【作者】 陈韵晴；

【导师】 王柯敏；

【作者基本信息】 湖南大学 ， 分析化学， 2006， 硕士

【摘要】 微芯片技术是当今化学与生命科学研究领域发展最快的重要前沿之一。一维微流控微珠阵列是本研究小组近年来发展起来的一种新型的生物分析芯片,这种分析芯片在阵列设计的灵活性、液流控制的简便性、试样的低消耗方面具有优异的性能。在此基础上,针对微珠表面的抗体固定化所面临的问题以及蛋白质分析的特点和要求,本论文重点研究了微珠表面的抗体修饰方法和蛋白质样品制备方法,优化了分析条件,并应用于蛋白质的表达分析。研究工作包括三个方面的内容:一、微流控通道的内壁改性处理及抗体微珠的制备。本文考察了聚二甲基硅氧烷(PDMS)微通道内壁对蛋白质的非特异性吸附,并对抗体微珠的制备进行了研究,制备了基于抗体微珠的一维蛋白质阵列芯片。通过对微通道内壁的改性处理,PDMS对蛋白质的非特异性吸附得到了有效的抑制。在抗体微珠的制备中,商品化小鼠单抗试剂中普遍添加的高浓度保护蛋白(常用牛血清白蛋白,即BSA)严重干扰了抗体的固定化。通过采用离心超滤对超微量抗体试剂进行预处理,大幅度改善了以BSA为保护蛋白的抗体试剂的抗体固定化效果。此外,本文对利用蛋白A改善抗体微珠的制备进行了考察和摸索。二、蛋白质检测条件的控制与优化。包括细胞总蛋白样品的制备、微珠上抗原-抗体反应及微珠洗涤条件、进样方式的选择与改进。本文对优化后的检测条件进行了考察,结果表明,一维蛋白质阵列芯片的性能得到了有效的改善。三、一维微流控蛋白质微珠阵列在蛋白质表达分析中的应用。本文以P53蛋白为检测对象,获取了一维蛋白质阵列芯片的灵敏度和检测限,也获得了实际样品A549细胞P53蛋白的拷贝数信息。本文以表达较稳定的β-Actin作为内参照,考察了不同细胞系的P53、c-Myc蛋白质表达差异、抗癌药物介导的同种细胞系的蛋白质差异表达,并用Western blotting方法进行了验证,两种方法得到了一致的分析结果。此外,本文对一维蛋白质阵列芯片应用于颈部恶性肿瘤分型开展了初步的研究。更多还原

【Abstract】 Microchip technology has become one of the most important areas in the field of chemistry and life sciences. One-dimensional (1-D) microfluidic bead arrays are kinds of novel biomolecular microanalysis platform that we proposed. These microchip platforms have excellent advantages in flexible array design, easy fluidic control and low consumption of the samples and reagents. In this thesis, based on this platform, modification of the antibodies on the bead surface and experimental optimization have been further investigated and have been applied for protein analysis successfully. This thesis includes three major parts as follows:1. Denaturalization of the inner wall of the microfluidic channels and preparation of the antibody-modified microbeads. In our investigation, non-specific protein adsorption on the inner wall of the PDMS micro-channels can be successfully restrained via proper modification of the inner wall. In antibody-beads preparation, the antibody immobilization was influenced greatly by protective proteins such as bovine serum albumin (BSA), which are commonly added at a high concentration to prevent the commercial antibody products from titer decrease. A simple pretreatment protocol based on ultra-filtration was carried out and was proved to be efficient for the BSA removal. Approach of protein A linkage for the improvement of antibody immobilization was also investigated.2. Optimization of experimental conditions. This part includes preparation of total protein samples from the culture cells, on-bead immuno-binding reaction and wash step, and choice of sampling. The optimized conditions were investigated and the results show that the performance of the 1-D protein arrays has been improved.3. Quantitative analysis of protein expression. The sensitivity and limit of detection was gained for the detection of P53 protein using the 1-D protein arrays. Also, we determined P53 copy number in average single A549 cell. In the illustration of differential protein expression analysis, a three-protein profiling of P53, c-Myc andβ-Actin was demonstrated. Pharmaceutical-mediated differential expressions of P53 and c-Myc in several kinds of cell lines were investigated and the reliability of the 1-D protein arrays were verified by Western blotting. In addition, preliminary work on neck malignant tumor typing has been carried out.更多还原