基于纳米界面DNA杂交链反应的单细胞内蛋白激酶分析研究

The assay of protein kinases in single cells is of great significance for further understanding the molecular mechanism in biochemical research and development of disease diagnosis and targeted therapy, which is a research hot spot and difficult point. Here, this project intends to amplify the protein kinase signal in situ via DNA hybridization chain reaction in order to maximize the sensitivity of protein kinase detection. By virtue of the unique properties of upconversion nanoparticle/dye doped silica complex and the signal amplification of DNA hybridization chain reaction, the basic mechanism of the biological molecule assembling on the nano-interfaces will be explored from the nano-scale level. The viaration regularity of the properties of polypeptides and interactions among polypeptides, nanomaterials and DNA nanostructures during the phosphorylation process is explored, accordingly elaborate novel ratio biosensing protocols of protein kinases, realizing the estimation of protein kinases in single cell. Meanwhile, with the high drug loading of nanomaterials and the structural controllability of DNA nanostructures, the fabricated nanoprobes will also be applied for protein kinase-mediated tumor targeted therapy. This project is not only necessary for further understanding the molecular mechanism of signal transduction, but also beneficial for the clinical diagnosis and targeted therapy.

单细胞内蛋白激酶的高灵敏分析对于生物化学研究及重大疾病的诊断与药物靶向治疗等都具有重要意义，也是目前研究的热点和难点。本项目拟通过纳米界面DNA杂交链式反应对蛋白激酶检测信号进行原位放大，旨在大幅提高蛋白激酶检测灵敏度，实现单细胞内蛋白激酶的活性分析。借助上转换荧光纳米粒子/染料掺杂二氧化硅复合材料的优异性质和DNA杂交链式反应的信号放大能力，研究纳米尺度界面上生物分子组装的作用机制，探索多肽底物磷酸化过程中自身性质变化及与纳米材料和DNA结构间的相互作用改变导致的光学信号变化规律，籍此开发蛋白激酶活性的比率型生物传感新方法,实现单细胞水平蛋白激酶活性的放大检测及荧光成像分析；同时利用纳米材料的载药能力及DNA纳米结构的结构可控性，实现蛋白激酶调节的药物可控释放与肿瘤靶向治疗。本项目的实施不仅对研究分子层面细胞内信号传导提供重要的参考价值，而且为与蛋白激酶有关的医疗和药物研发提供理论依据。

蛋白激酶是包括癌症在内的各种疾病的丰富生物标志物和治疗靶点。然而，在根据激酶过度激活实施治疗的同时，在体内检测激酶活性仍然具有挑战性。该项目构建了一个纳米中介器-效应器级联系统，通过DNA放大机制原位放大激酶催化的磷酸化过程，实现了激酶活性成像和体内激酶响应性药物释放，为癌症治疗提供了一种激酶特异性的感知和治疗范例；利用红光发射的上转换纳米粒子与Cy5.5掺杂的可降解介孔硅之间的发光共振能量转移，设计构筑了一种近红外双发射的可降解纳米载体，实现了治疗性蛋白的体内递送及可视化成像；基于“吸收竞争机制”制备了AuNCs@MnO2核壳纳米颗粒并实现了对细胞内谷胱甘肽的近红外成像；合成了可调色有机硅量子点并用于细胞及肿瘤靶向成像以及体内过氧化氢动态成像。该项目借助功能纳米材料与生物材料的智能组装，实现了肿瘤特异性标志物的成像与治疗监测，对于开发新型的光学传感体系用于癌症诊疗相关研究具有非常重要的意义。