Rapid, Sensitive Detection of Neurotransmitters at Microelectrodes Modified with Self-assembled SWCNT Forests

Carbon nanotube (CNT) modification of microelectrodes can result in increased sensitivity without compromising time response. However, dip coating CNTs is not very reproducible and the CNTs tend to lay flat on the electrode surface which limits access to the electroactive sites on the ends. In this study, aligned CNT forests were formed using a chemical self-assembly method, which resulted in more exposed CNT ends to the analyte. Shortened, carboxylic acid functionalized single-walled CNTs were assembled from a dimethylformamide (DMF) suspension onto a carbon-fiber disk microelectrode modified with a thin iron hydroxide-decorated Nafion film. The modified electrodes were highly sensitive, with 36-fold higher oxidation currents for dopamine using fast-scan cyclic voltammetry than bare electrodes and 34-fold more current than electrodes dipped in CNTs. The limit of detection (LOD) for dopamine was 17 ± 3 nM at a 10 Hz repetition rate and 65 ± 7 nM at 90 Hz. The LOD at 90 Hz was the same as a bare electrode at 10 Hz, allowing a 9-fold increase in temporal resolution without a decrease in sensitivity. Similar increases were observed for other cationic catecholamine neurotransmitters, and the increases in current were greater than for anionic interferents such as ascorbic acid and 3,4-dihydroxyphenylacetic acid (DOPAC). The CNT forest electrodes had high sensitivity at 90 Hz repetition rate when stimulated dopamine release was measured in Drosophila . The sensitivity, temporal resolution, and spatial resolution of these CNT forest modified disk electrodes facilitate enhanced electrochemical measurements of neurotransmitter release in vivo.     

Quantitative aspects of ohmic microscopy

The potential difference between two microreference electrodes, Δφ(sol), immersed in an aqueous sulfuric acid solution was monitored while performing conventional cyclic voltammetric experiments with a Pt disk electrode embedded in an insulating surface in an axisymmetric cell configuration. The resulting Δφ(sol) vs E curves, where E is the potential applied to the Pt disk electrode were remarkably similar to the voltammograms regardless of the position of the microreference probes. Most importantly, the actual values of Δφ(sol) were in very good agreement with those predicted by the primary current distribution using Newman's formalism (Newman, J. J. Electrochem. Soc.1966, 113, 501-502). These findings afford a solid basis for the development of ohmic microscopy as a quantitative tool for obtaining spatially resolved images of electrodes displaying nonhomogenous surfaces.     

Voltammetric detection of mercury and copper in seawater using a gold microwire electrode

A procedure is presented by which mercury and copper are determined simultaneously in seawater and dilute acid (0.01 M HCl) by anodic stripping voltammetry using gold microwire electrodes. It was found that anion (halide) adsorption is the cause for a gradual decrease in the height and potential of the mercury peak. The effect is eliminated by including an anion desorption step in the analysis at -0.8 V prior to each scan. This step was found to greatly improve the stability of the scans and enabled the use of background subtraction. Advantages of the microwire electrodes were a low roughness of the surface, without a need for pretreatment, and a very small diffusion layer (2 microm with stirring). Under the optimized voltammetric conditions, the detection limits were 6 pM mercury and 25 pM copper using 300-s deposition. These values are well below those reported previously for other electrodes including rotating disk electrodes. Measurements of the influence of the major anions I-, Br-, Cl-, SO4(2-), F-, HCO3-, and B(OH)4 on the response for mercury showed that bromide and chloride are predominantly responsible for the underpotential deposition mechanism of mercury in seawater. The method was applied to coastal water samples from Liverpool Bay.     

Cell-based indicator to visualize picomolar dynamics of nitric oxide release from living cells

We report a novel cell-based indicator that is able to visualize picomolar dynamics of nitric oxide release from living cells. Cells from a pig kidney-derived cell line (PK15) endogenously express soluble guanylate cyclase (sGC), which is a receptor protein for the selective recognition of NO. Binding of NO by sGC causes the amplified generation of guanosine 3',5'-cyclic monophosphate (cGMP). To make the PK15 cells into NO indicators, the cells are transfected with a plasmid vector encoding a fluorescent indicator for cGMP and fluorescence resonance energy transfer is recorded at 480 +/- 15 and 535 +/- 12.5 nm upon excitation of the cells at 440 +/- 10 nm. The cell-based indicator exhibits exceptional sensitivity (detection limit of 20 pM), selectivity, reversibility, and reproducibility. The outstanding sensitivity of the present indicator has led us to uncover an oscillatory release of picomolar concentrations of NO from hippocampal neurons. We present evidence that Ca2+ oscillations in hippocampal neurons underlie the oscillatory NO release from the neurons during neurotransmission. We also have succeeded in visualizing the extent of diffusing NO from single vascular endothelial cells. The present cell-based indicator provides a powerful tool to uncover picomolar dynamics of NO that regulates a wide range of cell functions in biological systems.     

