基于纳米界面生物分子作用的蛋白激酶活性检测新方法研究

Protein kinases are ATP-dependent phosphotransferases that deliver a single phosphoryl group from the γ position of ATP to the hydroxyls of serine (Ser), threonine (Thr), or tyrosine (Tyr) in protein substrates. The abnormal up- and down regulations of protein kinase activity are closely related to many diseases including cancer, diabetes and neurodegenerative diseases. Therefore, the detection of protein kinase-catalyzed phosphorylation as well as its inhibition by potential inhibitors has attracted substantial research interest. In this project, a series of high quality functional nanomaterials and nanostructures will be designed and synthesized for preventing biological molecules nonspecific adsorption through specially modifying their nano-surfaces; The substrate biological molecules which are targeted by the specific protein kinases will be designed, selected and coupled on the nano-interfaces; The basic mechanism of the biological molecule adsorbing, assembling and folding on the nano-interfaces would be further considered from the nano-scale level. Furthermore, the high specifically trapping and recognizing of the potential target molecules can be realized by improving the anti-interference and sensitivity of kinase detection method. Then, the important physical and chemical properties of energy transfer and material transmission are effectively converted into photoelectric signals through the photoelectric effects of nanometer materials in the biomolecular recognition process. Finally, the rapid, sensitive and real-time methods will be established for detecting protein kinase activity and screening inhibitors. Based on these new established methods, it would be valuable for exploring phosphorylation mechanism, and revealing pathogenesis of the major diseases and drug screening from the molecular level.

蛋白激酶的功能是将ATP的γ磷酸基转移到底物的特定氨基酸残基上，催化底物磷酸化。蛋白激酶活性的抑制或过表达与癌症、糖尿病、神经退行性疾病等重大疾病密切相关。因此，研究检测蛋白激酶活性及其抑制剂已成为生命科学的前沿研究领域。本项目拟设计和合成一系列高质量的功能纳米材料和纳米结构界面；设计和筛选具有被靶标蛋白激酶特异性识别的底物生物分子，实现生物分子在纳米界面上的偶联固定化；深入研究纳米尺度界面上生物分子吸附、组装和折叠的基本机制，提高激酶检测方法的抗干扰能力和灵敏度，实现靶标分子的高特异性捕获与识别，并通过纳米材料的光电效应将生物识别过程的能量传递和物质传输等重要的物理化学特性有效转换成光电信号，构建基于荧光分析、电化学传感和表面等离子共振耦合等技术的蛋白激酶活性检测以及激酶抑制剂筛选新方法，从分子水平上探索磷酸化修饰分子机制，对揭示重大疾病的发病机制以及筛选治疗药物具有重要价值。

蛋白激酶表达异常与癌症、糖尿病、神经退行性疾病等重大疾病密切相关，其活性检测及抑制剂筛选是生命科学的前沿研究领域。基于此，本项目构建了生物识别分子/纳米材料界面体系，研究了界面体系的影响因素和两者的相互作用机制，利用纳米材料的光电效应将磷酸化过程有效转换成光或电信号，构建了快速灵敏的蛋白激酶/抑制剂光学及电化学分析传感平台。（1）将CK2的底物多肽共价修饰于GQDs表面，通过磷酸化后Zr4+诱导GQDs聚集的荧光变化，建立了CK2活性检测及抑制剂筛选新方法。此外，以CK2底物多肽原位合成荧光金纳米簇（peptide-AuNCs），通过磷酸化后Zr4+诱导peptide-AuNCs聚集的荧光变化，实现了CK2活性检测及抑制剂分析。（2）利用羧肽酶Y（CPY）诱导的peptide-AuNCs荧光猝灭效应，结合磷酸化对CPY剪切的抑制机制，以peptide-AuNCs为探针建立了PKA “turn on” 荧光检测法。在此基础上，以PKA和CK2的底物多肽为模板合成多色荧光金属纳米簇，利用磷酸化前后CPY对纳米簇荧光的影响规律，在单一激发波长下即可实现PKA和CK2的同时检测和可视化分析。此外，以peptide-AuNCs和CdSe/ZnSQDs@SiO2作为双发射比率荧光探针，构建了一种可视化且灵敏的比率荧光法用于PKA检测。（3）基于抗磷酸化丝氨酸抗体与磷酸化多肽的特异性作用，通过链霉亲和素-生物素反应将酶标记的AuNPs连接到电极表面，建立了基于电致化学发光（ECL）的PKA活性分析法。此外，以固定在电极表面的GQDs为ECL发光试剂，利用抗原-抗体识别作用，将抗体标记的氧化石墨烯（GO）捕获到电极表面，利用GO与GQDs的能量转移机制建立CK2活性传感平台。（4）以GQDs和鲁米诺分别为阴极和阳极ECL发光试剂，基于AuNPs对GQDs阴极信号的减弱及对鲁米诺阳极信号的增强作用，建立了双电位比率ECL传感平台用于PKA的灵敏检测。（5）探讨了磷酸化底物对应激酶类型和磷酸化位点预测模型的理论构建。本项目已按计划完成，发表SCI论文31篇，授权发明专利7项，申请发明专利4项。