基于时间分辨荧光技术蛋白激酶活性分析新方法

Protein phosphorylation catalyzed by protein kinase is one of the most ubiquitous and important post-translational modification，and it plays an important role in many physiological process such as cell proliferation, differentiation and metabolism. Aberrant phosphorylation is closely related with a variety of human diseases, especially cancer. Therefore, developing potent and specific analytical methods of protein kinases are of great significance. In recent years, lanthanide luminescence nanomaterial has attracted growing interests due to their unique optical characteristics such as large stokes shifts, long luminescence lifetimes and narrow emission bands, and has been widely used in the time-resolved luminescence bioassays to solve the background interference of complex biological fluids, improving the analytical performance of biosensors greatly. Based on this, this project aims to exploit a series of sensitive and specific sensing platform of protein kinases based on lanthanide phosphate fluorescence nanomaterial, investigate the energy conversion, material transport mechanism related to the sensor process, reveal the sensor rule of protein kinases identification, develop a series of simple, quick, economic and sensitive protein kinase sensors for the real samples such as serum or cell lysates analysis, provide new methods for the diagnosis and prognosis of the associated diseases. The further investigation on the interaction between inhibitors and enzymes provides new ideas for the early diagnosis of cancer, diseases course monitoring and anti-cancer drug screening.

蛋白质磷酸化是生物体内最普遍、最重要的蛋白质翻译后修饰形式之一，它在细胞增殖、分化和代谢等生理过程调控中担任着重要角色，磷酸化异常与许多疾病，特别是癌症有着密切的关系。因此，开发简便、快速、高灵敏的蛋白激酶活性分析方法具有重要意义。近些年来，稀土发光材料因其大的斯托克位移、长荧光寿命等优异的光学性能被广泛地应用于生物分析中，将它们与时间分辨荧光分析技术相结合，可以有效解决生物样品自发荧光和背景干扰问题，提高检测的灵敏度与精确度。本项目以稀土磷酸盐荧光纳米材料作为传感元件，以蛋白激酶和磷酸化为研究对象，深入研究传感界面上生物靶标分子/纳米传感材料之间相互作用、能量转换以及物质输送等相关机理，揭示蛋白质磷酸化识别传感规律，开发一系列简便、快速、经济、灵敏度高的时间分辨荧光检测新方法，并将这些方法应用于血清或细胞裂解液等实际样品的检测，为癌症的早期诊断、病程监测、抗癌药物开发等提供新思路。

稀土发光材料因其大的斯托克位移、线性发射、长荧光寿命等优异的光学性能在化学传感领域的应用，可以有效解决生物样品自发荧光和背景干扰问题，提高检测的灵敏度与精确度，受到国内外科研工作者的关注。针对目前蛋白激酶活性检测中半导体量子点（QDs）、碳点、石墨烯量子点（GODs）等传统荧光探针存在的重金属毒性、背景干扰严重、光量子产率低等不足，本项目以稀土磷酸盐荧光纳米材料作为传感元件，基于CePO4与氧化石墨烯、碳量子点等纳米材料之间的能量转移机制，发展对蛋白激酶特异性、灵敏性检测的方法，为激酶抑制剂筛选及药物开发提供可靠的研究手段。成功开发了一种双信号比率荧光探针用于蛋白激酶活性及其抑制剂的传感方法，同时利用磷酸根对稀土离子的高亲和性，发展了一步法蛋白磷酸激酶活性检测，利用稀土Ce3+→Tb3+之间高效的能量传递效率，极大地提高了激酶检测灵敏度。将这些方法应用于血清实际样品的检测，为抑制剂的筛选提供依据。