Amperometric fluidic microchip array sensing device for nitric oxide determination in solution

In this paper, we report on the first use of an amperometric fluidic microchip array for the examination of nitric oxide in solution. The array chip is composed of 36 working platinum electrodes on a glass substrate. The electrodes have a diameter of 50 μm and are separated by 500 μm. The array chip is integrated within a flowing cell to obtain a fluidic-type sensing device. Two preliminary tests were performed. The first one consisted in assessing the fluidic set-up by using potassium ferrocyanide as test analyte. The second test was aimed at achieving the modification of the surface of the working electrodes by electrodepositing nickel tetrasulfonated phthalocyanine and Nafion® layers to show that the fluidic sensing device can be adapted to the analysis of nitric oxide in solution.     

Scanning electrochemical microscopy of model neurons: constant distance imaging

Undifferentiated and differentiated PC12 cells were imaged with the constant-distance mode of scanning electrochemical microscopy (SECM) using carbon ring and carbon fiber tips. Two types of feedback signals were used for distance control: the electrolysis current of a mediator (constant-current mode) and the impedance measured by the SECM tip (constant-impedance mode). The highest resolution was achieved using carbon ring electrodes with the constant-current mode. However, the constant-impedance mode has the important advantages that topography and faradaic current can be measured simultaneously, and because no mediator is required, the imaging can take place directly in the cell growth media. It was found that vesicular release events do not measurably alter the impedance, but the depolarizing solution, 105 mM K+, produces a dramatic impedance change such that constant-distance imaging cannot be performed during application of the stimulus. However, by operating the tip in the constant-height mode, cell morphology (via a change in impedance) and vesicular release could be detected simultaneously while moving the tip across the cell. This work represents a significant improvement over previous SECM imaging of model neurons, and it demonstrates that the combination of amperometry and constant-impedance SECM has the potential to be a powerful tool for investigating the spatial distribution of neurotransmitter release in vitro.     

Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase.

Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its inability to bind oxygen, and its high affinity for nitric oxide make it well-suited for sensor applications. The heme domain has been labeled with a fluorescent reporter dye and changes in this dye's intensity are observed based on the sGC heme domain's characteristic binding of nitric oxide. The current sensors are prepared with 100-microns optical fiber but could also be prepared using submicrometer fiber tips. These sensors have fast, linear, and reversible responses to nitric oxide and are unaffected by numerous common interferents, such as oxygen, nitrite and nitrate. The sensor limit of detection is 1 microM nitric oxide. Glutathione has been shown to decrease the sensitivity of the sensor; however, the sensor response remains linear and can be calibrated on the basis of the glutathione concentration present in the biological environment of interest. The sensors have been used to measure extracellular nitric oxide production by BALB/c mouse macrophages. Minimal nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 microM in a given cell culture.     

Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry

Single-cell mass spectrometry (MS) is a rapidly emerging field in metabolic investigations. The inherent chemical complexity of most biological samples poses analytical challenges when using MS platforms to measure sample content without prior chemical separation. Here, a single-cell capillary electrophoresis (CE) system was coupled with electrospray ionization (ESI) MS to enable the simultaneous measurement of a vast array of endogenous compounds in over 50 identified and isolated large neurons from the Aplysia californica central nervous system. More than 300 distinct ion signals (m/z values) were detected from a single neuron in the positive ion mode, 140 of which were selected for chemometric data analysis. Metabolic features were evaluated among six different neuron types (B1, B2, left pleural 1 (LPl1), metacerebral cell (MCC), R2, and R15) chosen for their various physiological functions. The results indicated chemical similarities among some neuron types (B1 to B2 and LPl1 to R2) and distinctive features for others (MCC and R15 cells). The quantitative nature of the MS platform allowed the comparison of metabolite levels for specific neurons. The CE-ESI-MS approach for examination of individual nanoliter-volume cells as described herein is readily adaptable to other volume-limited samples.     

