Neomycin, but Not Neamine, Blocks Angiogenic Factor Induced Nitric Oxide Release through Inhibition of Akt Phosphorylation.

Angiogenesis, the growth of new blood vessels, is a critical factor of carcinogenesis. Neomycin and neamine, two drugs blocking the nuclear translocation of angiogenin (ANG), have been proven to inhibit tumour growth in vivo. However, the high toxicity of neomycin prevents its therapeutic use, thus indicating that the less toxic neamine may be a better candidate. Endothelial cells were cultured on a biocompatible multiple microelectrode array (MMA). The release of NO evoked by ANG or vascular endothelial growth factor (VEGF) was detected electrochemically. The effects of neomycin and neamine on ANG- and VEGF-induced NO releases have been investigated. Neomycin totally blocks NO release for concentrations down to the pM range, probably through the inhibition of the Akt kinase phosphorylation, as revealed by confocal microscopy. On the other hand, both ANG- and VEGF-induced NO releases were not significantly hindered by the presence of high concentrations of neamine. The inhibition of the Akt pathway and NO release are expected to lead to a severe decrease in tissue growth and repair, thus indicating a possible cause for the toxicity of neomycin. Furthermore, the data presented here show that ANG- and VEGF-induced NO releases are not dependent on the nuclear translocation of angiogenin, as these events were not abolished by the presence of neamine.

Paper-Based Polymer Electrodes for Bioanalysis and Electrochemistry of Neurotransmitters.

This article describes the preparation and characterization of PEDOT-coated paper electrodes. Their specific behavior was investigated, especially the impact of electrode shape on the electrochemical properties. It was found that different electrode geometries promote different results, largely because of a potential drop in the bulk of the electrode material. More importantly, the suitability of these substrates for bio- and neurochemical analyses was investigated. The paper electrodes were found to offer better resistance to both protein and neurotransmitter foulings, in comparison to a planar electrode. Interestingly, long paper electrodes were more stable during sustained oxidation of dopamine and serotonin than short ones, possibly because of the conjunction of surface passivation and potential drop allowing for the site of oxidation to move along the electrode as fouling progresses.

Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer.

Fluorescence in situ hybridization (FISH) is one of the recommended techniques for human epidermal growth factor receptor 2 (HER2) status assessment on cancer tissues. Here we develop microfluidics-assisted FISH (MA-FISH), in which hybridization of the FISH probes with their target DNA strands is obtained by applying square-wave oscillatory flows of diluted probe solutions in a thin microfluidic chamber of 5 µl volume. By optimizing the experimental parameters, MA-FISH decreases the consumption of the expensive probe solution by a factor 5 with respect to the standard technique, and reduces the hybridization time to 4 h, which is four times faster than in the standard protocol. To validate the method, we blindly conducted HER2 MA-FISH on 51 formalin-fixed paraffin-embedded tissue slides of 17 breast cancer samples, and compared the results with standard HER2 FISH testing. HER2 status classification was determined according to published guidelines, based on average number of HER2 copies per cell and average HER2/CEP17 ratio. Excellent agreement was observed between the two methods, supporting the validity of MA-FISH and further promoting its short hybridization time and reduced reagent consumption.

Super-Resolution Imaging of a Dielectric Microsphere Is Governed by the Waist of Its Photonic Nanojet.

Dielectric microspheres with appropriate refractive index can image objects with super-resolution, that is, with a precision well beyond the classical diffraction limit. A microsphere is also known to generate upon illumination a photonic nanojet, which is a scattered beam of light with a high-intensity main lobe and very narrow waist. Here, we report a systematic study of the imaging of water-immersed nanostructures by barium titanate glass microspheres of different size. A numerical study of the light propagation through a microsphere points out the light focusing capability of microspheres of different size and the waist of their photonic nanojet. The former correlates to the magnification factor of the virtual images obtained from linear test nanostructures, the biggest magnification being obtained with microspheres of ∼6-7 µm in size. Analyzing the light intensity distribution of microscopy images allows determining analytically the point spread function of the optical system and thereby quantifies its resolution. We find that the super-resolution imaging of a microsphere is dependent on the waist of its photonic nanojet, the best resolution being obtained with a 6 µm Ø microsphere, which generates the nanojet with the minimum waist. This comparison allows elucidating the super-resolution imaging mechanism.

