【作者】 盖宏伟；

【导师】 林炳承； 马银法；

【作者基本信息】 中国科学院研究生院（大连化学物理研究所） ， 分析化学， 2005， 博士

【摘要】 单分子检测可以归属于分析化学领域的前沿技术之一,它不但实现了某种意义上可称之为最高灵敏度的分子检测,而且有可能实时监测反应途径和追踪大分子在执行生理功能时的结构变化,因此有望发现在其他常规实验中难以发现的实验现象。单分子荧光成像是实现单分子检测的手段之一。本论文主要针对单分子荧光成像检测的实现及其在溶液体系中的应用开展研究。主要包括:自行搭建了一套激光诱导荧光单分子成像装置,分别完成了多染料标记的单个λDNA分子在普通宽场和隐失场中的荧光成像及单个染料─罗丹明6G分子在隐失场中的荧光成像。建立了多染料标记分子和单染料标记分子的单分子荧光成像方法。这两种对象被分别应用到流体力学测速研究和分子相互作用研究中。在考察了微通道中静压力驱动样品的可行性之后,建立了在单分子水平上同时测量微通道中主体流速和近壁流速的方法。以175nm荧光颗粒和λDNA分子为对象在一次实验中同时测定了微通道中主体流速和近壁约300nm处的流速。主体流速和近壁流速测定的实验结果符合经典流体力学计算公式。观察到了微球与通道壁面的碰撞和微球进出隐失场时的速度变化。以单个异硫氰酸荧光素(FITC)标记的系列寡核苷酸分子和单个2-氨基吖啶酮(2-AMAC)标记的系列多糖分子为对象,观察分子个数和分子量的关系,发现了隐失场中检测到的分子个数随分子量的增大而减少。因相互作用而结合的分子复合物的分子量大于相互作用前的分子分子量,隐失场中检测到的分子复合物的分子个数应小于分子反应物的分子个数。据此提出了一种无需分离而鉴定分子相互作用的新方法。以肝素和G-CSF的相互作用验证了这一方法的可靠性。更多还原

【Abstract】 Single molecules detection (SMD) is one of the important techniques in the field of analytical chemistry. SMD is one of methods with the highest sensitive detection. It can be used to monitor the reaction pathway and trace the conformations dynamic of large biomolecules. The fluorescence imaging of single molecules is one of ways to detect individual molecules. It was realized and applied in this thesis, which was described as follows.A single molecules fluorescence imaging instrument was built up. It was calibrated by imaging singleλDNA molecules and Rhodamine 6G. Thus, the fluorescence imaging methods for single molecules labeled with multi-dyes and single dye were respectively realized. These two kinds of molecules were independently applied to studies of hydrodynamic and molecules interactions.After the validity of hydrostatic pressure used in microchannel was confirmed, a laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory. The collisions of microbeads with channel walls were directly observed. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.Series samples of oligonucleotides labeled with single Fluorescin isothiocyanate更多还原