Determination of NO production in melanoma cells using an amperometric nitric oxide sensor

This report describes the development of a simple, easy-to-fabricate, sensitive, and stable amperometric nitric oxide sensor. The sensor is based on metal-phthalocyanine mediators that are immobilized on a glassy carbon, Nafion(TM)-modified electrode. The detection of NO is based on the metal-phthalocyanine catalysis of NO oxidation at an anodic potential of +0.8 V. Measurements were performed with electrodes that had been modified using Co-, Mn-, Fe-, Cu-, or Ni-phthalocyanine or with metal-free, H-phthalocyanine. Comparing the responses to NO addition of the different metal-phthalocyanine-modified electrodes revealed that the Ni complex showed the highest catalytic activity. The activity of the Fe-phthalocyanine complex was high but less then that of the Ni complex. Mn- and Co-phthalocyanine were much less active. In a set of control experiments in which the phthalocyanine had been replaced by a metal-free, H-substituted phthalocyanine the response to NO addition was very low, clearly indicating that the metal center of the phthalocyanine is involved in the catalysis. Mediators were attached to the electrodes by solvating the mediator in organic solvent and then dropping the solution onto glassy carbon electrodes. The electrodes were then covered with a layer of Nafion(TM), which serves as a protective and permselective membrane. The Ni-phthalocyanine electrode was used to monitor NO production by the enzyme NO synthase and by the melanoma M2R cell line. The results suggest that the sensor can not only detect NO that is formed by the enzyme NO synthase, the producer of NO in-vivo, but also detect NO formed in-situ.