【作者】 钟华；

【导师】 张书圣；

【作者基本信息】 青岛科技大学 ， 应用化学， 2011， 博士

【摘要】 生物传感对于研究生物体内分子及细胞的结构和功能、生命活动的规律和本质、疾病的诊断、抗癌抗衰老等药物的设计、环境监测等都具有十分重要的意义。本文将DNA识别元件、适体识别元件与性能优良的纳米材料相结合,设计和研究了几种新型生物功能化探针,可用于癌细胞和靶DNA的特异性识别及检测。主要包括：1.设计了一种细胞外超分子网状DNA/量子点(QD)鞘膜探针,可用于癌细胞的高亮度的荧光成像。该超分子网状DNA/QD鞘膜是由修饰了Ramos细胞适体的DNA纳米线作为骨架,层层自组装大量的DNA/CdTe QD探针构建而成。其中,DNA纳米线骨架上负载的Ramos细胞适体可以特异性识别Ramos细胞并缠绕到细胞表面,同时,DNA纳米线骨架与DNA/CdTe QD探针发生多层自组装,形成超分子网状DNA/CdTe QD鞘膜,将细胞牢牢包裹于其中。由于该超分子网状DNA/QD鞘膜携带大量的荧光量子点,具有极高的荧光强度,是一种的生物相容性良好的荧光成像材料。对构建的DNA纳米线和超分子网状DNA/QD鞘膜进行了结构表征,并将该材料用于Ramos细胞的荧光成像,获得了高荧光亮度的细胞成像图。该材料也成功的应用于混合细胞系中Ramos细胞的识别,进一步证明了它良好的选择性。2.构建了一种新型的基于超分子网状DNA/CdTe QD鞘膜探针的电化学细胞传感器,可用于癌细胞的高灵敏高选择性定量检测。以Ramos细胞作为靶细胞,考察了方法的可行性。该电化学细胞传感器的构建原理为：将一端生物素修饰的Ramos适体固定到亲和素标记的聚苯乙烯微孔板上,由于适体对Ramos细胞具有很强的特异性识别能力,当加入细胞样品后,Ramos细胞被适体牢牢的固定在微孔板底部,再加入超分子网状DNA/CdTe QD鞘膜探针的合成原料,在细胞表面形成致密的超分子鞘膜探针。由于该超分子鞘膜是由DNA与CdTe QD发生多层自组装而成,负载了大量的CdTe QD标记物,具有显著的信号放大作用。将膜上的CdTe QD用稀酸溶解后得到Cd2+溶液,利用灵敏的差分脉冲阳极溶出伏安法(DPASV)对Cd2+进行定量检测。在优化的实验条件下,该电化学细胞生物传感器检测Ramos细胞的线性范围是10到1000个细胞,检测限为10个细胞。另外,该方法也成功的用于混合细胞系中Ramos胞的检测,证明该方法不仅灵敏度高,还具有良好的选择性,具有潜在的应用价值。3.研制了一种新颖的信号放大型荧光探针,可用于癌细胞的高亮度荧光成像以及靶DNA的可视化检测。该探针以表面羧基化的聚苯乙烯微球为载体,共价结合DNA连接链,并以该连接链为起始端,自组装DNA纳米线/CdTe QD探针构建而成。由于DNA纳米线末端包含靶分子的识别序列,可特异性的与靶分子结合,确保探针的良好选择性；DNA纳米线中间部分含有大量的CdTe QD探针结合序列,可负载大量的荧光标记物,因此可产生显著的信号放大效应。本工作分别以细胞适体和靶DNA的互补序列为识别元件,构建了可用于癌细胞的高亮度荧光成像的功能化探针和靶DNA的可视化检测的探针。在癌细胞的荧光成像中,该探针表现出了良好的特异性和荧光成像能力；在DNA的可视化检测中,利用这种探针可以直接观测到50 fM的靶DNA杂交反应。4.研制了一种新型一对一识别型三联金纳米粒子DNA探针,并构建了高灵敏的电化学DNA生物传感器。其工作原理为：在金电极表面固定巯基修饰的捕获DNA,之后分别与靶DNA和三联金纳米粒子DNA探针杂交,形成“三明治”式的DNA杂交复合物。探针上的三个金纳米粒子修饰了大量信号DNA,可以吸附大量的电化学指示剂[Ru(NH3)6]3+,利用计时电量法可实现靶DNA的电化学检测。该方法具有以下优点：一个三联金纳米粒子DNA探针只有一条靶DNA的识别链,因此可实现一对一的靶DNA识别和杂交,避免了传统DNA探针对靶DNA的消耗,提高了方法的灵敏度和选择性；三个金纳米粒子具有显著的信号放大效应,进一步提高了灵敏度。在优化的实验条件下,检测靶DNA的线性范围是1×10-16M～1×10-14 M,检测限为53 aM(3σ)。此外,对两碱基错配DNA以及非互补DNA检测结果表明,该方法具有良好的选择性。综上所述,该电化学DNA传感器灵敏度高,选择性好且操作简便,具有潜在的应用价值。更多还原

