Direct detection of S-nitrosothiols using planar amperometric nitric oxide sensor modified with polymeric films containing catalytic copper species

The direct amperometric detection of S-nitrosothiol species (RSNOs) is realized by modifying a previously reported amperometric nitric oxide gas sensor with thin hydrophilic polyurethane films containing catalytic Cu(II)/(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of S-nitrosothiols generates NO(g) in the thin polymeric film at the distal tip of the NO sensor. Three different species are examined to create the catalytic layer: (1) a lipophilic Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and (3) small (3-mu m) metallic Cu-0 particles. All three catalytic layers yield reversible amperometric response in proportion to the concentration of S-nitrosothiols (e.g., nitrosocysteine, nitrosoglutathione, S-nitroso-N-acetylcysteine, S-nitrosoalbumin) present in the aqueous test solution. Sensitivity toward the different RSNO species is dependent on the respective catalytic rates of decomposition of the RSNO species by reactive Cu(I), accessibility of the species into the polyurethane layer containing the catalyst, the level of reducing agents (ascorbate) used in solution to help generate reactive Cu(I) species, and the concentration of metal ion complexing agents present in the test solution (e.g., EDTA). Under optimized conditions, all RSNO species can be detected at <= 1 mu M levels, with sensor lifetimes of at least 10 days for the sensors based on Cu(II)-phosphate and Cu-0 particles. It is further shown that the new RSNO sensors can be used to assess the NO-generating ability of fresh blood samples by effectively detecting the total level of reactive RSNO species present in such samples.