【作者】 石岩；

【导师】 陆祖康；

【作者基本信息】 浙江大学 ， 光学工程， 2007， 博士

【摘要】 基于微流通道的生物分析技术是目前生物检测的研究热点，它涉及微细加工、生物工程、分析化学、高分子科学、光学工程等多个学科领域。我国在相关技术及其应用的研究中虽然已有很大发展，某些方面已达到或接近国际先进水平，但在相关科学仪器的关键技术如快速，高灵敏度检测研究方面，仍明显落后于世界先进水平。本论文研究应用于悬浮荧光微球阵列和毛细管电泳DNA分析的微流通道系统及其快速高灵敏度荧光检测技术。研制的基于微流通道的悬浮荧光微球阵列并行检测系统，采用脉冲氙灯对微流通道中二维分布的流动荧光微球连续曝光，并用CCD进行多微球成像，对获取的荧光图像进行微球识别，实现并行检测，首次获得了微流通道中直径6μm荧光微球的动态图像。系统的检测限可达荧光分子密度10μm^-2以下，检测速度可达每秒钟30000个荧光微球，比流式细胞术检测提高1～2个数量级。研制的毛细管电泳DNA分析系统，采用488nm或532nm激光激发毛细管中电泳分离的染色DNA，以f-theta物镜收集荧光并由光电倍增管进行共焦探测。利用实验优化的电泳条件，在系统上进行了双链DNA片段样品的单碱基分辨分离；以有效长度14cm的毛细管完成了STR基因位点的电泳分析，在23分钟内使全部基因位点基本达到基线分离，检测速度比同类的ABI 310仪器快2倍。模拟与实验研究毛细管微流通道中DNA电泳荧光检测的荧光和噪声空间分布，发现筛分介质的瑞利散射噪声和毛细管内外壁的折反射噪声相比可以忽略，最佳荧光收集方向位于与激发光成75°方向。论文提出了一种微芯片上微流通道检测的新结构，该结构以棱镜耦合激发光，以椭球反光碗将荧光会聚到微芯片外的远焦点，缩小荧光发散角，理论上物镜工作距离要求可以降至无限小，荧光收集效率可以提高6.3倍。更多还原

【Abstract】 Biological analysis technology based on microfluidic channel is a focus for scientists in the field of biological detection, which covers multi-disciplines such as microfabrication, biological engineering, analytical chemistry, polymer science, optical engineering and so on. Correlative technologies and applications have developed greatly in China. Some research results have reached or have been closed to international top level. But in some key technologies such as high speed detecting and high sensitivity detecting, our research still has a large gap with the international top level. This dissertation carries on the research on fluorescence detecting technology with fast speed and high sensitivity used for suspension fluorescence microspheres’ array and capillary electrophoresis system for DNA analyzing.The parallel detecting technology for suspension fluorescence microspheres’ array in this dissertation adopted a pulsed Xenon lamp to expose flowing fluorescence microspheres serially, which had a 2D distributing in microfluidic channel. CCD was used to capture images of multi-microsphere at the same time. Microspheres were recognized in fluorescence images and parallel detection was achieved. Dynamic images of fluorescence microspheres with diameter of 6 microns were captured for the first time in the system. Limit of detection of the system was fewer than 10 fluorescence molecules per square micron. Detecting speed could reach 30000 fluorescence microspheres per second, which was higher than that of flowing cytometry by 1～2 orders of magnitude.DNA analyzing system by capillary electrophoresis in the dissertation adopted a laser of 488nm or 532nm to excite fluorophore labeling DNA separated by electrophoresis in a capillary. Fluorescence was collected by an f-theta lens and was detected by a PMT with confocal optical arrangement. The electrophoresis condition was optimized in experiments. Due to the system optimization, dsDNA fragments" were separated and single base pair resolution was achieved in the system. Electrophoresis analysis of Short Tandem Repeats loci in a capillary with effective length of 14cm could be completed. Resolution for all loci couldbe closed to baseline level in 23 minutes, which was shorter than half of the time used by ABI 310. Research on spatial distribution of florescence and noise indicated that compared with the noise caused by refraction and reflection from inner and outer walls of the capillary, noise reduced by Rayleigh scattering could be ignored. The optimal angle between the fluorescence collecting direction and the exciting laser beam was 75° .At last, a new architecture of fluorescence detecting for microfluidic channel in microfluidic chip was presented. In this design, a prism was utilized to couple exciting laser into the microfluidic channel and an elliptical reflector was used to focus fluorescence outside of the microfluidic chip and to reduce the divergent angle of fluorescence. Theoretical requirement for working distance of objective for this structure could be reduced to be infinitely short. Fluorescence collection efficiency was improved for about 6.3 times.更多还原