Project Summary: Background and Challenge: Peroxynitrite (OONO-) emerged as a potent cytotoxic compound and has been implicated in a host of pathophysiological conditions. Peroxynitrite is the primary product of the in vivo reaction of nitric oxide and superoxide anion-radical. The multifaceted physiologic reactions of this compound are directly implicated in a number of pathologies including cardiovascular disease, immune response, chronic inflammation, and sepsis, to cite a few. According to recent statistics by the American Heart Association, just cardiovascular disease alone claims about 7 deaths every 4 minutes. On the other hand, sepsis affects 1.7 million adults in the United States each year and potentially contributes to more than 250,000 deaths. Just these two statistics are staggering and make the footprint of this deadly biological analyte an important priority. The common thread that links peroxynitrite to all cited pathologies is its potent reactivity toward most cellular components including DNA, proteins, and lipids in cell membranes. Substantial oxidations and other transformations of proteins, DNA, and lipids contribute to the disruption of key cellular functions. Assessing peroxynitrite’s deleterious effects and examining hypotheses of its potential signaling roles cannot be achieved without first accurately measuring and monitoring its concentration. This task is however inherently difficult due to low submicromolar concentrations under physiologic conditions coupled with its high reactivity. Sensitive and accurate measurement of peroxynitrite is crucial in order to shed light on the illusive pathophysiologic roles of this metabolite. Some of the known detection methods for peroxynitrite include oxidation of fluorescent probes, EPR spectroscopy, chemiluminescence, immunohistochemistry, and probe nitration; however, these are more difficult to apply for real-time quantification due to their inherent complexity. The electrochemical detection of peroxynitrite is a simpler and more convenient technique for application in biological settings. However, a systematic development of the right electrode interface that enhances the sensitivity and selectivity for this molecule is lacking. Recently, several synthetic organic selenides have been prepared as antioxidants in medicinal chemistry. Electrochemical data in our hands showed that some organoselenium compounds have specific redox activity with peroxynitrite in solution. For these reasons, we believe that an electrode interface decorated with organoselenides attached to the surface will potentially serve as catalytic entities for mediated PON electrocatalytic determination. Our proposal: In this work, we propose to develop a functional thin film material based on defined organic selenides chemically attached on graphite electrodes and use this interface in sensitive electrochemical determination of peroxynitrite. This bottom-up interface design approach is innovative because it allows us to design an electrocatalytic interface for the detection and determination of peroxynitrite driven by molecular and electronic properties of the organic selenides used. This is driven by the overall hypothesis that the redox-rich organoselenium compounds will allow us to use them as active redox catalytic centers tethered to the electrode surface to electrocatalytically measure PON in solution. The work will pursue three specific aims including: 1) developing a selenide-decorated electrode for PON determination using a reference compound, followed by 2) generating a library of selenides with varying substituents on the selenium catalytic center and test the catalytic properties of the resulting modified interface towards PON determination; and finally 3) miniaturizing the best performing catalytic interfaces by transferring the process to ultramicroelectrodes (carbon fiber) to prepare a single-body PON microsensor for use under biological settings. Significance: The successful development of a reliable PON microsensor will not only enable in-situ measurement of this reactive stress marker under biological setting but will also shed light on obscure mechanisms through which this potent species operates under many disease states.

Project Narrative: Peroxynitrite is a biological metabolite that is physiologically active and extremely toxic. It is involved in the progression of a number of diseases with high morbidity and mortality rates, including cardiovascular disease which is still the number 1 killer disease in the United States. Understanding the clinical footprint of this molecule is not fully possible without having a reliable tool to measure its small and changing concentrations under biological settings. Our long-term goal in this proposal is to develop a peroxynitrite sensor that can be deployed to measure transient changes of the concentration of this metabolite in biological systems in order to be able to understand and shed light on the far-reaching clinical implications of this toxic metabolite.