Synthetic Melanin Films as Potential Interfaces for Peroxynitrite Detection and Quantification

Peroxynitrite (PON) is a highly reactive oxygen-nitrogen species that facilitates both oxidation and nitration reactions. PON is the primary product of the reaction of superoxide anion-radical with nitric oxide radical. It has been well established that PON is a major noxious molecule, which is implicated in a host of pathophysiological conditions. Recently, PON has emerged as a new member of the nitroxidative array of reactive metabolites. Early clinical reports have emphasized the deleterious physiological reactivity of PON with many cellular targets including DNA, proteins, and lipids at cell/tissue levels. Melanin is a natural pigment that has many physiologic functions including the neutralization of highly reactive oxidative species. Tyrosine and its derivative 5,6-dihydroxyindole (DHI) are precursors of eumelanin, a brown or black forms of melanin that is also photo-stable. Recent studies have suggested that melanin can act as antioxidative therapy to scavenge reactive oxygen and nitrogen species including PON. In this project, we examine the chemical interaction between synthesized PON and polymerized films of DHI as a model of synthetic melanin. First, we studied the electrochemical characteristics of polymerized 5,6-dihydroxyindole on graphite electrodes, and then monitored the changes after adding aliquots of PON. This part of the work reports primarily on chemical changes within the electro-polymerized films of melanin on the electrode. We also studied the rates of chemical decay of PON in the presence of the transparent Indium Tin Oxide (ITO) electrodes coated with melanin films using absorbance spectroscopy. Ultraviolet-visible spectroscopy showed a significant difference between the decomposition rates of PON alone and in the presence of DHI films. The melanin films were characterized with Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) to image and determine the surface chemical composition of the synthetic film. X-ray Photoelectron Spectroscopy (XPS) has been used to detect and analyze the change on the synthetic melanin films coated ITO slides before and after exposure to PON. Finally, we report on the possibility of using DHI films as a platform for the quantitative detection of PON in solutions using amperometry and impedance