SYNTHESIS OF NITROXIDES FOR USE AS PROCATIONIC LABELS AND THEIR INCORPORATION INTO NAFION FILMS

A nitroxide label was covalently attached to amphetamine and two antidepressants, nortriptyline and desipramine, by forming an amide linkage between the amino group on each of these compounds and 3-carboxyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy. This nitroxide label was also attached to be biologically important compound biotin, but a spacer group, N,N'-dimethyl-1,6-hexanediamine, was needed to make two amide linkages between the carboxylic acids on the nitroxide and biotin. These nitroxide-labeled substances undergo a one-electron reversible oxidation between 0.5 and 0.6 V (vs. Ag/AgCl) at a naked glassy carbon (GC) elecrode and between 0.3 and 0.4 V at a GC electrode coated with a polyanionic film of Nafion. When a 0.8-V potential is applied to a GC/Nafion electrode in the presence of an aqueous buffer solution of one of these nitroxides, the nitroxide preconcentrates in the film in its oxidized form, the oxoammonium cation. Subsequent scanning of the potential in the negative direction using square wave voltammetry produces a reduction wave with a relatively large peak current making it possible to detect almost nanomolar quanitities of the procationic nitroxide. This preconcentration of procationic nitroxides makes this redox label useful in a recently developed analytical technique that combines immunoassay with Nafion-modified electrodes.