【作者】 逯超亮；

【导师】 常津；

【作者基本信息】 天津大学 ， 材料学， 2010， 硕士

【摘要】 单分散微米级聚苯乙烯微球具有形貌规则、粒径均一、良好的表面反应能力和易于功能化等优点,在免疫技术和疾病早期诊断等领域具有很高的研究和应用价值。在液相生物芯片的应用中,如何制得高性能聚苯乙烯微球并实现编码,对液相生物芯片实现快速、高通量检测起着至关重要的作用。本文研究了单分散、粒径可控的功能化聚苯乙烯微球的制备,探讨了不同的微球编码方式,实现了在免疫技术方面的初步应用。首先,以分散聚合制备的直径2μm左右的聚苯乙烯微球为种子,通过种子聚合的方法制备了粒径在5-15μm的羧基化聚苯乙烯微球,并对种子聚合的机理、共聚的可行性进行了探讨,同时探索了单体溶胀时间、苯乙烯的加入量、甲基丙烯酸的加入量等反应条件对微球粒径和形貌的影响;通过酶联免疫反应评价了微球的表面反应能力和羧基的活性。其次,通过在种子聚合单体溶胀阶段加入致孔剂甲苯,制备出多孔聚苯乙烯微球,探讨了交联度和甲苯用量对微球表面孔径大小和分布的影响;用不同发射波长的量子点对多孔微球进行荧光编码,制备出量子点荧光编码微球,并对多孔荧光微球荧光性能进行了相应的表征;用多孔的量子点编码荧光微球进行免疫反应实验,并通过光纤光谱仪对免疫反应后微球的荧光光谱进行扫描分析。扫描电子显微镜分析表明,制得的微球形貌规则、粒径高度均一,流式细胞术分析表明,不同粒径的微球分属不同的点群并能够完全分开,因此可通过微球粒径差异实现编码;红外谱图和核磁谱图证明,在种子聚合中甲基丙烯酸通过共聚的方法被成功引入到了微球表面,电导滴定测得聚合物微球表面羧基含量为0.3mmol/g,免疫反应实验进一步证实微球表面羧基具有良好的反应活性。量子点荧光编码微球荧光谱图表明可基于微球中量子点的最大发射峰位实现编码;激光共聚焦显微镜表征显示,相比于无孔致密的荧光微球,多孔荧光微球内部荧光强度分布更加均匀,证明量子点通过多孔微球的孔道渗入了微球内部且均匀分布;免疫反应实验证明多孔荧光微球能够稳定地结合抗原(人IgG),并且能够特异识别相应的抗体(量子点标记的羊抗人IgG)。本课题的研究成果为液相生物芯片中编码微球的研发提供了重要的理论支持和实验依据,在生物识别和检测领域具有广阔的应用前景。更多还原

【Abstract】 There is extensive research and using value of monodisperse micron-sized polystyrene microspheres in immunological technique and early diagnosis of diseases. It is attributed to many advantages of the microspheres such as good spherical shape, uniform size, high surface reaction capability, and easily to be functionalized. The preparation of high-performance encoded beads play a critical role in the field of suspension array biochip for rapid and multiplex detection.In this work, the preparation of monodisperse functionalized polystyrene microspheres, encoding methods and their applications in immunological technique were investigated. Firstly, the 2μm polystyrene seeds were synthesized by dispersion polymerization. And carboxylated polystyrene microspheres with the diameter in the range of 5-15μm were prepared via seed polymerization. The mechanism of seed polymerization and the copolymerization feasibility of styrene with methacrylic acid (MAA) were studied. The parameters influencing the diameter and morphology of microspheres, including the monomer swelling time, the content of styrene and MAA, were investigated. The reactivity of carboxyl group on the surface of microspheres was confirmed by enzyme linked immunosorbent assay (ELISA). Secondly, for producing porous polystyrene microspheres, toluene as the porogen was added into the system during the monomer swelling stage of seed polymerization. The effect of the toluene content and the crosslinking degree on the size and distribution of pore on the microsphere surface were investigated. Quantum Dot-encoded fluorescent microspheres were successfully obtained by tagging quantum dots (QDs) with different fluorescence emission peaks into porous microspheres. The fluorescent properties of the porous quantum dot-encoded fluorescent microspheres were characterized. The porous quantum dot-encoded fluorescent microspheres were used in immunoreactions and their fluorescence spectra were obtained by optical fiber optic spectrograph.The monodisperse and spherical microspheres was observed in the scanning electronic microscope (SEM) photographs. In flow cytometry analysis, the microspheres with various sizes can be completely separated into different groups, which show the diameter-encoded method can be validly employed in the multiplexing detection systems. The results of FTIR and 1HNMR indicated that the MAA monomer was successfully copolymerized to the microspheres. The content of carboxyl group on the microsphere surface was 0.3mmol/g via conductometric titration. The high reactivity of carboxyl group on the surface of microspheres was well established by immunoreactions. The fluorescence photographs and spectrums showed that the quantum dot-encoded fluorescent microspheres were effectively encoded using QDs with different emission wavelengths. The fluorescent intensity distribution of quantum dot-encoded fluorescent microspheres was characterized by the laser confocal fluorescence microscopy. The results showed that the QDs permeated into the porous microspheres effectively via the interior pores, offering a more homogeneous interior fluorescent intensity than the non-porous ones. The immunoreactions showed that the porous quantum dot-encoded fluorescent microspheres can be sensitized by antigen (human IgG) stably and capture the antibody specifically (QDs labled goat-anti-human IgG).This work is beneficial to fabrication of polymer beads for suspension array biochip, and this robust study can be further utilized to the field of immunological technique and early diagnosis of diseases.更多还原