Synthesis,characterization, biodegradability and biocompatibility of a temperature-sensitive PBLA-PEG-PBLA hydrogel as protein delivery system with low critical gelation concentration

Temperature-sensitive hydrogels were designed using a series of A-B-A triblock copolymers consisting of poly (ethylene glycol) (PEG) with different molecular weights as the hydrophilic block B and poly (β-butyrolactone-co-lactic acid)(PBLA) with varying block lengths and composition as the hydrophobic block A. The triblock copolymers were synthesized by ring-opening polymerization (ROP) of β-BL and LA in bulk using PEG as an initiator and Sn(Oct)₂ as the catalyst. Their chemical structure and molecular characteristics were determined by NMR, GPC and DSC, and the relationship between structure and phase behaviors in aqueous solutions was investigated as well. It was found that the phase behaviors in aqueous solutions including critical micelle concentration (CMC), sol-gel-sedimentation phase transition temperature, gel window width and critical gelation concentration (CGC) are largely dependent on the molecular weight and block length ratio of PEG/PBLA. Most importantly, they show a very low CGC ranging from 4 to 8?wt% because of the introduction of β-BL. Furthermore, the biodegradability and biocompatibility of the hydrogels were evaluated. Finally, lysozyme as a model protein was used to evaluate the ability to deliver protein drugs in a sustained release manner and biologically active form. All results demonstrated that the temperature-sensitive in situ forming hydrogel has a promising potential as sustained delivery system for protein drugs.     

Graphite nanoplatelets/multiwalled carbon nanotubes hybrid nanostructure for electrochemical capacitor

Recently, the focus on carbon based nanostructures for various applications has been due to their novel properties such as high electrical conductivity, high mechanical strength and high surface area. In the present work, we have investigated the charge storage capacity of modified graphite nanoplatelets and hybrid structure of graphite nanoplatelets-multiwalled carbon nanotubes (MWNTs). These MWNTs can be used as spacers to reduce the possibility of restacking of graphite nanoplatelets and hence increases the surface area of the hybrid carbon nanostructure thereby high degree of metal oxide decoration is achieved over the hybrid structure. MWNTs were prepared by catalytic chemical vapor deposition technique and further purified with air oxidation and acid treatment. Graphite was treated with conc. nitric acid and sulphuric acid in the volumetric ratio of 1:3 for 3 days and these modified graphite nanoplatelets were further stirred with MWNTs in equal weight ratio to form hybrid nanostructure. Further, ruthenium oxide (RuO2) nanoparticles were decorated on this hybrid structure using chemical route followed by calcination. RuO2 decorated hybrid carbon nanostructure was characterized by using X-ray diffraction, Electron microscopy and Raman spectroscopy. The performance of the hybrid structure based nanocomposite as electrochemical capacitor electrodes was analyzed by studing its capacitive and charge-discharge behaviours using cyclic voltammetry and chronopotentiometry techniques and the results have been discussed.     

Voltammetric measurement of oxygen in single neurons using platinized carbon ring electrodes.

Naflon-coated ultrasmall platinum ring electrodes have been implanted in the giant dopamine neuron of the pond snail Planorbis corneus, and the oxygen concentration inside these single neurons has been estimated. Experimental data suggest that the intracellular oxygen level in the identified dopamine neuron of P. corneus is approximately 0.032 mM. The oxygen concentration immediately outside the cell (ca. 10 microns away from the cell) is 0.041 mM. Furthermore, staircase voltammetry can be used to monitor dynamic changes in oxygen concentration inside the cell after bathing with Ringer's solution saturated with air/oxygen. Data obtained for intracellular oxygen concentrations suggest that intracellular oxygen consumption is increased following potassium chloride-induced stimulation of these cells.     

Dual microelectrodes for distance control and detection of nitric oxide from endothelial cells by means of scanning electrochemical microscope

Dual Pt disk microelectrodes consisting of a 10-microm distance sensor and a 50-microm nitric oxide sensor were prepared. The 50-microm electrode was modified with Ni(4-N-tetramethyl)pyridyl porphyrin enclosed in the polymer network of a negatively charged electrodeposition paint. This paint prevented the dissolution of the otherwise soluble porphyrin in the aqueous test medium due to charge interactions. It also denied negatively charged ions in the analyte solution access to the electrode surface by electrostatic repulsion, thereby preventing interference from anions such as nitrite, nitrate, and ascorbate. With the aid of a scanning electrochemical microscope, it was possible to use the distance sensor by recording the negative feedback effect on the reduction of molecular oxygen to "guide" the nitric oxide sensor to various known distances from a layer of adherently growing human umbilical vein endothelial cells for the detection of nitric oxide released from the cells upon stimulation with bradykinin. The use of the distance sensor made it possible to preserve the integrity of the adherently growing cells concomitantly with the modified electrode by preventing the deterioration of the modifying layer during the distance adjustment step.     

