Vesicle Collision Protocols for the Study of Quantum Size and Exocytotic Fraction Released.

We review the methods of vesicle impact electrochemical cytometry, intracellular impact electrochemical cytometry, and single cell amperometry and their application to measuring the storage of neurotransmitters in cellular vesicles. We provide protocols to measure vesicle content, the release of catecholamines, and from there the fraction of transmitter released in each exocytosis event. The focus here has been a combination of methods to evaluate factors related to neuronal function at the cellular level and implications in, for example, cognition.

Monitoring H2O2 inside Aspergillus fumigatus with an Integrated Microelectrode: The Role of Peroxiredoxin Protein Prx1.

Peroxiredoxins (Prx) are important proteins involved in hydroperoxide degradation and are related to virulence in several pathogens, including Aspergillus fumigatus. In this work, in vivo studies on the degradation of hydrogen peroxide (H2O2) in the microenvironment of A. fumigatus fungus were performed by using an integrated Pt microelectrode. Three A. fumigatus strains were used to confirm the role of the cytosolic protein Prx1 in the defense mechanism of this microorganism: a wild-type strain, capable to expressing the protein Prx1; a Δprx strain, whose gene prx1 was removed; and a genetically complemented Δprx1::prx1+ strain generated from the Δprx1 and in which the gene prx1 was reintroduced. The fabricated microelectrode was shown to be a reliable inert probe tip for in situ and real-time measurements of H2O2 in such microenvironments, with potential applications in investigations involving the mechanism of oxidative stress.

In-vivo electrochemical monitoring of H2O2 production induced by root-inoculated endophytic bacteria in Agave tequilana leaves.

A dual-function platinum disc microelectrode sensor was used for in-situ monitoring of H2O2 produced in A. tequilana leaves after inoculation of their endophytic bacteria (Enterobacter cloacae). Voltammetric experiments were carried out from 0.0 to -1.0V, a potential range where H2O2 is electrochemically reduced. A needle was used to create a small cavity in the upper epidermis of A. tequilana leaves, where the fabricated electrochemical sensor was inserted by using a manual three-dimensional micropositioner. Control experiments were performed with untreated plants and the obtained electrochemical results clearly proved the formation of H2O2 in the leaves of plants 3h after the E. cloacae inoculation, according to a mechanism involving endogenous signaling pathways. In order to compare the sensitivity of the microelectrode sensor, the presence of H2O2 was detected in the root hairs by 3,3-diaminobenzidine (DAB) stain 72h after bacterial inoculation. In-situ pH measurements were also carried out with a gold disc microelectrode modified with a film of iridium oxide and lower pH values were found in A. tequilana leaves treated with bacteria, which may indicate the plant produces acidic substances by biosynthesis of secondary metabolites. This microsensor could be an advantageous tool for further studies on the understanding of the mechanism of H2O2 production during the plant-endophyte interaction.

The use of a gold disc microelectrode for the determination of copper in human sweat.

A novel approach of using a gold disc microelectrode to analyze sweat samples for copper ions by anodic square wave stripping voltammetry (SW stripping voltammetry) is described. Sweat was collected from the lower back of four subjects after physical exercise and the sample volume required for the determinations was 100 µL. Under the optimized conditions, the calibration plot was linear over the range 1-100 µmol L(-1) Cu(II) with a limit of detection of 0.25 µmol L(-1). The precision was evaluated by carrying out five replicate measurements in a 1 µmol L(-1) Cu(II) solution and the standard deviation was found to be 1.5%. Measurements were performed by inserting the microelectrode into sweat drops and Cu(II) concentrations in the analyzed samples ranged from 0.9 to 28 µmol L(-1). Values obtained by the proposed voltammetric method agreed well with those found using graphite furnace atomic absorption spectroscopy (GFAAS).

Voltammetric performance and application of a sensor for sodium ions constructed with layered birnessite-type manganese oxide.

The preparation and electrochemical characterization of a carbon paste electrode modified with layered birnessite-type manganese oxide for use as a sodium sensor is described. The effects of powder synthesis process (sol-gel and redox precipitation) for birnessite on the electrochemical activity of the sensor was investigated by cyclic voltammetry. The carbon paste electrode modified with birnessite-type manganese oxide that was synthesized by the sol-gel method showed a best electrochemical for sodium ions. The detection is based on the measurement of anodic current generated by oxidation of Mn(III) to Mn(IV) at the surface of the electrode and consequently the sodium ions extraction into the birnessite structure. The best voltammetric response was obtained for an electrode composition of 15% (w/w) birnessite oxide in the paste, a TRIS buffer solution of pH 8.0 and a scan rate of 50 mV s(-1). A sensitive linear voltammetric response for sodium ions was obtained in the concentration range of 7.89x10(-5) to 3.49x10(-4) mol L(-1) with a slope of 37.5 microA L mmol(-1) and a detection limit (3sigma/slope) of 3.43x10(-5) mol L(-1) using cyclic voltammetry. Under the working conditions, the proposed method was successfully applied to determination of sodium ions in urine samples.

An Electrochemical Sensor Based on Nanostructured Hollandite-type Manganese Oxide for Detection of Potassium Ions.

The participation of cations in redox reactions of manganese oxides provides an opportunity for development of chemical sensors for non-electroactive ions. A sensor based on a nanostructured hollandite-type manganese oxide was investigated for voltammetric detection of potassium ions. The detection is based on the measurement of anodic current generated by oxidation of Mn(III) to Mn(IV) at the surface of the electrode and the subsequent extraction of the potassium ions into the hollandite structure. In this work, an amperometric procedure at an operating potential of 0.80 V (versus SCE) is exploited for amperometric monitoring. The current signals are linearly proportional to potassium ion concentration in the range 4.97 × 10(-5) to 9.05 × 10(-4) mol L(-1), with a correlation coefficient of 0.9997.