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

A Nafion (R)(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 N-2, both of which reduced the NO signal to baseline levels. Results indicated enhanced NO sensitivity at the Nafion (R)(5/2)sensor (1.67 +/- 0.08 nA mu M-1) compared to bare Pt (1.08 +/- 0.20 nA mu M-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 (R) layers was reduced to 2/1 and an insulating outer layer of poly(o-phenylenediamine) added. When tested under physiological conditions of 37 degrees C the response time of the Nafion (R)(5/2) sensor improved to 14.00 +/- 2.52 s. In addition, the NO response was not affected by physiological concentrations of O-2 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 (R)(5/2) sensor implanted in the striatum of freely-moving rats, thus supporting reliable in vivo recording of NO.