用于线粒体中活性氧定量检测的FRET比率型双光子荧光探针的构建及生物成像应用研究

Quantification detection of reactive oxygen species (ROS) in mitochondria with high sensitivity and selectivity is imperative for elucidating its functions in health or disease states. Owing to increased penetration depth, minimized fluorescence background, and less light scattering and tissue injury, two-photon fluorescent probes have been developed for detecting ROS in mitochondria. But most previously reported two-photon probes were designed on the basis of single- emission intensity changes, which could be affected by instrumental efficiency, environmental conditions, and the concentration of probe molecules. To address the issues, this project will be designed for developing two-photon fluorescent probes based on fluorescence resonance energy transfer for quantification of ROS in mitochondria. In this project, naphthalene derivative with excellent two-photon characteristics will be employed as energy donor, 1,8-naphthalimide derivative with recognition group will be used as energy acceptor, and the cationic pyridinium moieties will be utilized as mitochondria-targeted functional group and connecting group between naphthalene derivative and 1,8-naphthalimide derivative. And the probe will be applied to ratiometric quantitative detection of ROS in mitochondria of living cells, tissues and living animals. Furthermore, the probe will be used to directly visualize overgeneration of ROS during inflammation and ischaemia-reperfusion injury at cell, tissue and organ levels, respectively. Therefore, the FRET-based two-photon fluorescent probe will be a potential biological tool to explore the roles of ROS in mitochondria under different physiological and pathological settings. This project will provide evidence for the early diagnosis of related diseases with ROS in mitochondria.

线粒体中活性氧的浓度水平与生物体生理和病理进程密切相关。双光子荧光探针由于具有较深的组织穿透能力、较低的背景荧光与自吸收干扰等优点，已被广泛用于生命体内活性氧检测。而目前，大多数活性氧双光子荧光探针属于增强型或淬灭型，易受到探针分子浓度和其所处环境等因素的影响，难以满足线粒体内活性氧定量检测的要求。本项目拟基于荧光共振能量转移(FRET)机理构建比率型双光子小分子荧光探针用于线粒体活性氧的定量检测，采用具有优良的双光子光学性能的萘类染料作为能量供体，具有4-位易修饰的1,8-萘酰亚胺类染料作为能量受体，吡啶阳离子作为连接基团和线粒体靶向基团，构建系列活性氧荧光探针，实现活细胞、组织、活体线粒体中活性氧的定量检测，并应用于炎症和缺血再灌注损伤等病理模型中的活性氧浓度水平的监测，以期为研究线粒体活性氧浓度水平与生物体生理和病理进程的关联以及相关疾病的早期诊断提供有价值的理论依据和技术支持。

细胞器（溶酶体、内质网、线粒体）内活性氧的浓度水平与生物体生理和病理进程密切相关。荧光探针已经成为细胞器活性氧浓度水平监测的有效方法。而目前，大多数细胞器靶向活性氧荧光探针属于增强型或淬灭型，易受到探针分子浓度和其所处环境等因素的影响，难以满足细胞器内活性氧定量检测的要求。本项目利用比率型荧光探针具有可以避免如探针浓度、激发光源稳定性、仪器的灵敏度、光漂白和背景荧光等引起的检测误差等优点，围绕靶向细胞器的活性氧分析与成像研究的现实需求，选取溶酶体、内质网和线粒体内次氯酸为研究对象，构建了一系列新型的溶酶体靶向的比率型次氯酸荧光探针、内质网靶向的比率型次氯酸荧光探针、线粒体靶向的增强型次氯酸荧光探针、基于香豆素-萘酰亚胺类的ICT-FRET比率型次氯酸荧光探针。项目组研究了这一系列探针在体外和细胞中对次氯酸的检测与荧光成像性能，取得了良好的效果。上述探针具有优异细胞器靶向性能、良好的光稳定性、高选择性、高灵敏度、响应时间快、工作pH范围宽等优点，为研究溶酶体、内质网和线粒体内活性氧浓度水平与生物体生理和病理进程的关联以及相关疾病的早期诊断提供有价值的理论依据和技术支持。