of AA by the negative electric field at the holding potential and the irreversible redox reaction. In mouse-brain slices, cavity CNPEs detected exogenously applied dopamine, showing they do not clog in tissue. Thus, cavity CNPEs are promising neurochemical sensors that provide spatial resolution on the scale of hundreds of nanometers, which is useful for small model organisms or for locations near specific cells.
Assuntos
Carbono/química , Dopamina/análise , Técnicas Eletroquímicas/métodos , Animais , Ácido Ascórbico/química , Encéfalo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microeletrodos , Nanoestruturas/química , OxirreduçãoRESUMO
Carbon fiber micro- and nanoelectrodes have been extensively used to measure dopamine and other neurotransmitters in biological systems. Although the radii of some reported probes were âª1 µm, the lengths of the exposed carbon were typically on the micrometer scale, thus limiting the spatial resolution of electroanalytical measurements. Recent attempts to determine neurotransmitters in single cells and vesicles have provided additional impetus for decreasing the probe dimensions. Here, we report two types of dopamine sensors based on carbon nanopipets (CNP) prepared by chemical vapor deposition of carbon into prepulled quartz capillaries. These include 10-200 nm radius CNPs with a cavity near the orifice and CNPs with an open path in the middle, in which the volume of sampled solution can be controlled by the applied pressure. Because of the relatively large surface area of carbon exposed to solution inside the pipet, both types of sensors yielded well-shaped voltammograms of dopamine down to ca. 1 nM concentrations, and the unprecedented voltammetric response to 100 pM dopamine was obtained with open CNPs. TEM tomography and numerical simulations were used to model CNP responses. The effect of dopamine adsorption on the CNP detection limit is discussed along with the possibilities of measuring other physiologically important analytes (e.g., serotonin) and eliminating anionic and electrochemically irreversible interferences (e.g., ascorbic acid).
Assuntos
Técnicas Biossensoriais/métodos , Carbono/química , Dopamina/análise , Microeletrodos , Nanotubos de Carbono/química , Adsorção , Técnicas Biossensoriais/instrumentação , Humanos , Limite de DetecçãoRESUMO
We demonstrate the feasibility of attaining femtomolar limits of quantitation in electroanalysis. The method employed is based on electrocatalytic amplification, where small quantities of metal deposit performed on a carbon electrode causes a large increase in the observed current, for example, for the oxidation of water. We show calibration curves at the femtomolar level for cobalt, nickel, and lead ions on carbon ultramicroelectrodes (CUMEs), ca. 500 nm radii. The CUME was biased at a potential where the ion would deposit as the metal oxide, MOx, and a high concentration of species that is oxidized at the deposit is present in solution. Blips were observed in the amperometric i-t response, and their frequency scaled linearly with the concentration of ions at the femtomolar level. From these results, the limits of quantitation for cobalt, nickel, and lead ions were reported at 10 s of femtomolar level for the first time.
RESUMO
The properties of nanoparticles (NPs) are determined by their size and geometric structures. A reliable determination of NP dimension is critical for understanding their physical and chemical properties, but sizing ultrasmall particles on the order of nanometer (nm) scale in the solution is still challenging. Here, we report the size measurement of PtNP at nanometer resolution by in situ scanning electrochemical microscopy (SECM), performed with the electrochemical generation and removal of H2 bubble at a reasonably small distance between tip and substrate electrodes in 200 or 500 mM HClO4 solution. A series of different PtNPs or nanoclusters were electrodeposited and in situ measured in the solution, proving the concept of sizing ultrasmall particles using tip generation/substrate collection mode of SECM. This technique could be also used for investigations of other supported ultrasmall metal nanocluster systems.
RESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas Eletroquímicas , Metástase Neoplásica , Espécies Reativas de Nitrogênio/análise , Espécies Reativas de Oxigênio/análise , Carbono/química , Eletrodos , Humanos , Tamanho da Partícula , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Investigating the collisions of individual metal nanoparticles (NPs) with electrodes can provide new insights into their electrocatalytic behavior, mass transport, and interactions with surfaces. Here we report a new experimental setup for studying NP collisions based on the use of carbon nanopipettes to enable monitoring multiple collision events involving the same NP captured inside the pipet cavity. A patch clamp amplifier capable of measuring pA-range currents on the microsecond time scale with a very low noise and stable background was used to record the collision transients. The analysis of current transients produced by oxidation of hydrogen peroxide at one IrOx NP provided information about the origins of deactivation of catalytic NPs and the effects of various experimental conditions on the collision dynamics. High-resolution TEM of carbon pipettes was used to attain better understanding of the NP capture and collisions.
RESUMO
We report on imaging of local electric field on an electrode surface with plasmonic electrochemical impedance microscopy (P-EIM). The local electric field is created by putting an electrode inside a micropipet positioned over the electrode and applying a voltage between the two electrodes. We show that the distribution of the surface charge as well as the local electric field at the electrode surface can be imaged with P-EIM. The spatial distribution and the dependence of the local charge density and electric field on the distance between the micropipet and the surface are measured, and the results are compared with the finite element calculations. The work also demonstrates the possibility of integrating plasmonic imaging with scanning ion conductance microscopy (SICM) and other scanning probe microscopies.