Nitric oxide monitoring in brain extracellular fluid: characterisation of Nafion^®-modified Pt electrodes in vitro and in vivo

A Nafion^®(5 pre-coats/2 dip-coats)-modified Pt sensor developed for real-time neurochemical monitoring has now been characterised in vitro for the sensitive and selective detection of nitric oxide (NO). A potentiodynamic profile at bare Pt established +0.9 V (vs. SCE) to be the most appropriate applied potential for NO oxidation. The latter was confirmed using oxyhaemoglobin and N₂, both of which reduced the NO signal to baseline levels. Results indicated enhanced NO sensitivity at the Nafion^®(5/2) sensor (1.67 ± 0.08 nA µM⁻¹) compared to bare Pt (1.08 ± 0.20 nA µM⁻¹) and negligible interference from a wide range of endogenous electroactive interferents such as ascorbic acid, dopamine and its metabolites, NO₂⁻ and H₂O₂. The response time of 33.7 ± 2.7 s was found to improve (19.0 ± 3.4 s) when the number of Nafion^® layers was reduced to 2/1 and an insulating outer layer of poly(o-phenylenediamine) added. When tested under physiological conditions of 37 °C the response time of the Nafion^®(5/2) sensor improved to 14.00 ± 2.52 s. In addition, the NO response was not affected by physiological concentrations of O₂ despite the high reactivity of the two species for each other. The limit of detection (LOD) was estimated to be 5 nM while stability tests in lipid (phosphatidylethanolamine; PEA) and protein (bovine serum albumin; BSA) solutions (10%) found an initial ca. 38% drop in sensitivity in the first 24 h which remained constant thereafter. Preliminary in vivo experiments involving systemic administration of NO and L-arginine produced increases in the signals recorded at the Nafion^®(5/2) sensor implanted in the striatum of freely-moving rats, thus supporting reliable in vivo recording of NO.