Selenium-based electrocatalytic sensors for sensitive peroxynitrite detection in biological media: a bottom-up approach for functional interface design

项目摘要： 生物学背景和挑战：过氧亚硝酸盐（OONO-）是一种强效细胞毒性化合物 与一系列病理生理状况有关过氧亚硝酸盐是 一氧化氮与超氧阴离子自由基的体内反应。多方面的生理反应， 该化合物直接涉及许多病理学，包括免疫应答、慢性 炎症、败血症和心血管疾病，仅举几例。仅仅是心血管疾病 每4分钟约有7人死亡，败血症每年影响美国170万成年人， 导致超过25万人死亡将过氧亚硝酸盐与所有引用的 病理学是其对大多数细胞成分的有效反应性，包括DNA，蛋白质和脂质， 细胞膜蛋白质、DNA和脂质的大量氧化和其他转化有助于 关键细胞功能的破坏。 评估过氧亚硝酸盐的有害影响和检查其潜在假设的任务 信号角色是非常困难的。主要原因是准确测量和监测的方法 由于在生理条件下低亚微摩尔浓度 再加上它的高反应性。过亚硝酸根的电化学检测是一种简单， 更方便的技术应用于生物环境。然而，系统的发展 缺乏增强该分子的灵敏度和选择性的正确电极界面。在 我们正在进行的工作是开发用有机硒化物修饰的电极界面， 到表面，作为敏感和选择性PON电催化测定的催化实体。 本行政补充文件的要求： 申请资金用于购买电化学扫描探针显微镜设备， 对于完成当前NIH项目的具体目标2和具体目标3的各个方面至关重要。的 设备型号为ElProScan ELP电化学扫描显微镜及相关配件。 SECM设备是一种独特的扫描探针显微镜系统， 超微型电极作为扫描探针，以便以高空间分辨率和精确度测量局部 在感兴趣的衬底（在这种情况下，衬底中的衬底）上的电流测量（活性/电子交换） 活单细胞的脂质体形式，其在刺激时产生过氧亚硝酸盐）。 该请求是为了在目前NIGMS资助的赠款范围内支持工作： 所要求的设备将用于在该区域生成高分辨率地形图像。 过氧亚硝酸盐释放导致的电化学活性形式（参见随附的2页 研究策略）。该仪器将以多种方式使用：1）它将用于测试小型化 传感器的基础上最活跃的硒化物在这项工作中开发。2)该设备还将用于 测试最佳传感器，用于活单细胞上的PON测量。3)我们还将使用 仪器上的细胞集合。所有这些方法都将支持具体目标2的各个方面， 具体目标3如研究策略部分所述。 我们要求的电化学扫描探针显微镜是兼容的，将安装在我们的 Olympus倒置显微镜这将使我们能够轻松、可重复地验证 我们的微型硒基过氧亚硝酸盐传感器对生物源的性能 过氧亚硝酸盐。该设置将允许我们同时进行扫描和电流测量， 在生物材料上具有高的时间和空间分辨率，例如活的单细胞和 细胞以及细胞样的脂质膜（NOS-负载脂质体），如我们在我们的母体NIH中所描述的 正在进行的项目。

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.