具有荧光淬灭效应的HAp纳米晶簇在DNA氧化损伤可视化检测中的应用基础研究

The visual detection of DNA oxidative damage has important practical value for early determination of related disease and evaluation of antioxidant effect for the treatment owing to the advantages of rapid, convenient and intuitive. Nanostructured hydroxyapatite (HAp) agglomerate has been widely used in the field of biological sensing technology, attributed to its unique crystal structure and excellent biocompatibility. Based on the previous researches, the nanostructured HAp agglomerate with fluorescence quenching capability fabricated by hydrothermal-biomineralization is proposed to construct a new fluorescence-detecting platform by combining the DNA molecular beacon technology in this project, aimed for the visual detection of DNA oxide damage. The main contents of include the effect of biomineralization conditions to the microstructure and fluorescence quenching capability of HAp agglomerates; the influence of homogeneity of HAp material on the self-assembly of DNA beacons; and the stability of constructed system applied for DNA oxide damage detection in complex environment. Through the above researches, it is expected to achieve the new method for constructing novel fluorescence detection system based on the biomineralized HAp nanomaterial, and clarify the regulatory mechanism of fluorescence quenching effect along with its influence on the detection of DNA oxidative damage biomarker in the novel fluorescence detection system. This research will provide solid experimental foundation for the establishment of new visual DNA oxide damage determination system.

可视化检测DNA氧化损伤以其快速、便捷、直观等优点在相关重大疾病的早期检测及药物治疗效果评估等方面具有重要的应用价值。羟基磷灰石（HAp）纳米晶簇因其特殊的晶格结构和良好的生物相容性而被广泛用于生物传感领域。申请人拟在前期的研究工作基础上，采用生物矿化法制备具有荧光淬灭效应的HAp纳米晶簇，并与核酸分子信标技术结合构建新型荧光检测体系，用于DNA氧化损伤的可视化检测。主要研究内容包括生物矿化合成条件对HAp纳米晶簇的微观结构以及荧光淬灭效应的影响机制；材料的均质性调控对核酸探针表面自组装的影响规律；复杂环境中所构建体系用于DNA氧化损伤检测的稳定性等。通过上述研究，建立基于生物矿化制备HAp纳米晶簇构建新型成荧光检测体系的新方法，并阐明该新型荧光检测体系中荧光淬灭效应的调控机制及其对于DNA氧化损伤标志物检测的影响规律。研究将为建立DNA氧化损伤标志物可视化检测新方法提供坚实的实验基础。

8-羟基脱氧鸟苷是目前DNA 氧化损伤最常用的生物标志物，对其进行检测可以评估生物体 氧化损伤和修复的程度、氧化应激与DNA 损伤的相互关系，进而评价抗氧化剂治疗DNA 氧化损 伤的效果。羟基磷灰石（HAp）以其特殊的晶体结构而广泛用于生物传感领域，其物化特性与微观结构密切相关。项目负责人采用水热－生物矿化技术制备出具有荧光淬灭新特性的HAp 材料，并结合DNA分子信标技术，将其构建成荧光检测平台，用于DNA氧化损伤的可视化检测。研究结果表明，天然碳酸钙生物质模板在生物矿化环境中对于HAp纳米晶簇微观晶体结构有显著影响，其中经过5天矿化处理后的海螺壳内外表面和鸡蛋壳内表面具有较好的荧光淬灭-复原特性；不同微观结构HAp晶簇对于荧光淬灭-复原性质的影响主要是晶格结构和表面官能团的作用，其中以CaHPO4为主要成分的基体材料比其它磷酸钙类物质具有更好的荧光淬灭特性。通过上述研究，基本建立了模板对生物矿化制备HAp纳 米晶簇微观结构的调控机制；获得了微观结构对HAp纳米晶簇荧光淬灭机制的影响规律，以及复杂环境下该检测平台对于生物标志物（8-OH-dG及miRNA等）检测的影响规律，对于该检测平台在临床医学、生物医学等相关领域的实际应用奠定了实验基础。