【作者】 杨柳；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2008， 博士

【摘要】 生物大分子所构筑的细胞表面(细胞壁、细胞膜)是细胞与外界环境间进行物质和能量交换的屏障,也是一些物理、化学因素作用于细胞的靶点之所在。在纳米水平上研究细胞表面的基本结构及其受外界环境作用前后的变化,对于认识细胞表面结构与功能之间的关系,全面评价药效并对药物合成路线提供参考都具有重要意义。另一方面,单碱基错配的检测对于从分子水平上阐明多种疾病形成的原因,实现基因水平的治疗都是至关重要的前提条件。发展具有高灵敏度、高选择性的单碱基错配检测方法势在必行。上述两个研究领域都已经成为生物分析化学研究的前沿和热点。本论文以上述科学问题为研究目标,充分利用原子力显微镜(AFM)能够在大气及液下高分辨成像和对微弱相互作用力敏感的优势,表征了不同药物以及脉冲电场(PEF)对细菌细胞壁表面形态和构成的影响,考察了细胞在大气氛和液下成像的条件,发展了基于力测定并结合发夹型探针的高灵敏、高选择性的单碱基错配检测方法。主要内容归纳如下:1、基于AFM大气氛中成像,研究天然与半合成内酰胺类抗生素对大肠杆菌形态的影响。选择具有微小分子结构差别的天然(青霉素)和半合成(阿莫西林)抗生素,分别对大肠杆菌进行作用。利用AFM大气氛中制样简便、轻敲模式成像分辨率高、样品损伤性小的优势,对药物的作用效果进行了表征。观察到这两种药物在细菌表面造成的纳米水平上小孔状损伤的密度和发生位点均存在区别,从形态学角度解释了阿莫西林比青霉素药效强的原因。作为对照的氧氟沙星未使细菌表面出现显著的损伤。该结果在纳米水平上证明了不同抗生素的药效,同时也表明AFM能够为研究药物分子结构与作用效果的关系提供新的表征手段。2、结合AFM液下成像和力测定两种技术,研究了PEF对细菌表面结构与构成的影响。对通过静电吸附固定于基底上的细菌样品,原位施加不同剂量的PEF,表征了菌体形态及其与AFM探针的相互作用力的变化。结果表明随着PEF剂量的增加,细菌表面损伤的程度逐渐增大,具体表现为细菌数目的减少、相互作用力的增大以及力曲线上出现多粘附相互作用特征区。对照实验表明相互作用力的变化是由于细胞壁外层的肽聚糖分子网络状结构被破坏,从而使内膜中的蛋白组分暴露所致。该结果在分子水平上证明了PEF对细菌表面的影响,同时也表明AFM是研究细胞表面组分变化与外界物化刺激之间相互关系的强有力的工具。3、气氛中及生理环境下AFM细胞成像的实现及条件优化。首先,比较了三种常用的固定剂对三种细胞在大气氛中成像效果的影响。其中,0.5%的戊二醛具有对细胞细微结构的最佳保留能力。在此基础上,进一步考察了扫描速度和不同培养基环境对细胞液下成像的影响并进行了条件优化。贴壁生长的肾系膜细胞和肾小管上皮细胞均可在培养基中直接进行成像,细胞骨架清晰。该结果为将AFM成像和力测定及其联用技术应用到细胞水平搭建了一座桥梁。4、AFM力谱分析用于增强发夹型探针的单碱基错配检测能力。我们将探针与完全互补和一系列含有不同类型单碱基错配的互补序列分别修饰到针尖与基底上,通过测量探针与完全互补以及错配序列之间的相互作用力,对单碱基错配进行检测。不同于荧光检测时灵敏度受到碱基错配位置和序列中GC含量的显著影响,这种分子水平的相互作用在检测时不受错配碱基类型和位置的影响,均可实现高灵敏度检测。该方法解决了在利用荧光方法检测单碱基错配时,发夹型探针存在的序列依赖性问题,大大增强了发夹型探针的错配检测能力。更多还原

【Abstract】 Cell envelope, including cell wall and cell membrane is the interface between cell and outer environment for material and energy exchange. It also provides protection of cell organelle and target sites from chemical or physical stimulations. The investigation of cell envelope at the nanometer level is very important in understanding the relationship between its structure and function, comprehensively evaluating the drug efficacy and providing evidence toward the development of medicines. On the other hand, the detection of single base mismatch is critical to illuminate the nosogenesis of some disease and realize the gene therapy, which appeals us to develop convenient, accurate and sensitive assays. Both of the above two issues are the promising directions in bio-analytical chemical research, and deserve devotion of analytical chemists.In this dissertation, utilizing the advantage of the atomic force microscopy (AFM), including the characterization of the surface topography at the atomic level and the measurement of intermolecular forces as low as 10-12 N (pN)grade, we investigated the effect of different antibiotics and the pulsed electric field (PEF) on cell envelope, established the optimized condition for the cell imaging and improved the detection ability toward the single base mismatch on the basis of the hairpin probe. The main researches of this dissertation are summarized as follows:1. AFM study of different effects of natural and semisyntheticβ-lactam on the cell envelope of E.coli. The distinct effect of amoxycillin and penicillin has been investigated using tapping mode imaging in air. The results indicated that although amoxycillin and penicillin could both induce nanoporous damage to