Amperometric determination of nitric oxide derived from pulmonary artery endothelial cells immobilized in a microchip channel

Abstract

A simple method for immobilizing a confluent layer of bovine pulmonary artery endothelial cells (bPAECs) in microchip-based channels is described. The microchips are prepared from poly(dimethylsiloxane) and have channel dimensions that approximate resistance vessels in vivo. The reversibly sealed channels were coated with fibronectin (100 µg ml⁻¹) by aspiration. The bPAECs, which were introduced in the same manner, became attached to the fibronectin coating in about 2 h. The microchip could then be resealed over a micromolded carbon ink electrode (24 µm width × 6 µm height). Coating the carbon microelectrode with a 0.05% Nafion solution selectively blocked nitrite (10 µM) from being transported to the electrode surface while nitric oxide (NO, 10 µM) was amperometrically measured. Upon stimulation with adenosine triphosphate (ATP, 100 µM) the immobilized bPAECs produced and released micromolar amounts of NO. This NO production was effectively inhibited when the immobilized cells were incubated with L-nitro-arginine methyl ester (L-NAME), a competitive inhibitor for nitric oxide synthase. Moreover, once the immobilized bPAECs were no longer able to produce NO, incubation with L-arginine allowed for further ATP-stimulated NO production.