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

项目总结： 生物学背景和挑战：过氧亚硝酸盐(Oono-)是一种有效的细胞毒性化合物 这与一系列的病理生理状况有关。过氧亚硝酸盐是亚硝酸根的主要产品 体内一氧化氮和超氧阴离子自由基的反应。多方面的生理反应 这种化合物直接与许多病理有关，包括免疫反应、慢性 炎症、败血症和心血管疾病，仅举几例。仅心血管疾病就声称 在美国，每年每4分钟约有7人死亡，脓毒症影响170万成年人， 造成超过25万人死亡。将过氧亚硝酸盐与所有引用的 病理是它对大多数细胞成分的强烈反应，包括DNA，蛋白质和脂类 细胞膜。蛋白质、脱氧核糖核酸和脂类的大量氧化和其他转化起了作用。 对关键细胞功能的破坏。 评估过氧亚硝酸盐的有害影响和检验其潜在可能性的假设的任务 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 由于生理条件下亚微摩尔浓度低，其浓度自然很难达到。 条件加上它的高反应性。过氧亚硝酸盐的电化学检测是一种更简单、更简单的方法 更方便的技术在生物环境中的应用。然而，一个系统的发展 缺乏增强该分子灵敏度和选择性的右电极界面。在 在母公司的资助下，我们开发了用有机硒化合物装饰的电极接口 作为催化实体的表面，用于灵敏和选择性的PON电催化测定。 应本行政副刊的要求： 需要资金购买电化学扫描探针显微镜(SECM)设备， 对于完成目前国家卫生研究院项目的具体目标2和具体目标3至关重要。这个 设备型号为ElProScan ELP电化学扫描显微镜及相关附件。 SECM设备是一种独特的扫描探针显微镜系统，它使用了显微和 超微电极作为扫描探头，实现高空间分辨率和高精度的局部测量 感兴趣底物(本例中为底物)上的电流测量(活度/电子交换) 在刺激下产生过氧亚硝酸根的活单细胞脂质体的形式)。 该请求是为了在目前NIGMS资助的赠款范围内很好地支持工作： 所要求的设备将用于生成高分辨率地形成像 过氧亚硝酸盐释放导致的电化学活动形式(见所附2页 研究战略)。该仪器将以多种方式使用：1)它将用于测试微型化 基于本工作中开发的最活跃的硒化物的传感器。2)设备也将被使用 测试用于在活的单细胞上测量PON的最佳传感器。3)我们还将使用 在细胞集合上使用仪器。所有这些方式都将支持特定AIM2的各个方面，特别是 具体目标3，如研究战略部分所述。 我们要求的电化学扫描探针显微镜是兼容的，并将安装在我们的 现有奥林巴斯倒置显微镜。这将使我们能够轻松且可重复地验证 我们的小型化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，并 具有高时间和空间分辨率的生物材料，如活的单细胞和 细胞以及我们在母公司NIH中描述的细胞样脂膜(加载一氧化氮合酶的脂质体) 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.