【作者】 潘春锋；

【导师】 姚守拙； 郭慢丽；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 硕士

【摘要】 准确、灵敏地诊断白血病对于临床医生选择合适的治疗方法至关重要。目前白血病诊断的常规技术往往耗时、成本高、检测仪器昂贵,所以,开发一些检测成本低,使用仪器简单,并能提供灵敏、准确的诊断结果的方法是即时诊断领域的发展目标。适体是一种被视为化学合成抗体的单链DNA/RNA构成的分子探针,在生物医学领域具有广阔的应用潜力。本论文在基于适体的肿瘤细胞检测方面开展了一些研究工作。另外,构建纳米复合材料用于发展骨组织工程意义重大。本论文的主要研究工作如下:1.将对人急性白血病细胞(CCRF-CEM)具有特异性识别能力的巯基修饰适体(sgc8c)自组装到金电极表面,开发了一种简单、方便、低成本、无标记的早期检测白血病细胞的电化学传感器。用循环伏安和电化学阻抗表征了修饰电极,并通过计时库仑分析得到了电极表面的适体密度。所制备的适体传感器经CCRF-CEM细胞孵育后,电极界面上的电子转移电阻(Ret)显著增大;且传感器对CCRF-CEM细胞具有良好的选择性。Ret值和细胞浓度对数值呈线性关系,检测线性范围为1×10~4-1×107 cells/mL,检测限为6×103 cells/mL。2.利用无标记金纳米颗粒(AuNPs)做指示剂,开发了一种可视比色检测肿瘤细胞的方法。该分析主要是基于单链DNA能够展开并暴露出碱基部分,吸附到AuNPs表面,从而使得AuNPs在高盐浓度下保持稳定。当将CCRF-CEM细胞悬浮液和单链寡核苷酸适体sgc8c孵育后,适体可选择性地结合到目标细胞表面,使得离心上清液中DNA的浓度降低,导致AuNPs在高盐浓度下失去稳定并发生团聚,而非目标细胞RAJI和随机序列DNA不会引起AuNPs溶液颜色的变化。该方法在肉眼比色分析和光谱分析方面都具有很好的灵敏度。3.通过溶剂挥发法,将碳纳米管(CNTs)掺杂到壳聚糖-明胶(chitosan–gelatin, CG)复合材料中,显著增强了CG复合材料的机械性能,将扩大其在骨组织工程中的应用前景。用扫描电镜、接触角测试和拉伸测试表征了CG/CNTs材料的表面形貌、亲水性和机械性能。CNTs均匀地分散在CG膜的基质中;与CG膜相比,仅添加0.4% CNTs,纳米复合材料的杨氏模量就增强了180%。利用人成骨细胞MG-63研究了复合材料的生物相容性,显微镜观察、吖啶橙染色和MTT研究结果表明,CG/0.4% CNTs不会影响细胞的贴壁、铺展和增殖。更多还原

【Abstract】 Accurate, sensitive methods for leukemia diagnosis facilitate the selection of effective therapeutic pathways by clinicians. Current methods are time-consuming, expensive, and require advanced instrumentation. Some more cost-effective methods requiring simple or no instrumentation yet still providing great sensitivity and accuracy would be ideal for point of care diagnosis. Aptamers, single-stranded DNA/RNA probes, are poised to become a chemist’s antibody and have the potential to serve as molecular probes for a variety of biomedical applications. In this thesis, a series of studies were carried out based on aptamer for the detection of cancer cells. On the other hand, the construction of nanocomposites is of great significance for promoting the development of bone tissue engineering. The main work of this thesis is summarized as follows:1. An electrochemical biosensor was developed for CCRF-CEM acute leukemia cells based on the thiol-terminated aptamer (sgc8c) self-assembled onto gold electrode surface as recognition probe. The biosensor provided a simple, convenient, low-cost and label-free method for early leukemia diagnosis. The modified electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface density of aptamers was determined by chronocoulometric method. Upon incubation of the aptamer-modified electrode with CCRF-CEM cells, the electron-transfer resistance (Ret) increased substantially. The selectivity of the sensor was excellent. A linear relationship between Ret and logarithmic value of CCRF-CEM cells concentration was found in the range of 1×10~4 to 1×10~7 cells/mL, with a detection limit of 6×10~3 cells/mL.2. A direct visualization method for cancer cell detection was developed by using unmodified gold nanoparticle (AuNPs) as indicator. The assay is essentially based on that ss-DNA can uncoil sufficiently to expose its bases, then absorb onto the AuNPs surface, and can make AuNPs stabilization at high salt concentration. The incubation of target CCRF-CEM cells with oligonucleotide strand aptamer sgc8c made the aptamer concentration in centrifugal supernatant significantly decrease. AuNPs lost the stabilization and aggregated under high salt concentration, while RAJI control cells and random sequence DNA did not elicit change in color. The assay showed excellent sensitivity with both the naked eye and based on absorbance measurements.3. In this study, biopolymer chitosan-gelatin/multiwalled carbon nanotubes nanocomposites (CG/CNTs) have been successfully prepared by a simple solution-evaporation method. The carbon nanotubes enhanced the mechanical properties of the CG films for the application in bone engineering. The morphology, hydrophilicity and mechanical properties of the CG/CNTs nanocomposites have been characterized with field emission scanning electron microscopy (SEM), contact angle and tensile tests. The CNTs were homogeneously dispersed throughout the chitosan-gelatin matrix. When compared with chitosan-gelatin film, the tensile modulus of the nanocomposites was greatly improved by about 180% with incorporation of only 0.4 wt % of CNTs into the chitosan-gelatin matrix. The growth behaviors of MG-63 osteoblastsic cells on CG and CG/CNTs films were investigated with morphology observation, acridine orange (AO) staining, and MTT assay. It was found that the CG/0.4% CNTs can not significantly influence the attachment, spreading and proliferation of the MG-63 cells.更多还原