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 N(2), 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 microM(-1)) compared to bare Pt (1.08 +/- 0.20 nA microM(-1)) and negligible interference from a wide range of endogenous electroactive interferents such as ascorbic acid, dopamine and its metabolites, NO(2)(-) and H(2)O(2). 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 degrees 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 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(5/2) sensor implanted in the striatum of freely-moving rats, thus supporting reliable in vivo recording of NO.

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Various Nafion ® coating procedures were examined in order to design a simple and reproducible coating method to maximise permselective characteristics, and thus eliminate signals from electroactive interferents, in sensors designed for direct in vivo measurements in the brain. Interferents investigated included ascorbic acid (AA), the principal endogenous electroactive interferent present in the brain, and uric acid. Application of the Nafion ® (5% commercial solution) using a thermally annealing procedure involving 5 pre-coats, and 2 subsequent dip-bake layers resulted in elimination of interferent signals. It also produced complete blocking of the signal for the neurotransmitter dopamine. The optimum time and temperature for annealing was found to be 5 min at 210 °C. An examination of shelf life over two weeks indicated negligible AA interference over this period. Preliminary investigations with respect to the potential use of these Nafion ® -modified Pt electrodes in the design of implantable, first generation, peroxide detecting biosensors indicated that the modified electrode had no effect on O 2 permeability but did produce a significant decrease in H 2 O 2 sensitivity. While this may preclude their use in biosensor development they may be more suitable for detection of gaseous neurochemicals such as nitric oxide.

The increasing scientific interest in nitric oxide (NO) necessitates the development of novel and simple methods of synthesising NO on a laboratory scale. In this study we have refined and developed a method of NO synthesis, using the neutral Griess reagent, which is inexpensive, simple to perform, and provides a reliable method of generating NO gas for in-vivo sensor calibration. The concentration of the generated NO stock solution was determined using UV-visible spectroscopy to be 0.28±0.01 mmol L À1 . The level of NO 2 À contaminant, also determined using spectroscopy, was found to be 0.67±0.21 mmol L À1 . However, this is not sufficient to cause any considerable increase in oxidation current when the NO stock solution is used for electrochemical sensor calibration over physiologically relevant concentrations; the NO sensitivity of bare Pt-disk electrodes operating at +900 mV (vs. SCE) was 1.08 nA lmol À1 L, while that for NO 2 À was 5.9·10 À3 nA lmol À1 L. The stability of the NO stock solution was also monitored for up to 2 h after synthesis and 30 min was found to be the time limit within which calibrations should be performed.