Spatial resolution of single-cell exocytosis by microwell-based individually addressable thin film ultramicroelectrode arrays.

We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-µm-width square ultramicroelectrodes, 25 3-µm-width square ultramicroelectrodes, or 36 2-µm-width square ultramicroelectrodes, all inside a 40 × 40 µm square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 µm square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-µm-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.

Magnetic particle-scanning for ultrasensitive immunodetection on-chip.

We describe the concept of magnetic particle-scanning for on-chip detection of biomolecules: a magnetic particle, carrying a low number of antigens (Ag's) (down to a single molecule), is transported by hydrodynamic forces and is subjected to successive stochastic reorientations in an engineered magnetic energy landscape. The latter consists of a pattern of substrate-bound small magnetic particles that are functionalized with antibodies (Ab's). Subsequationuent counting of the captured Ag-carrying particles provides the detection signal. The magnetic particle-scanning principle is investigated in a custom-built magneto-microfluidic chip and theoretically described by a random walk-based model, in which the trajectory of the contact point between an Ag-carrying particle and the small magnetic particle pattern is described by stochastic moves over the surface of the mobile particle, until this point coincides with the position of an Ag, resulting in the binding of the particle. This model explains the particular behavior of previously reported experimental dose-response curves obtained for two different ligand-receptor systems (biotin/streptavidin and TNF-α) over a wide range of concentrations. Our model shows that magnetic particle-scanning results in a very high probability of immunocomplex formation for very low Ag concentrations, leading to an extremely low limit of detection, down to the single molecule-per-particle level. When compared to other types of magnetic particle-based surface coverage assays, our strategy was found to offer a wider dynamic range (>8 orders of magnitude), as the system does not saturate for concentrations as high as 10(11) Ag molecules in a 5 µL drop. Furthermore, by emphasizing the importance of maximizing the encounter probability between the Ag and the Ab to improve sensitivity, our model also contributes to explaining the behavior of other particle-based heterogeneous immunoassays.

Development of an organic lateral resolution test device for imaging mass spectrometry.

An organic lateral resolution test device has been developed to measure the performance of imaging mass spectrometry (IMS) systems. The device contains periodic gratings of polyethylene glycol (PEG) and lipid bars covering a wide range of spatial frequencies. Microfabrication technologies were employed to produce well-defined chemical interfaces, which allow lateral resolution to be assessed using the edge-spread function (ESF). In addition, the design of the device allows for the direct measurement of the modulation transfer function (MTF) to assess image quality. Scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to characterize the device. TOF-SIMS imaging was used to measure the chemical displacement of biomolecules in matrix-assisted laser desorption/ionization (MALDI) matrix crystals. In a proof-of-concept experiment, the platform was also used to evaluate MALDI matrix application methods, specifically aerosol spray and sublimation methods.

Single cell amperometry reveals glycocalyx hinders the release of neurotransmitters during exocytosis.

The diffusional hindrance of the glycocalyx along the cell surface on exocytotic peaks, observed with single cell amperometry, was investigated. Partial digestion of the glycocalyx with neuraminidase led to the observation of faster peaks, as shown by varied peak parameters. This result indicates that diffusion of small molecules in the partially digested glycocalyx is 2.2 faster than in the intact glycocalyx. Similarly, neutralization of the negative charges present in the cell microenvironment led to faster peak kinetics. The analysis of the vesicular efflux indicates that the diffusion coefficient of dopamine at the cell surface is at most 45% of the diffusion coefficient in free solution. This study shows that the glycocalyx plays an important role in the diffusion kinetics of processes along the cell surface, including exocytotic events.

Individually addressable thin-film ultramicroelectrode array for spatial measurements of single vesicle release.