【Abstract】 Biosensors are developed for the study of the structure, the function and the molecular interaction of molecules in organisms, which plays important roles in the development of clinical, pharmaceutical, and environmental applications. Combined DNA recognition, aptamer recognition with nanoparticles (NPs) with excellent properties, several novel bio-functionalized DNA/nanoparticles probes were constructed for the specific recognition and detection of target DNA and cancer cell. The experimental protocols could be summarized as follows:1. Acted as a cage-type cellular probe, an extracellular supramolecular reticular DNA/quantum dot (QD) sheath was developed for the high-intensity fluorescence microscopy imaging of Ramos cells. The extracellular supramolecular reticular DNA/QD sheath was constructed from layer-by-layer self-assembly of DNA/CdTe QD probes and DNA nanowire frameworks functionalized with Ramos cell-binding aptamer. The DNA/QD sheath formed specifically and quickly on the surface of Ramos cells at physiological temperature. And the assembly of large numbers of DNA/CdTe QD probes on the surface of Ramos cells produced exceedingly high fluorescence intensity. Using the extracellular supramolecular reticular DNA/QD sheath as the cellular probe, Ramos cells could be distinctly observed and easily distinguished from a mixture of multiple cancer cells by fluorescence microscopy imaging. This strategy presents a promising platform for convenient evaluation of cancer cell.2. Based on the extracellular supramolecular reticular DNA/QD sheath probe, a sandwich-type electrochemical cellular sensor was developed for the accurate quantitative analysis of Ramos cells. The electrochemical cellular sensor was constructed using Ramos cell-binding aptamer-immobilized polystyrene microwell plates to capture Ramos cells and then assembling the supramolecular reticular DNA/QD sheath on the surface of Ramos cells. A sensitive differential pulse anodic stripping voltammetry (DPASV) detection system was used to quantify the concentration of Cd2+ produced by the acid dissolution of DNA/CdTe QD probes. Under the optimized conditions, Ramos cells could be detected quantitatively in the range from 10 to 1000 cells with a detection limit of 10 cells. This strategy can be widely used in cell recognition, cancer imaging and detection, and also shows applied potential in cell surface engineering.3. A high-intensity polystyrene microbead probe that was functionalized with supramolecular DNA/CdTe QD nanowires was developed for the direct fluorescence microscopy imaging of DNA hybridization event. The supramolecular DNA/CdTe QD nanowires capable of loading a large number of CdTe QDs were formed on the surface of polystyrene microbeads through hybridization of DNA molecules and produced exceedingly high fluorescence intensity so that single polystyrene microbead probe could be easily observed by fluorescence microscopy imaging. Using the high-intensity polystyrene microbead probe, target DNA as low as 50 fM could be recognized. In addition, combined cell-specific aptamer recognition element with the probe, cancer cell could be easily distinguished by fluorescence microscopy imaging. The proposed method provided an alternative strategy for the rapid, visible determination of DNA hybridization and cancer cell.4. A new kind of one-to-one recognition DNA probe based on triple Au nanpparticles (Tri-AuNPs) was developed. The DNA biosensor based on the one-to-one recognition Tri-AuNPs DNA probe was constructed by immobilizing capture DNA on the gold electrode and hybridization with target DNA, which further hybridized with the only one single strand DNA on one terminal of the DNA probe. Electrochemical signals of hexaammineruthenium(Ⅲ) ([Ru(NH3)6]3+) bound to the reporter DNA via electrostatic interactions were measured by chronocoulometry (CC). The sensitivity of the DNA biosensor was achieved through the signal amplification by one-to-one recognition Tri-AuNPs DNA probe. Under the optimized experimental conditions, target DNA could be determined in a linear range from 1×10-16 M to 1×10"14 M. The detection limit of 53 aM of target DNA was obtained. The resulting DNA biosensor exhibited ultrasensitivity, high selectivity, and good reusability as a promise alternative technique for other bioassays.更多还原