Project Summary: Biological Background and the Challenge: Peroxynitrite (OONO-) is a potent cytotoxic compound that 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, immune response, chronic inflammation, sepsis, and cardiovascular disease, to cite a few. just cardiovascular disease alone claims about 7 deaths every 4 minutes and sepsis affects 1.7 million adults in the United States each year and contributes to more than 250,000 deaths. 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. The task of assessing peroxynitrite’s deleterious effects and examining hypotheses of its potential signaling roles is very difficult. The primary reason is that methods for measuring and monitoring accurately its concentration are inherently difficult due to low submicromolar concentrations under physiologic conditions coupled with its high reactivity. 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. In the ongoing work of the parent grant we develop electrode interfaces decorated with organoselenides attached to the surface that serve as catalytic entities for sensitive and selective PON electrocatalytic determination. The request of this Administrative Supplement: Funds are requested to purchase an Electrochemical Scanning Probe Microscope (SECM) Equipment that is critical for the completion of aspects of specific Aims 2 and Specific Aim3 of the current NIH project. The equipment model is the ElProScan ELP electrochemical scanning microscope and related accessories. The SECM equipment is a unique scanning probe microscope system that uses micro- and ultramicrolectrodes as scanning probes in order to measure with high spatial resolution and accuracy local current measurement (activity/election exchange) over substrates of interest (in this case substrates in the form of liposomes of live single cells that generate peroxynitrite upon stimulation). The Request is to Support Work Well Within the Scope of the Current NIGMS-funded Grant: The requested equipment will be used in a manner to generate high-resolution topographic imaging in the form of electrochemical activity as a result of peroxynitrite release (see the accompanying 2-page Research Strategy). The instrument will be used in a variety of ways: 1) it will be used to test miniaturized sensors based on the most active selenides as developed in this work. 2) The equipment will also be used to test the most optimal sensors for PON measurement over live single cells. 3) We will also use the instrument over collections of cells. All of these ways will support aspects of Specific Aim2 and particularly Specific Aim3 as explained in Research Strategy section. The electrochemical scanning probe microscope that we request is compatible and will be mounted on our existing Olympus inverted microscope. This will allow us to easily and reproducibly validate the performance of our miniaturized selenide-based peroxynitrite sensors on biological sources of peroxynitrite. The setup will allow us to carry out scanning and current measurement at the same time and with high temporal and spatial resolution on biological materials such as live single cells and collections of cells as well lipid membranes of cell-like (NOS-Loaded Liposomes) as we described in our parent NIH project that is underway.

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. This supplement is to acquire an electrochemical scanning probe microscope that will be used to validate and use miniaturized peroxynitrite sensors to detect oxidative stress marker at the level of live single cells.