Porous silicon based negative electrodes for lithium ion batteries

Various structures of macroporous silicon intended for the fabrication of lithium ion battery anodes have been studied. Macroporous membranes were prepared by the photoelectrochemical etching of n-type silicon wafers possessing various resistances, followed by the removal of the substrate. The porosity was increased via additional oxidation with the subsequent etching of oxide. The electric contacts on the membrane were fabricated by depositing copper with a titanium sublayer and soldering the structure to a supporting molybdenum disk. The electrochemical characteristics of anodes were studied in a cell with a lithium counterelectrode. These measurements showed that the obtained porous silicon electrodes possess a high capacity for lithium insertion (up to 50 mAh/cm²) and admit more than 20 charge/discharge cycles.     

On-chip amperometric measurement of quantal catecholamine release using transparent indium tin oxide electrodes

Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis, but microfabricated devices offer the promise of higher throughput. Here we report development of a microchip device that uses transparent indium tin oxide (ITO) electrodes to measure quantal exocytosis from cells in microfluidic channels. ITO films on a glass substrate were patterned as 20-mum-wide stripes using photolithography and wet etching and then coated with polylysine to facilitate cell adherence. Microfluidic channels (100 mum wide by 100 mum deep) were formed by molding poly(dimethylsiloxane) (PDMS) on photoresist and then reversibly sealing the PDMS slab to the ITO-glass substrate. Bovine adrenal chromaffin cells were loaded into the microfluidic channel and adhered to the ITO electrodes. Cells were stimulated to secrete by perfusing a depolarizing "high-K" solution while monitoring oxidation of catecholamines on the ITO electrode beneath the cell using amperometry. Amperometric spikes with charges ranging from 0.1 to 1.5 pC were recorded with a signal-to-noise ratio comparable to that of carbon-fiber electrodes. Further development of this approach will enable high-throughput measurement of quantal catecholamine release simultaneously with optical cell measurements such as fluorescence.     

Neural cell growth on TiO2 anatase nanostructured surfaces

Titanium oxides have anti-inflammatory activity and tunable electrochemical properties that make them attractive materials for biomedical applications. This work investigated the compatibility of nanometric coatings of low-temperature phases of TiO₂ with neurons in 4-day and 10-day cultures, using different cell densities to quantify cell survival and neurite extension. TiO₂ films were prepared by sol–gel and thermal treatment (250–450 °C) of hydrolyzed titanium tetra-isopropoxide on electrically conducting or insulating slides. The conducting substrates were not passivated by the nanometric oxide layer and could be used as electrodes. Characterization of the films showed nano-structured TiO₂ containing exclusively Ti⁺⁴ valence in anatase and amorphous phases. When coated with polylysine, all films permitted good neuron attachment and survival for at least ten days in culture. However, they generally reduced neurite growth compared to cell culture borosilicate glass, with dendrites more affected than axons. The analyses of surface topography, hydrophilicity, charge and chemical composition revealed that TiO₂ chemistry was the main factor responsible for neurite inhibition.     

Deckschichtbildung und Korrosionsverhalten des hochsiliciumlegierten austenitischen Sonderstahls X 2 CrNiSi 18 15 in Salpetersäuren

The Formation of Surface Films and the Corrosion Resistance of the Silicon Containing Austenitic Steel X 2 CrNiSi 18 15 in Nitric Acids The oxide layers consist of two parts: the lower one is mainly Croxide, the upper one is SiO₂. ESCA and AAS measurements were made in order to study the formation of the surface layer on the austenitic steel X2 CrNiSi 18 15 in nitric acids. A film, which is chromium oxide, is formed first. On top of this film a second film consisting of SiO₂ grows. The elements nickel and manganese are not found in the formation of the oxide films. Immediately below the oxide layer the steel is enrichened with chromium and depleted of iron. In order to find the in some cases very small corrosion rates (5 · 10^?5 mm · a^?1) in a reproducible manner, the amounts of iron, chromium and nickel which had been dissolved were measured by means of the AAS method as a function of time. For stationary samples apparent activation energies of 65.2 kJ/Mol (azeotropic nitric acid) and 37.5 kJ/Mol (highly concentrated nitric acid), respectively, were found. These data confirm the assumption that the corrosion rate is determined by reactions at the phase boundaries. No appreciable influence of the flow velocity on the corrosion rate was detected.     

