Profiling H2O2 from single COS-7 cells by means of scanning electrochemical microscopy.

We report quantitative determination of extracellular H2O2 released from single COS-7 cells with high spatial resolution, using scanning electrochemical microscopy (SECM). Our strategy of depth scan imaging in vertical x-z plane was conveniently utilized to a single cell for obtaining probe approach curves (PACs) to any positions on the membrane of a live cell by simply drawing a vertical line on one depth SECM image. This SECM mode provides an efficient way to record a batch of PACs, and visualize cell topography simultaneously. The H2O2 concentration at the membrane surface in the center of an intact COS-7 cell was deconvoluted from apparent O2, and determined to be 0.020 mM by overlapping the experimental PAC with the simulated one having a known H2O2 release value. The H2O2 profile determined in this way gives insight into physiological activity of single live cells. In addition, intracellular H2O2 profile was demonstrated using confocal microscopy by labelling the cells with a luminomphore, 2',7'-dichlorodihydrofluorescein diacetate. The two methodologies have illustrated complementary experimental results of H2O2 detection, indicating that H2O2 generation is centered at endoplasmic reticula.

Analyzing the influence of alloying elements and impurities on the localized reactivity of titanium grade-7 by scanning electrochemical microscopy.

Scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX) was applied to investigate the grain boundaries on ASTM grade-7 titanium (Ti-7) with a freshly polished surface, and the results showed that the alloying element, Pd, and the impurity, Fe, cosegregated to grain boundaries. Scanning electrochemical microscopy (SECM) was used to study the variations in reactivity on Ti-7 exposed to an aerated neutral solution of 0.1 M NaCl. Locations that possessed an enhanced reactivity compared to the oxide-covered (TiO(2)) surface of the grains on SECM images were proposed to be the boundaries. These areas were further activated by the application of a cathodic bias, and interconnection of the active locations allowed the construction of "grain boundary maps". Variations in surface reactivity were quantitatively analyzed by fitting probe approach curves (PACs) to curves simulated with a model based on finite element analyses using the platform of COMSOL multiphysics software. The difference in reactivity between active grain boundaries and oxide-covered grains was up to a factor of 100 on freshly polished surfaces. This difference decreased to a factor of 10-15 after longer-term exposure of the Ti-7 to the aerated solution, indicating partial passivation of the Pd/Fe-stabilized beta-phase in the grain boundaries. PAC analyses of oxide-covered grains showed that the reactivity increased logarithmically as the bias potential to the Ti-7 was decreased, consistent with reduction of the insulating TiO(2) layer to a more conductive TiOOH layer.

Effects of cadmium on photosynthetic oxygen evolution from single stomata in Brassica juncea (L.) Czern.

Scanning electrochemical microscopy (SECM) was utilized to investigate photosynthetic oxygen evolution from single stomata in leaves of live Brassica juncea (L.) Czern cultured in nutrient solution to which 0.2 or 0.01 mM CdC12 had been added. The bulk leaf surface serves as an insulator normally; therefore, a typical negative feedback was observed on the probe approach curves (PACs) when the probe approached epidermal cells. When the probe tip approached an open stoma, a higher tip current was detected due to the O2 release from this stoma. Thus, SECM can be used to map the O2 concentration profile near the leaf surface and study stomatal complex structure size and density. The oxygen release from single stomata was also analyzed by comparison of experimental PACs with those simulated by COMSOL multiphysics software (version 3.4). In addition to an increase in the stomatal complex size and a decrease in the complex density, the Cd accumulation caused up to a 26% decrease in photosynthetic rate determined at the level of a single stoma. The O2 evolution was also monitored by recording the tip current vs time when a tip sat above the center of a stoma. Periodic peaks in O2 release-time curves were observed, varying from 400 to 1600 s. The opening and closing activities of single stomata were also imaged by SECM.

High performance liquid chromatographic determination of platinum in blood and urine samples of cancer patients after administration of cisplatin drug using solvent extraction and N,N'-bis(salicylidene)-1,2-propanediamine as complexation reagent.

A high performance liquid chromatographic (HPLC) procedure has been developed for the determination of cisplatin, based on the pre-column derivation of platinum(II) with reagent N,N'-bis(salicylidene)-1,2-propanediamine (H2SA2pn). The neutral platinum complex was extracted, concentrated in an organic solvent and then injected (5 microl) on a reverse phase HPLC column, Varian Micro-Pak SP C-18, 5 microm (150 mm x 4.0 mm i.d.). The complex was eluted isocratically using a ternary mixture of methanol/acetonitrile/water (40/30/30, v/v/v) at a flow rate of 1.0 ml/min and was determined by a UV detector set at 254 nm after elution. A detection limit was found to be 4.0 ng per injection. The amounts of platinum in blood serum and urine of cancer patients after administration of cisplatin were observed in a range of 221-298 ng/ml and 43-97 ng/ml with relative standard deviation (R.S.D.) of 3.6-4.6% and 3.5-4.8%, respectively. Preliminary metabolism profiles of Pt concentrations in blood and urine from the patients were established.

Cadmium-induced plant stress investigated by scanning electrochemical microscopy.

In vivo oxygen evolution above single stomata in Brassica juncea has been used to investigate, for the first time, the effect of Cd-induced stress as imaged by scanning electrochemical microscopy (SECM). SECM images showed a clear stomatal structure-a pore, whose aperture is modulated by two guard cells, serving as the conduit for the oxygen produced. Lower stomatal density and larger stoma size were found in plants treated with 0.2 mM CdCl2 compared with control plants. Either the introduction of Cd caused a slower cell replication in the plane of the epidermis, hence fewer stomata, and/or the number of open stomata was reduced when plants were under Cd-stress. Oxygen evolution above individual stomatal complexes in Cd-treated plants was lower than that from control plants, as determined from the electrochemical current above the middle of each stoma. All guard cells under illumination were swollen, indicating that the stomata were open in both control and treated plants. Thus, decreased oxygen evolution in response to Cd cannot be attributed to simple closing of the stomata, but to a lower photosynthetic yield. SECM provides an excellent tool for monitoring the effects of Cd on photosynthetic activity at the scale of individual stomata.