Thin-film platinum ultramicroelectrode arrays (MEAs) with subcellular microelectrodes were developed for the spatial measurement of neurotransmitter release across single cells or clusters of single cells. MEAs consisting of 16, 25, and 36 square ultramicroelectrodes with respective widths of 4, 3, and 2 µm were fabricated on glass substrates by photolithography, thin-film deposition, and reactive ion etching. The electrodes in each MEA are tightly defined in a 30 µm × 30 µm square, which is potentially useful to measure exocytosis across a single cell or clusters of single cells. These MEAs have been characterized with scanning electron microscopy and cyclic voltammetry and show excellent stability and reproducibility. Culturing PC12 cells on top of the MEAs has been achieved by modifying the array with a poly(dimethylsiloxane) chamber and coating a thin layer of collagen IV on top of the electrode surface. The electrochemical response to dopamine has been characterized after coating the surface with the cell-adhering molecules and then with cells attached. Amperometric detection demonstrates that individual exocytotic events can be recorded at these arrays with spatial resolution for dynamic electrochemical measurements near 2 µm. In contrast to previous single-cell experiments, the effect of dopaminergic drugs on imaging single vesicle exocytotic release from PC12 cell clusters is presented at cell clusters incubated with the dopamine precursor and Parkinson's therapy agent, L-3,4-dihydroxyphenylalanine, and at cell clusters incubated with the vesicular monoamine transport inhibitor, reserpine. The results of electrochemical imaging demonstrate that the drug effect on PC12 cell clusters is consistent with previous single-cell experiments.

Carbon nanotube fiber microelectrodes show a higher resistance to dopamine fouling.

We have compared the properties and resistance to DA fouling of a carbon nanotube fiber (CNTF) microelectrode to a traditional carbon fiber (CF) microelectrode. These two materials show comparable electrochemical activities for outer-sphere and inner-sphere redox reactions. Although the CNTF might have a higher intrinsic RC constant, thus limiting its high-frequency behavior, the CNTF shows a significantly higher durability than the CF in terms of electrode stability. During constant oxidation of 100 µM DA, the signal measured by the CNTF microelectrode shows a 2-h window over which no decrease in current is observed. Under the same conditions, the current obtained at the CF microelectrode decreases by almost 50%. A model of the fouling process, assuming the formation of growing patches of insulator on the surface, has been compared to the data. This model is found to be in good agreement with our results and indicates a growth rate of the patches in the 0.1-2 nm s(-1) range.

Evaluating the diffusion coefficient of dopamine at the cell surface during amperometric detection: disk vs ring microelectrodes.

During exocytosis, small quantities of neurotransmitters are released by the cell. These neurotransmitters can be detected quantitatively using electrochemical methods, principally with disk carbon fiber microelectrode amperometry. An exocytotic event then results in the recording of a current peak whose characteristic features are directly related to the mechanisms of exocytosis. We have compared two exocytotic peak populations obtained from PC12 cells with a disk carbon fiber microelectrode and with a pyrolyzed carbon ring microelectrode array, with a 500 nm ring thickness. The specific shape of the ring electrode allows for precise analysis of diffusion processes at the vicinity of the cell membrane. Peaks obtained with a ring microelectrode array show a distorted average shape, owing to increased diffusion pathways. This result has been used to evaluate the diffusion coefficient of dopamine at the surface of a cell, which is up to an order of magnitude smaller than that measured in free buffer. The lower rate of diffusion is discussed as resulting from interactions with the glycocalyx.

Biological applications of the electrochemical sensing of nitric oxide: fundamentals and recent developments.

Nitric oxide (NO) is a unique cellular messenger linked to a number of important biological processes. Its free radical nature, small size and fast diffusivity make it highly reactive and membrane permeable. Unfortunately, its reactivity, coupled with the inherent complexity of in situ biological measurements, makes it a challenge to detect. For the past 20 years, electrochemical methods have been used to investigate the role of NO in a number of biological processes, including vascular physiology, immune response, neuronal mediation, tissue growth and oxidative stress. This review examines the biological applications of electrochemical NO sensors and the technologies used to elucidate different physiological phenomena associated with this unique biomolecule with a specific focus on the developments and innovations reported in the last 3 years.