Effects of bioactive ceramics on the pathogenesis of rat vascular smooth muscle cells treated with phorbol 12-myristate 13-acetate

Vascular smooth muscle cells (VSMCs) play a pivotal role in vascular injury through proliferation and migration. Pro-inflammatory cytokines and cyclooxygenase (COX)-2 and nitric oxide synthase (NOS) are highly associated with the pathogenesis of VSMCs. We investigated the effect of bioactive ceramics on the expression of inflammatory cytokines, COX-2, and inducible NOS (iNOS) induced by phorbol 12-myristate 13-acetate (PMA) in rat VSMCs. The ceramics inhibited mRNA expression of IL-1β, TNF-α, IL-6, COX-2, and iNOS. Prostaglandin release was also diminished by the ceramics. The bioactive ceramics effect on cytokines, COX-2, and iNOS expression was achieved by inhibition of NF-κB activity. Interestingly, the ceramics-induced up-regulation of expression of endothelial NOS resulted in an increase of nitric oxide production. Thus, bioactive ceramics may have dual effects on the pathogenesis of VSMCs by regulation of NF-κB activity and NO production.     

Ratiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c' and the detection of extra- and intracellular macrophage nitric oxide.

Ratiometric and lifetime-based sensors have been designed for cellular detection of nitric oxide. These sensors incorporate cytochrome c', a hemoprotein known to bind nitric oxide selectively. The cytochrome c' is labeled with a fluorescent reporter dye, and changes in this dye's intensity or fluorescence lifetime are observed as the protein binds nitric oxide. The ratiometric sensors are composed of dye-labeled cytochrome c' attached to the optical fiber via colloidal gold, along with fluorescent microspheres as intensity standards. These ratiometric sensors exhibit linear response, have fast response times (< or = 0.25 s), and are completely reversible. The sensors are selective over numerous common interferents such as nitrite, nitrate, and oxygen species, and the limit of detection is 8 microM nitric oxide. The lifetime-based measurements are made using free, dye-labeled cytochrome c' in solution and have a limit of detection of 30 microM nitric oxide. The use of these two techniques has allowed measurement of intra- and extracellular macrophage nitric oxide. Employing the ratiometric fiber sensors gave a multicell culture average extracellular nitric oxide concentration of 210 +/- 90 microM for activated macrophages, while an average intracellular concentration of 160 +/- 10 microM was determined from the lifetime-based measurements of dye-labeled cytochrome c' in the macrophage cytosol. Microscopic adaptation of the lifetime-based methods described here would allow direct correlation of intracellular nitric oxide levels with specific cellular activities, such as phagocytosis.     

Bench-top method for fabricating glass-sealed nanodisk electrodes, glass nanopore electrodes, and glass nanopore membranes of controlled size

A simple benchtop method of fabricating glass-sealed nanometer-sized Au and Pt disk electrodes, glass nanopore electrodes, and glass nanopore membranes is reported. The synthesis of all three structures is initiated by sealing the tips of electrochemically sharpened Au and Pt microwires into glass membranes at the end of a soda lime or lead glass capillary. Pt and Au nanodisk electrodes are obtained by hand polishing using a high-input impedance metal oxide semiconductor field effect transistor (MOSFET)-based circuit to monitor the radius of the metal disk. Proper biasing of the MOSFET circuit, based on a numerical analysis of the polishing circuit impedance, allows for the reproducible fabrication of Pt disk electrodes of radii as small as 10 nm. Significantly smaller background currents in voltammetric measurements are obtained using lead glass capillaries, a consequence of the lower mobility of Pb(2+) (relative to Na(+)) in the glass matrix. Glass nanopore electrodes and glass nanopore membranes are fabricated, respectively, by removal of part or all of the metal sealed in the glass membranes. The nanostructures are characterized by atomic force microscopy, steady-state voltammetry, and ion conductivity measurements.     

Simultaneous electrochemical measurements of oxygen and dopamine in vivo.

Fast-scan cyclic voltammetry, a demonstrated analytical method for the in vivo detection of catecholamine neurotransmitters, is extended to the simultaneous determination of molecular oxygen (O2). Cyclic voltammograms were recorded at a scan rate of 400 V/s at carbon-fiber disk electrodes coated with a perfluorinated ion-exchange material. The peak current for O2 occurs near -1.2 V under these conditions. In flow-injection experiments, these electrodes respond to step changes in dopamine and O2 with a half-rise time of less than 200 ms. The voltammetric peak current is independent of flow rate, indicating a diffusion-limited response unaffected by convection. Several compounds present in the in vivo matrix (adenosine, glutathione, and NAD and glutamic, lactic, and uric acids) were tested and shown not to interfere with the voltammetric signal for O2. These electrodes maintain a stable response in vivo for at least 6 h. They have been used to measure transient increases in both dopamine and O2 in the extracellular fluid of the caudate nucleus of an anesthetized rat in response to an electrical stimulus.