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Its relevance has been leading to the development of different analytical methodologies to assess the temporal and spatial fluxes of NO under the complex biological milieu. Third\u2010generation electrochemical biosensors are promising tools for <jats:italic>in loco<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> NO quantification and, over the past years, heme proteins and porphyrins have been used in their design. Since there are some limitations with the biorecognition element directly adsorbed onto the electrode surface, nanomaterials (carbon nanotubes, gold nanoparticles, etc.) and polymers (cellulose, chitosan, nafion\u00ae, polyacrylamide, among others) have been explored to achieve high kinetics and better biosensor performance. 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