【作者】 吴娟芳；

【导师】 罗国安；

【作者基本信息】 清华大学 ， 化学， 2006， 硕士

【摘要】 毛细管电泳和在其基础上发展起来的微流控芯片技术以微型化、自动化、高效性与便携化的独特优势十分适合生命分析化学的要求,本论文围绕其热点问题和发展中的关键问题进行有益探索。采用毛细管区带电泳(CZE)系统地对小檗碱和自己设计的两类不同中间碱基序列的17mer双链DNA(dsDNA)的相互作用和作用选择性进行分析。Kenndler模型分析结果显示,小檗碱对dsDNA只有微弱的结合能力,与中间碱基序列为GGCC的dsDNA相比,小檗碱易于与中间序列为AATT的dsDNA相结合,与后者的结合常数Ka约为(1.0±0.7)×103L/mol。实验还表明,在简单的电泳条件下能将17mer的单链DNA(ssDNA)和其退火形成的dsDNA相分离,使得CZE有望成为除繁琐的平板电泳之外,又一考察DNA退火程度的方式。此外,实验中还对比考察了含寡聚核苷酸的样品在内壁未经处理的毛细管和线性聚丙烯酰胺涂层后的毛细管中电泳行为的差异,显示其在非涂层柱中表现出强烈的吸附现象,充分证明了涂层柱在分析这类样品时的极大优势。微型化的特点对微流控芯片的检测灵敏度提出了更高的要求,进行样品预富集是有效手段之一。采用化学镀的方法对径迹-蚀刻技术得到纳米孔道的聚碳酸酯滤膜(PCTE)进行修饰,以缩小孔径和改变膜表面的带电性质。以SEM、TEM技术对化学镀后滤膜的表面和孔道内部形貌特征进行表征,发现化学镀后的滤膜纳米孔道呈现中间粗、两头细的雪茄状;原始标称孔径为100nm的PCTE滤膜在化学镀1h左右,表面孔径迅速达到30nm左右,此后的下降速度则极为缓慢。以光刻和注塑法制作了上层为聚二甲基硅氧烷PDMS盖片、下层为玻璃底板、中间夹有含经过化学镀PCTE滤膜的夹心型微流控芯片,用于在线样品预富集。以别藻蓝蛋白(APC)为例,采用共焦激光诱导荧光技术为检测方法和简单的电压切换方式,进行在线预富集,在90s内快速获得了约80倍的富集倍数。进一步实验又考察了洗脱电压对富集后APC出峰的影响,证实了以分子大小为机理的富集机制。更多还原

【Abstract】 Capillary electrophoresis (CE) and CE based microfluidic chip fit for the bioanalytical chemistry quite well by right of their unique characteristics of micromation, automation, high efficiency and portability. This work has done some meaningful exploration on some key problems within this field.Two double stranded DNA (dsDNA) with different central base pairs were designed to systematically investigate the binding affinity and sequence specificity of berberine with dsDNA by capillary zone electrophoresis (CZE). The data analysis with the Kenndler model proved only low affinity between dsDNA and berberine and suggested some weak binding preference of berberine for AATT-containing to GGCC-containing dsDNA. The binding constant, Ka, between berberine and the former one is about (1.0±0.7)×103L/mol. In addition, the separation of single-stranded DNA (ssDNA) from dsDNA under simple electrophoretic conditions enabled CZE to be a potentially alternative tool to check the extent of DNA annealing, which is usually done by the time-consuming and labor-intensive slab electrophoresis. Besides, we compared the electrophoretic behaviors of dsDNA in an uncoated capillary and in a linear polyacrylamide coated capillary and the results suggested that there was a strong adsorption of dsDNA to the inner wall of uncoated capillary. All these above prove that the coated capillary has a large advantage in analyzing samples of this kind.The characteristic of micromation has put forward a higher requirement to improve the detection sensitivity of microfluidic chip. One of the most effective methods is to carry out sample preconcentration before analysis. The polycarbonate membrane with nanochannels created by track-etch treatment (PCTE) was electroless plated with Au in order to reduce the nanochannel ID and change the charge condition of the membrane surface. SEM and TEM were used to characterize the morphologies of the membrane surface and theinner wall of the nanochannels after electroless plating, where cigar-shaped nanochannels were observed with bottlenecks at both ends but a large ID in the middle. After an hour of plating, the ends ID of the nanochannel, which were 100nm originally, were soon reduced to about 30nm and then slowed down. Photolithography and injection molding were employed to fabricate a microfluidic chip with a polydimethylsiloxane (PDMS) cover plate and a glass bottom plate and an electroless plated PCTE membrane sandwiched between them. This microfluidic chip was later used in preconcentrating the sample of allophycocyanin (APC). By combining the confocal laser induced fluorescence detector and simple bias switching method with the microfluidic chip, an enrichment factor of almost 80 was quickly obtained within 90s. Further experiments were also done to measure the influence of the eluting bias on the APC eluting peak, which proved a molecular size based enrichment mechanism.更多还原