【作者】 任伟；

【导师】 李正平； 刘成辉；

【作者基本信息】 河北大学 ， 分析化学， 2014， 硕士

【摘要】 均相荧光传感器因其灵敏度高、数据易读取、样品量小、操作简便，在金属离子、核酸以及其他生物分子的选择性检测中被广泛应用。然而，受限于复杂生化体系中的强散射和高荧光背景，均相荧光传感器并不适用于诸如血清等实际样品的临床分析；此外，均相荧光传感器难以器件化、只能一次性使用、检测体系容易被污染等缺点也限制了其实际应用。以功能化的微球为生化反应及信号富集的载体构建各类生物传感技术，可方便地实现与复杂基质的分离而避免干扰，该类方法目前已成为对复杂介质中生物分子进行检测的重要手段。本论文以微球作为荧光信号富集基体，在DNA及蛋白激酶活性分析方面进行了积极的探索。高灵敏度脱氧核糖核苷酸（DNA）检测在遗传性疾病早期诊断和基因工程中起着重要作用，因此，对于快捷、简便的DNA检测方法的研究也在不断深入。本文以链霉亲和素（streptavidin，STV）修饰的磁性微球Dynabeads M-270为荧光富集载体，通过STV与生物素（Biotin）的特异性结合将目标DNA的茎环结构检测探针（Bio-H1）富集于M-270微球表面。当目标DNA存在时，Bio-H1的粘性末端会与其杂交配对，进而茎环结构被打开。经磁分离后，茎环结构展开所形成的新的粘性末端可以继续引发体系中荧光标记的两种茎环结构DNA交替发生杂交链式反应（Hybridization Chain Reaction，HCR），在微球表面富集大量荧光信号，达到了信号放大的目的。经流式细胞仪检测，该传感器的检测限达到了0.5pmol/L，比传统微球传感器灵敏度高三个数量级；与此同时，该传感器的特异性高，在复杂生化体系中的应用也得到了验证。蛋白激酶在细胞内的信号转导过程中扮演着重要的角色，由激酶活性过强所引起的蛋白磷酸化异常会导致包括癌症在内的很多疾病。因此，对蛋白激酶活性进行准确检测对于疾病诊断及靶向药物开发都具有重要意义。本文利用锆离子功能化的二氧化硅多孔微球为信号富集载体，成功构建了一种高灵敏度检测蛋白激酶活性的方法。微球表面修饰的锆离子可特异性识别并结合别蛋白激酶催化生成的磷酸化荧光底物多肽，而与未磷酸化的多肽无相互作用。因此，蛋白激酶活性与微球富集的荧光信号呈正相关。通过对微球表面的磷酸化多肽进行洗脱并检测其荧光强度，即可实现蛋白激酶活性的灵敏检测。该传感器操作简便、特异性高，避免了复杂生化体系对测定的影响，为生物样品中蛋白激酶的测定以及激酶抑制剂的筛选提供了新的途径。更多还原

【Abstract】 The properties of high sensitivity, easy readout, low sample volume and handy separationmake the homogeneous fluorescent sensors widely used in the specific detections of metalions, nucleic acids and other bio-molecules. However, a majority of the homogeneousfluorescent sensors are not suitable for the analysis of complex biological samples due to thestrong light scattering and autofluorescence background in the complex matrix such as serum;besides, the practical application of this kind of sensors is also limited by difficulties in devicemaking, disposable using and easy to be contaminated. In this regard, bio-sensors made byfunctionalized micro beads coupled with fluorescence labeling have become a fascinatingchoice for the detection of many kinds of bio-molecules, allowing extraction of the signalsfrom the sample matrix filled up with interference. This manner has become one of the mostimportant technologies for the detection of bio-molecules in complex matrix. In this thesis,micro beads are used as the fluorescence enriching matrix to investigate their applications inDNA and protein kinase activity detection.We established a new DNA detection method by combining the superparamagnetic beadsfor easy separation and signal collection, hybridization chain reaction (HCR) for enzyme-freeamplification and flow cytometer for powerful detection. In this design, thesuperparamagnetic streptavidin (STV)-functionalized micro beads Dynabeads M-270areapplied as the carrier of the fluorescence signals. The hairpin biotin-modified probe DNABio–H1could bind to the surface of the M-270via specific STV-biotin interaction. Upon theexistence of target, it will hybridize with the sticky end of Bio-H1to open the hairpinstructure. After the magnetic separation, the newly released sticky end of Bio-H1on the beadswill further induce the hybridization chain reaction (HCR) on the addition of two fluorescencelabeled hairpin structures. In this way, the fluorescence signals enriched on the micro beadsnumerously, the purpose of signal amplification achieved. The fluorescence signals of M-270are analyzed by flow cytometry. It is tested by flow cytometry that the detection limit of thissensor is0.5pmol/L, which is about3orders of magnitude higher than the tradition beadsbased sensors. The specificity of this sensor has also been evaluated as well as the application in complex matrix.Protein kinases play important roles in intracellular signal transduction pathways.Hyperactivity of some kinases and the following abnormal protein phosphorylation may leadto various human diseases including cancer. Therefore, protein kinases are regarded as afamily of extremely important molecular target for drug therapy. In this article, wesuccessfully design a protein kinase activity detection sensor by Zr4+functionalized SiO2micro beads. The Zr4+on the surface of the micro beads could specifically bind thephosphorylated peptides, and does not affect on the non-phosphorylated ones. Hence, there isa positive correlation between the activity of the protein kinase and the fluorescence signalsenriched on the micro beads. The quantitative assay of the protein kinase activity could bedetermined by the elution of the micro beads. This sensor is of easy operation, high specificityand feasible in complex biological samples, providing a new way in protein kinase detectionand screening of potential protein kinase inhibitors.更多还原