Nitric oxide synthase in confined environments: Detection and quantification of nitric oxide released from cells and modified liposomes using a sensitive metal catalyst-PEDOT modified carbon fiber electrode.

摘要

Nitric oxide (NO) is a freely diffusible, gaseous free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response, and inflammatory response. NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. A deficiency in NO plays a role in hypertension, hyperglycaemia, and arteriosclerosis, among other pathological states. Conversely, increased NO levels contribute to arthritis, septic shock, and hypotention. Therefore, measuring and quantifying NO production in biological systems and matrices may be vital in elucidating physiological and pathological processes. The goal of this work is to develop an ultra-sensitive and selective electrochemical sensor taking advantage of NO-sulfur chemistry. In particular, electropolymerizing 3,4-ethylenedioxythiophene (EDOT) monomers on the surface of our electrodes yield a suitable sulfur-based polymer PEDOT to be used as an affinity matrix for NO sensing. In other work, we have shown that the ruthenium (Ru) mediates the catalytic oxidation of NO. In this work, we tried to achieve improved sensitivity by combining both Ru nanoparticles and PEDOT using the layer-by-layer (LBL) modification method. Further, to eliminate interferences the Ru-PEDOT-Ru modified carbon fiber was coated with a nafion layer, which acts as an anionic filter. We used our NO-sensor to accurately monitor NO release from mouse embryonic fibroblast cells as well as isolated single human umbilical vein endothelial cells.;A second part of this work focused on testing the performance of our sensors in characterizing NO release from liposomes with confined NOS enzyme. Liposomes are spherical, closed, self-assembled phospholipids, which enclose part of the surrounding solvent in their interior. Liposomes can enclose an aqueous medium separate from the external aqueous medium. Therefore, liposomes can be used as carriers of enzymes (NOS in this case) without negative impact on the molecular function of the encapsulated biomolecule and can be considered as a promising way to transport and deliver NO to targets. We prepared NOS-loaded liposomes and characterized encapsulation efficiency of various procedures. We also tested the performance of our NO-sensors to monitor and quantify NO release from NOS-loaded liposomes as potential vehicles for on-site NO delivery.