Nanodiamond Quantum Sensors for Free Radical Detection

摘要 活性氧物种(ROS)是人类健康的关键介质，但当调控不当时，可能会导致 许多疾病(如心血管疾病、帕金森氏病、阿尔茨海默病、癌症、 唐氏综合征、白内障、几种神经系统疾病等)。而ROS的生物效应被认为是 由他们的空间(亚细胞定位)和时间(暴露时间)水平决定，详细 了解细胞内特定部位的ROS浓度及其与疾病发病机制的关系 目前下落不明。造成这种情况的主要原因是商业上没有实验工具来检测和 以足够的灵敏度和空间和时间分辨率表征特定细胞位置的ROS。 电子顺磁共振(EPR)被认为是明确化学物质的金标准 用自旋捕获法鉴定ROS。然而，实施的技术障碍很高，而且 事实证明，在生物环境中开发基于EPR的ROS成像是困难的。 基于染料与ROS反应时荧光发射变化的方法更容易获得 而不是EPR。虽然这种光学方法可以很容易地与细胞成像相结合，但当前的 实施充满困难，包括在ROS检测中缺乏特异性、毒性问题、 由探针本身产生的人工ROS，由于高水平的背景而导致的信号可变性 荧光，重要的是，光漂白。该第一阶段提案通过以下方式推进了ROS检测领域 开发一种新的基于纳米钻石(ND)的明亮荧光ROS传感器家族，该传感器将结合 EPR自旋捕获的专一性和信息内容，以及由 光学探头，不存在光毒性和光漂白问题。我们的商业化之路 组建了一支在ND加工和商业化、尖端ROS开发方面具有专业知识的团队 电子顺磁共振和化学合成方面的检测方案，以及自由基生物学和氧化应激方面的专业知识。 第一阶段的目的是演示由自旋反应组成的ROS传感器的原理证明原型。 分子构筑在ND表面，关联荧光和EPR数据。ROS传感器将被设置为 在体外测试，以检测由黄嘌呤氧化酶系统产生的超氧阴离子自由基，然后检测和 RAW264.7巨噬细胞内ROS成像。ND相对于传统ROS光学探头的基准将是 目的论证ND-ROS传感器在延长观测周期和减少结果误差方面的优势。 可变性。这些新的ROS检测工具的商业化将使对特定地点的纵向研究成为可能 在细胞和组织中产生ROS，以促进对ROS和氧化应激在 疾病的发病机制和进展，否则是无法实现的。此外，适配的ND-NV基自旋 体外临床诊断探针具有很高的商业潜力。

Summary Reactive oxygen species (ROS) are key mediators in human health but when misregulated can contribute to the progression of many diseases (e.g., cardiovascular disease, Parkinson's disease, Alzheimer's disease, cancer, Down's syndrome, cataract, several neurological disorders, etc.). While biological effects of ROS are thought to be determined by their both spatial (subcellular localization) and temporal (duration of exposure) levels, detailed understanding of site-specific ROS intracellular concentrations and their relationship to the disease pathogenesis is currently missing. The main reason for this is the commercial unavailability of experimental tools to detect and characterize ROS at specific cellular locations with sufficient sensitivity and spatial and temporal resolution. Electron paramagnetic resonance (EPR) is considered to be the gold standard for unambiguous chemical identification of ROS by spin-trapping methods. However, the technical barriers for implementation are high, and efforts toward developing EPR-based imaging of ROS within a biological environment have proven difficult. Methods based on changes in fluorescence emission upon reactions of a dye with ROS are more accessible than EPR. While such optical methods can be readily combined with cellular imaging, the current implementations are riddled with difficulties including lack of specificity in ROS detection, toxicity concerns, artefactual ROS production by the probes themselves, signal variability due to high levels of background fluorescence and, importantly, photobleaching. This phase I proposal advances the field of ROS detection by developing a new family of nanodiamond (ND) based bright fluorescent ROS sensors that will combine the specificity and information content of EPR spin trapping with the advanced imaging capabilities enabled by optical probes without the problems of phototoxicity and photobleaching. Our pathway to commercialization assembles a team with expertise in ND processing and commercialization, development of cutting-edge ROS detection schemes in EPR and chemical synthesis, and expertise in free radical biology and oxidative stress. Phase I is aimed at demonstrating a proof-of-principle prototype ROS sensor which consists of spin-reactive molecules crafted on ND surface and correlating fluorescence and EPR data. The ROS sensor will then be tested in vitro to detect superoxide radical produced by a xanthine oxidase system and then detection and imaging ROS in RAW264.7 macrophages. Benchmarking of ND over conventional ROS optical probes will be aimed to demonstrate advantages of ND ROS sensors in extending the observation period and reducing results' variability. Commercialization of these new ROS detection tools will enable longitudinal studies of site-specific ROS production in cells and tissue to advance the understanding of the roles of ROS and oxidative stress in the pathogenesis and progression of diseases not otherwise achievable. Moreover, the adaption ND-NV-based spin probes to ex vivo clinical diagnostics has high a commercial potential.