光声光谱技术在血管细胞内生物活性物质研究中的应用探索

Bioactive substances (e.g. reactive oxygen species, ROS) not only play a regulating vascular homeostasis and remodeling capabilities, but also are the basis cause of many important cardiovascular diseases. In situ analysis and tracking of their generation, transformation, and metabolism in vascular cells has important scientific significance and application value to reveal the mechanism of vascular homeostasis and remodeling. Combining the interdisciplinary strengths of analytical chemistry, biochemistry, and photoacoustic spectroscopy, we proposed herein that bioactive substances, such as nitric oxide, superoxide and functional biomolecules, have their own PAS spectral fingerprint characteristics in various media including phosphate buffer saline solution and cell tissues. The objective and content of this research proposal thus will focus on the development and application of photoacoustic spectroscopy (PAS) method for detecting and monitoring functional biomolecules and ROS including superoxide, nitric oxide, hypochlorite, related enzymes, and etc. The application of the PAS technique in ROS and biomolecules monitoring can be achieved by recoding the dynamic PAS characteristics of all ROS and biomolecules during their whole life span and physiological processes of vascular cells. The resultant PAS characteristics will be explored as spectral indicators providing information for the existence, concentration level, and metabolism of ROS and biomolecules (including nitric oxide, superoxide, peroxynitrite, and etc). The overlap of PAS spectral of molecules and cell components (DNA, membrane, nucleus, mitochondria and enzymes) would make the rapid analysis and monitoring difficult to realize. Such problems will be resolved by comparing their frequencies, relaxing process and lifetime of the excited states, and modulation. Consequently, the interactions and transformation among these molecules in different stages of vascular cell life span can be reflected by monitoring the PAS spectral changes. Therefore, the application of developed PAS method would produce new methods and technologies for understanding the generation and trafficking of reactive and silent small molecules in vascular cells. Furthermore, the research results would also help to develop novel diagnostic and prognostic tools and innovate the treatments for the major health problems with vascular pathologies.

活性氧类生物活性物质不仅发挥着调节血管稳态与重构的功能，同时也是许多重要心血管疾病的基础诱因。识别、分析和跟踪它们在血管细胞内的水平、作用和代谢，对揭示血管稳态与重构的调控机制具有重要的科学意义和应用价值。本项目结合分析化学、生物化学、光声光谱等学科交叉优势，以血管细胞中一氧化氮、超氧阴离子、关键功能生物分子等生物活性物质的光谱特征和相互作用为研究对象，探索光声光谱在血管重构研究中运用的新方法和新技术。研究获取它们在不同环境介质中的吸收光谱基础数据和光声光谱特征参数，发展活性氧和生物分子的特征光声信号的提取和解析方法，实现功能生物分子氧化损伤修复过程中的光声光谱识别与跟踪，得到它们的光声光谱动态特征并建立相关的识别与跟踪模式，揭示活性氧与生物分子间的相互作用机制，为进一步理解血管细胞内活性物质的生理机制和致病机理提供新的光谱手段。预期成果将推动光声光谱技术在血管稳态与重构研究中的应用。

本项目根据计划书，经过三年的探索设计合成了新型的具有光声信号和荧光信号的有机分子探针、纳米材料及其功能复合物，结合新的光学识别原理与方法构建了荧光的光学调控开关，实现了对血管细胞中具有调控功能的活性氧类物质如NO、NO2、.OH、SO2、H2S、CO等和能量物质如ATP的高选择性、高灵敏检测。发展了针对细胞中微弱酸碱变化的高选择性、高灵敏检测方法技术。研究成果的主要发现点如下：（1）制备了具有光声信号的荧光量子点和过渡金属配合物，通过表面功能化修饰，提高了其生物相容性，获得针对氮氧化物、二氧化硫、硫化氢以及次氯酸的敏感的光谱基础数据和光声响应信号，用于分析估计这些活性物质在血管细胞氧化应激过程导致的损伤程度、含量及浓度变化。（2）通过巧妙设计和调控核酸适配体的结构，发展了DNA链之间的化学反应原理转化为光学信号输出的新方法，实现了对血管内皮细胞中能量物质三磷酸腺苷（ATP）的荧光光谱信号的灵敏识别，可用于考察三磷酸腺苷在肿瘤坏死因子引起血管内皮细胞损伤中可能的保护作用。（3）设计了具有质子化调控性能的功能有机分子，对细胞环境中微弱的酸碱波动识别灵敏并伴随光声与荧光信号的特征变化，为血管细胞状态监测提供重要数据。这些研究进展对于血管细胞内活性物质的光声光谱检测提供了光谱数据分析基础，开拓了血管细胞活性物质研究的技术途径。上述研究工作取得了多项具有一定创新性和系统性的研究成果，部分研究成果已经在Analytical Chemistry、Sensors and Actuators B、Analytica Chimica Acta、Talanta、Nano Research、Inorganic Chemistry、Langmuir、Analyst等国际期刊上发表SCI收录论文17篇（已标注该项目），获国家发明专利授权1项，申报国家发明专利4项。