Amperometric measurements at cells support a role for dynamin in the dilation of the fusion pore during exocytosis.

Dynamin is a GTPase mechanochemical enzyme involved in the late steps of endocytosis, where it separates the endocytotic vesicle from the cell membrane. However, recent reports have emphasized its role in exocytosis. In this case, dynamin may contribute to the control of the exocytotic pore, thus suggesting a direct control on the efflux of neurotransmitters. Dynasore, a selective inhibitor of the GTPase activity of dynamin, was used to investigate the role of dynamin in exocytosis. Exocytosis was analyzed by amperometry, thus revealing that dynasore inhibits exocytosis in a dose-dependent manner. Analysis of the exocytotic peaks shows that the inhibition of the GTPase activity of dynamin leads to shorter, smaller events. This observation, together with the rapid effect of dynasore, suggests that the blocking of the GTPase induces the formation of a more narrow and short-lived fusion pore. These results suggest that the GTPase properties of dynamin are involved in the duration and kinetics of exocytotic release. Interestingly, and in strong contrast with its role in endocytosis, the mechanochemical properties of dynamin appear to contribute to the dilation and stability of the pore during exocytosis.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.

Carbon-ring microelectrode arrays for electrochemical imaging of single cell exocytosis: fabrication and characterization.

Fabrication of carbon microelectrode arrays, with up to 15 electrodes in total tips as small as 10-50 µm, is presented. The support structures of microelectrodes were obtained by pulling multiple quartz capillaries together to form hollow capillary arrays before carbon deposition. Carbon ring microelectrodes were deposited by pyrolysis of acetylene in the lumen of these quartz capillary arrays. Each carbon deposited array tip was filled with epoxy, followed by beveling of the tip of the array to form a deposited carbon-ring microelectrode array (CRMA). Both the number of the microelectrodes in the array and the tip size are independently tunable. These CRMAs have been characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy, and electrogenerated chemiluminescence. Additionally, the electrochemical properties were investigated with steady-state voltammetry. In order to demonstrate the utility of these fabricated microelectrodes in neurochemistry, CRMAs containing eight microring electrodes were used for electrochemical monitoring of exocytotic events from single PC12 cells. Subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) were successfully detected with the CRMAs.

Electrochemical study of the intracellular transduction of vascular endothelial growth factor induced nitric oxide synthase activity using a multi-channel biocompatible microelectrode array.

BACKGROUND: Nitric oxide (NO) plays a major role in physiology as a biological mediator. NO has been identified in nervous, immune and vascular systems and is a critical parameter in numerous pathologies, such as cancer. This article describes the electrochemical biomeasurements of NO synthase (NOS) activity from cultured endothelial cells using a multiple microelectrode array. METHODS: Firstly, the effect of biocompatible fibronectin coating on electrochemical measurements was investigated. Secondly, endothelial cells were deposited on the fibronectin coated sensor and NO release was triggered with vascular endothelial growth factor (VEGF). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as an inhibitor of NO production, and different kinase blockers were investigated. Change in NOS activity was quantified using differential pulse voltammetry before and after addition of VEGF. RESULTS: Our results show that carefully applied layers of fibronectin have a very limited effect on electrochemistry and that VEGF induces an increase in NOS activity that is mainly mediated through the phosphatidylinositol 3 kinase (PI-3), and not by the extracellular signal-regulated kinases 1/2. Results obtained using electrochemical sensors were supported by wound healing assay demonstrating the critical role of phosphatidylinositol 3 kinase and extracellular signal-regulated kinases 1/2 for angiogenesis. CONCLUSION: Electrochemical study of the intracellular transduction of the VEGF signal leading to NO synthesis was achieved, showing the critical role of PI-3 kinase. GENERAL SIGNIFICANCE: This study presents an electrochemical sensor allowing measurements of NOS activity in cell cultures and tissue samples.

Effect of the doping level on the biological stability of hydrogenated boron doped diamond electrodes.

