【作者】 刘斌；

【导师】 张小玲；

【作者基本信息】 陕西师范大学 ， 分析化学， 2006， 硕士

【摘要】 细胞内的重要生理活性物质有很多，如钙，锌，铜，氧等。它们广泛分布于人体的细胞和体液之中，对人体的生命活动起着重要的作用。随着人们对胞内重要生理活性物质的重要作用以及他们与某些疾病发生和发展关系的认识越来越深入，胞内重要生理活性物质的研究已成为化学，生物学，基础和临床医学等学科的重要前沿热点研究课题。目前胞内重要生理活性物质的研究方法很多。人们常常根据各种方法的特点，按照所进行实验的具体要求而选择不同的测定方法。如生物发光蛋白法、有机显色剂法、离子传感器法、微电极法、核磁共振法，荧光探针法等。其中又以荧光探针法研究居多。传统的荧光探针具有反应迅速、测定灵敏度高、选择性好、线性范围宽等优点，但在生物医学研究应用中也存在一些难以克服的缺陷，如有可能产生的细胞毒副作用、与细胞内物质的螯合作用、与蛋白质的键合、指示剂泄露、光漂白效应等，因而对检测结果存在很大的干扰。另外，传统的荧光分子荧光标记方法，只能在生物分子的活性基团上连接少数的几个荧光分子，分析灵敏度有限。人们经过大量的研究提出了很多的解决方法，如双波长比率法，纳米微胶囊包被法等，尤其是纳米微胶囊包被法可以有效的解决上述问题。在很大程度上克服了传统荧光试剂的缺陷，荧光染料的稳定性显著提高，避免了荧光染料与生物活性物质直接接触所带来的副作用，而且光漂白现象也大大减少。为此，本论文利用我们实验室自行合成的新型Ca²⁺荧光探针STDBT和STDIn，采用纳米微胶囊包被法合成了两种细胞钙荧光纳米微胶囊传感器一生物包埋胶囊探针（probe encapsulated by biologicaUy localized embedding，PEBBLEs） STDBT和STDIn，并采用透射电镜、荧光光谱法、激光共聚焦显微镜和流式细胞仪等手段对所制备纳米胶囊探针的形态及粒径分布、光谱性能、对Ca²⁺的响应性能、发光稳定性及细胞标记性能等进行了系统的研究，与未包被的探针染料进行了对比。结果表明，所制备纳米胶囊探针粒径小（30nm），分布均匀，形态规则，表面圆润，发光性质稳定，对Ca²⁺响应灵敏，且通过与细胞简单共培育即可有效的进入细胞，其中STDBT纳米胶囊探针主要标记胞核区域，标记后显微镜下至少可观察2h以上。为胞核内Ca²⁺的测定研究提供有效的手段。 本研究论文主要包括以下两个部分： 一、概述 论文对纳米微胶囊包被法合成聚合物型荧光纳米微胶囊传感器—生物包埋更多还原

【Abstract】 Abstract Changes in intracellular ion concentrations accompany many processes in living cells, including transport, signaling, and enzyme function. One of the most widely studied ion, calcium, has been found to be ubiquitous in these and other cellular processes. Several methods have been developed for studying intracellular calcium. Optical fluorescence methods for analyzing intracellular ion concentrations have become widespread biological tools. Injection of fluorescent indicator dyes into a cell combined with dualwavelength (ratiometric) imaging, confocal microscopy, twophoton fluorescence, or fluorescence lifetime imaging has provided important insights into the concentration and spatial location of ions throughout single cells. One challenge for these optical methods has been that each dye must be chosen carefully and assessed for its ability to provide accurate and reliable information from within a cell. Factors such as toxicity to the cell, intracellular sequestration, protein binding, and leakage of the dye from the cell frequently complicate the interpretation of the results and must be considered. While there are indicator dyes, such as fura-2, that work very well within various cells, many fluorescent probes suffer from the problems listed above, thus limiting the number of dyes available for reliable intracellular measurements.The probes encapsulated by biologically localized embedding (PEBBLE) sensor was devised in order to provide a fluorescence based method for intracellular analysis that circumvents many of the above-mentioned problems. The PEBBLE is a submicrometer, spherical polymer that contains one or more fluorescent dyes that are trapped within the pores of the matrix. The polymer provides a protective coating for the dyes, so that they are not affected by the cell, thereby minimizing error from protein binding and membrane/organelle sequestration. Thus, PEBBLEs can be calibrated in vitro and used in vivo, with the response of the sensors remaining consistent regardless of environment. Multiple dyes can be combined in a single PEBBLE sensor, so that a reference standard can be used to minimize errors from uneven dye distributions throughout the cell. Another advantage of PEBBLEs is that they are small enough to be biocompatible, yet large enough to be retained in the cytoplasm of the cell with no leakage of the sensor out of the cell, even after several days. Furthermore, many toxic effects of the dye are eliminated.更多还原