Electrochemical high-content screening of nitric oxide release from endothelial cells
Abstract
Release of nitric oxide (NO) is of high importance for regulating endothelial cell functions during vasodilatation, vascular remodeling, and angiogenesis. Thus, a direct and reliable real-time method for NO detection that takes into account time-dependent variations of the NO concentration in the complex reaction within the diffusion zone above the cells is vital for obtaining information about the role of NO in intracellular endothelial signal transduction and its impact on the surrounding cells. In this study, the time course of vascular endothelial growth factor E (VEGF-E) stimulated NO release from transformed human umbilical vein endothelial cells (T-HUVEC) was investigated by means of metalloporphyrin-based NO sensors employed in an electrochemical robotic system. The NO sensor was obtained by electrochemically induced deposition of Ni(II) tetrakis(p-nitrophenylporphyrin) on a 50-microm diameter platinum disk electrode which was integrated, together with a 25-microm diameter platinum disk, in a double-barrel electrode arrangement. The second electrode was used as a guidance sensor for the automatic and highly reproducible positioning of the NO sensor at a known distance from a layer of adherently growing cells by using z-approach curves in the negative feedback mode of scanning electrochemical microscopy (SECM). The electrochemical robotic system allows the fully automated detection of NO with high sensitivity and selectivity to be performed in real time within 96-well…
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