Fouling of electrode surfaces by electrode reaction products or by biological spectator species is known to inactivate electrochemical sensors and thus limit their use in biological conditions. Here we present an investigation on the stability of boron doped diamond (BDD) electrodes with different levels of doping. Three different doping levels were used (0.1, 1 and 5% in the carbon phase). The highly doped (5%) BDD is of particular interest as it is here used for the first time for biological applications. Three different redox reactions were examined based on their electrode reaction characteristics: ruthenium(III) hexaammine (outer sphere), ferrocyanide (surface dependent), dopamine (adsorption mediated). The effect of albumin at blood concentration was studied. All results were compared with glassy carbon. There were no significant differences for the outer sphere electrochemistry, but all the BDDs showed improved resistance to fouling for the ferrocyanide oxidation. The electrocatalytic activity of BBD towards dopamine oxidation increased with increased boron content. However, this appears to be due to a larger number of defect sites which also increases the vulnerability to fouling by albumin and by electrode reaction products and the 5% BDD had similar properties to glassy carbon in this regard. These results suggest that it is possible to optimise the BDD performance for specific applications and that the large potential window for BDD may be due, at least in part, to its relatively poor electrocatalytic activity.

Angiogenin induces nitric oxide synthesis in endothelial cells through PI-3 and Akt kinases.

Angiogenesis, the formation of new blood vessels, is a critical but complex phenomenon modulated by numerous physicochemical conditions. Nitric oxide (NO) is a very well known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of angiogenin, a major angiogenic factor, and extracellular NO release. NO concentration was sensed electrochemically using a fibronectin-coated multiple microelectrode array. Angiogenin was shown to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. The effect of angiogenin on NOS was demonstrated using l-NAME, a competitive NOS inhibitor. Dose-time dependence was investigated, showing a stimulation threshold in the 250 ng/mL-1 microg/mL range and a maximal NO release after 30 min of exposure to angiogenin. To elucidate the very complex reactive pathway of angiogenin, we have used various selective inhibitors to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited the angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus. Similarly, NOS activity was stopped by blocking the PI-3/Akt kinase signaling transduction cascade, showing the importance of this pathway.

Automated phenotyping of Caenorhabditis elegans embryos with a high-throughput-screening microfluidic platform.

The nematode Caenorhabditis elegans has been extensively used as a model multicellular organism to study the influence of osmotic stress conditions and the toxicity of chemical compounds on developmental and motility-associated phenotypes. However, the several-day culture of nematodes needed for such studies has caused researchers to explore alternatives. In particular, C. elegans embryos, due to their shorter developmental time and immobile nature, could be exploited for this purpose, although usually their harvesting and handling is tedious. Here, we present a multiplexed, high-throughput and automated embryo phenotyping microfluidic approach to observe C. elegans embryogenesis after the application of different chemical compounds. After performing experiments with up to 800 embryos per chip and up to 12 h of time-lapsed imaging per embryo, the individual phenotypic developmental data were collected and analyzed through machine learning and image processing approaches. Our proof-of-concept platform indicates developmental lag and the induction of mitochondrial stress in embryos exposed to high doses (200 mM) of glucose and NaCl, while small doses of sucrose and glucose were shown to accelerate development. Overall, our new technique has potential for use in large-scale developmental biology studies and opens new avenues for very rapid high-throughput and high-content screening using C. elegans embryos.

Comparative study of poly(styrene-sulfonate)/poly(L-lysine) and fibronectin as biofouling-preventing layers in dissolved oxygen electrochemical measurements.

Oxygen is a major actor of many physiological, biological and industrial processes; as such, its monitoring is of paramount importance. The effects of protein biofouling on dissolved oxygen measurements are described. The consequences of protein adsorption on electrode kinetics and mass transport were quantified using cyclic voltammetry, AC impedance and rotating disc amperometry, firstly in a solution of albumin, secondly in a complex protein suspension of liver tissue. The effect of membrane coatings poly(styrene-sulfonate)/poly(L-lysine) and fibronectin were investigated. The relative significance on mass transport, surface diffusion and electrocatalysis were examined by comparing the experimental data for an outer-sphere redox couple, ruthenium hexaammine (III)/(II), with the physiologically significant electrocatalytic O(2) reduction reaction.