【作者】 白洁；

【导师】 李正平；

【作者基本信息】 河北大学 ， 分析化学， 2014， 博士

【摘要】 激酶在细胞增殖、分化和代谢等生理过程的调控中担任着重要的角色。众多的人类疾病与激酶的活性异常有着直接或者间接的关系。因此对激酶活性的分析检测对于生化研究、临床诊断及对重大疾病的药物靶向治疗等都具有重要意义。研究快速、简单、灵敏度高和特异性好的检测激酶活性的新方法具有广泛的应用前景。本论文以蛋白激酶和己糖激酶为研究对象，主要应用荧光共轭聚合物、功能性磁性微球、便携式血糖仪等生物传感器为技术平台，结合分子荧光、荧光成像等检测技术，研究建立了一系列检测激酶活性的新方法，主要内容如下：1．基于阴离子荧光共轭聚合物的荧光共振能量转移作用，发展了一种均相、通用型的新型生物传感器，用于蛋白激酶活性的检测。水溶性荧光共轭聚合物（PFPaa）带有氨基二乙酸的侧链，它与金属离子（Zr4+）形成配位化合物。荧光标记的肽底物在蛋白激酶的作用下被磷酸化，其磷酸基团与Zr4+特异性结合。以Zr4+为“桥梁”，PFPaa与标记在肽上的荧光基团被拉近，产生了强烈的荧光共振能量转移（FRET），并对荧光信号进行放大，由此可实现对蛋白激酶活性的高灵敏度检测。该方法操作简便，可检测0.0005U·μL-1~1U·μL-1的蛋白激酶，并可用于抑制剂的筛选。2．将TiO2修饰在Fe3O4/SiO2磁性微球表面。荧光标记的底物肽在蛋白激酶存在时被磷酸化，其磷酸基团与Ti4+特异性结合，从而被富集在磁性微球表面。通过对富集的磷酸化肽上标记的荧光进行检测或对磁性微球表面荧光成像进行分析，可以实现对蛋白激酶活性的高灵敏度检测。该方法对蛋白激酶活性的测定范围为0.0005U·μL-1~0.5U·μL-1，检出限为0.0001U·μL-1。该方法成功的应用于细胞裂解液中蛋白激酶活性检测及蛋白激酶活性抑制剂的筛选。此外对不同的蛋白激酶底物肽标记不同的荧光基团，还可以实现对不同蛋白激酶的同时检测。3．基于便携式血糖仪，建立了一种简单、快速、均相测定已糖激酶活性的方法。己糖激酶催化葡萄糖的磷酸化，磷酸化的葡萄糖在血糖仪上不产生响应信号，以商品化的便携式血糖仪作为实验信号测定设备，根据体系中剩余葡萄糖的含量，间接测得己糖激酶的活性。本方法的检测范围为10-6U·μL-1~10-2U·μL-1，这种新的分析方法也可应用于己糖激酶抑制剂的高通量筛选。更多还原

【Abstract】 Kinases play an important role in regulation of fundamental biological processes,including cell proliferation, differentiation and metabolism. Aberrations in kinase activitiescan result in a number of diseases. Therefore, sensitive and widely applicable assays formonitoring kinase activity are of great signifcance for further understanding the molecularmechanism in biochemical research, clinical diagnosis, and development of targeted therapy.In this study, we focus on the development of sensitive methods for detection of proteinkinase activity and hexokinase activity. This dissertation presents a series of new methods fordetection of kinase activity by using conjugated polymers, functionalized magneticmicrospheres and glucose meter technology integrated with molecular fluorescence andfluorescence images detection. The main contents are as follows:1. Anionic fluorescent conjugated polymer (PFPaa) coupled with the metal ion-mediatedFRET, can be used to design a versatile, homogeneous, and simple platform for detection ofprotein kinase activities. In conclusion, we have demonstrated that PFPaa can complex withZr4+and the complexed Zr4+can selectively recognize the phosphate group on the peptidesubstrates, resulting in e cient FRET from PFPaa to the fluorophores labeled on thephosphorylated peptides. Based on the light-harvesting property and amplification offluorescence signals, PFPaa can be used to design a versatile, homogeneous, robust platform todetect protein kinase activity with high sensitivity. Protein kinase A (PKA) activity can bequantitatively detected over a wide concentration range from0.0005U·μL-1~1U·μL-1. Thenew strategy provides a simple detection procedure, easy readout and cost e ective manner forprotein kinase assay, which shows great potential for high-throughput assay in clinicaldiagnostics and drug discovery applications.2. A simple, highly sensitive, and dual-readout fuorescent assay is developed for thedetection of protein kinase activity based on the specifc recognition utility of TiO2-coatedFe3O4/SiO2magnetic microspheres (TMSPs) for kinase-induced phosphopeptides. When thefuorophore-labeled substrate peptides are phosphorylated by the kinase reaction, they canbind specifcally to the TiO2layer of TMSPs by means of phosphate groups, resulting infuorophore enrichment on the TMSP surfaces. The accumulated fuorophores on the TMSPsare proportional to the kinase activity, and the fuorescence signal readout could be run through either direct fuorescent imaging of the TMSPs or measurement of the fuorescenceintensity by simply detaching the fuorescent phosphopeptides into the solution. The TMSPsexhibit extremely high selectivity for capturing phosphorylated peptides over thenonphosphorylated ones, resulting in an ultrahigh fuorescence signal-to-background ratio of42, which is the highest fuorescence change thus far in fuorescent assays for detection ofprotein kinase activities. Therefore, the proposed fuorescent assay presents high sensitivity,low detection limit of0.0001U·μL-1, and wide dynamic range from0.0005U·μL-1to0.5U·μL-1with protein kinase A (PKA) as a model target. Moreover, the TMSP-based fuorescentassay can simultaneously quantify multiple kinase activities with their specifc peptideslabeled with di erent dyes. This new strategy is also successfully applied to monitoringdrug-triggered PKA activation in cell lysates. Therefore, the TMSP-based fuorescent assay isvery promising in high-throughput screening of kinase inhibitors and in highly sensitivedetection of kinase activity, and thus it is a valuable tool for development of targeted therapy,clinical diagnosis, and studies of fundamental life science.3. We provide a simple, rapid, homogeneous method for measuring hexokinase activiteby quantitating the amount of glucose remaining in solution following a kinase reaction,which is monitored by the personal glucometer. The glucose level is correlated with theamount of glucose present and is inversely correlated with the amount of kinase activity. Thewide dynamic range was detected from10-6U·μL-1to10-2U·μL-1by using HK as a modeltarget. Moreover, this new method is also successfully applied to high-throughput screeningof HK inhibitors. Thus is a valuable tool for development of targeted therapy as well as thestudies of fundamental life science.更多还原