the envelope of E. coli, the distribution of the pores was different: those induced by penicillin were randomly distributed on the cell surface, while those induced by amoxycillin were far more numerous and mainly on the two ends of the cell. These findings could explain why the effect of amoxycillin is stronger than that of native penicillin. Ofloxacin was used as a control due to its inactive membrane, and no cell wall damages were observed. It has been demonstrated that AFM is a useful tool in discerning and verifying antibiotic mechanisms at nanometer level and can be helpful to explain the relationship between chemical structure and the function of antibiotics.2. The effect of PEF on the cell envelope components of S. epidermidis was investigated by the combination of AFM imaging and force measurement under PBS. The topography of bacteria and interaction force between bacterial envelope and tip was probed in situ before and after different dosages of applied PEF. The results showed that the introduction of PEF would decrease the amount of the immobilized cell and induce the multi-unbinding force between AFM tip and the cell surface, both of which were more obvious with the increasing dosage of PEF. It was deduced that PEF could induce the collapse of the peptidoglycan layer and then the exposure of the plasma membrane, which was further confirmed by the effect of the lysozyme and heat on the bacterial envelope. The above conclusions offered strong evidence for the bactericidal mechanism of PEF at the molecular level, and demonstrated that the AFM is a powerful technique to explain the relationship between the chemical component change of the cellular envelope and the external stimulation.3. cell imaging was realized in air and under liquid, and the experimental conditions were optimized. First, three kinds of cell fixed with different fixatives were imaged in air and the qualities of image were evaluated individually. Among them, 0.5% glutaraldehyde kept the fine structure of all cells. Then, all these cells were successfully imaged under physiological media. The effect of the scan rate and different medium on cell imaging was investigated. Both MC and NRK cell could be imaged with clear cell cytoskeleton in the culture medium and the duration was about one hour. These results paved the way for the further cell investigation based on AFM technique.4. Using force spectroscopy analysis to improve the properties of the hairpin probe. The sensitivity of hairpin-probe-based FRET fluorescence analysis was sequence-dependent in detecting single base mismatches with different positions and identities. Through modified tip and substrate with hairpin probe and complementary sequence individually, the detection sensitivity of single base mismatch was systematically investigated by force spectroscopy analysis. The uneven fluorescence analysis sensitivity was obviously influenced by the GC contents as well as the location of the mismatched base. However, force spectroscopy analysis displayed a high and even sensitivity in detecting differently mismatched targets. This could be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe and to enhance the probe ability in mismatch detection.更多还原