基于表面等离子体共振原理的病原体快速检测芯片的研制

发布时间：2018-02-04 15:20

本文关键词： SPR 生物传感器基因芯片病原体胶体金　出处：《重庆医科大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的:构建基于表面等离子体共振(Surface Plasmon Resonance,SPR)原理的病原体快速检测基因芯片。利用SPR传感器的快速、敏感、高效、不需标记及纯化等特点,建立一套完整的基因芯片系统检测技术,使之具有设备简单,灵敏度高,应用范围广,技术稳定易于掌握等优点,达到使基因芯片的检测技术能更适用于临床、普通实验室甚至更复杂环境的目的。方法:1.通过查阅文献报道以及临床调查,确定检测靶病原体。对各靶病原体的特征性核酸标志进行确定以及扩增引物的设计并对扩增条件进行优化确定;2.设计各靶病原体特异性寡核苷酸探针,利用生物信息学方法对探针进行计算机模拟筛选;3.在载玻片表面铺制具有SPR响应的50nm金膜层,并比较了常用两种不同直径胶体金溶液金膜铺制方法的效果;4.利用硫醇化合物表面单分子自组装层技术(Self-assembled Monolayer,SAM)固定探针,制备具有SPR响应的基因检测芯片,并对自组装时间,探针浓度等进行优化;5.将筛选出的检测探针、阳性对照探针以及阴性探针点利用SAM技术制成杂交芯片,利用酶标记化学发光法对各探针的杂交性能进行检测;6.利用SPR检测系统对所构建芯片的检测性能进行了测试;7.结合Chelex-100处理临床样品进行检测,与常规临床检测方法比较该芯片的检测特性。结果: 1.确定了7种临床常见的感染病原体及临床少见但重要的
[Abstract]:Objective: to construct surface Plasmon Resonance based on surface plasmon resonance (SPR). Based on the characteristics of SPR sensor such as fast sensitivity, high efficiency, no marking and purification, a set of complete gene chip system detection technology was established. It has the advantages of simple equipment, high sensitivity, wide range of application, stable and easy to grasp and so on. It can make the detection technology of gene chip more suitable for clinical, general laboratory and even more complex environment. Methods 1. The target pathogens were determined by consulting literature reports and clinical investigations. The characteristic nucleic acid markers of each target pathogen and the design of amplification primers were determined and the conditions of amplification were optimized. 2. The specific oligonucleotide probes for each target pathogen were designed, and the probes were screened by computer simulation using bioinformatics. 3. A 50nm gold film with SPR response was prepared on the slide surface, and the results of two kinds of gold film preparation methods of colloidal gold solution with different diameters were compared. 4. Self-assembled Monolayerian SAM (Self-assembled Monolayerian SAM) immobilized probe was developed by using the monolayer layer technique on the surface of mercaptan compounds. A gene detection chip with SPR response was prepared and the self-assembly time and probe concentration were optimized. 5. The hybridization microarray was made by using SAM technique, the detected probe, the positive control probe and the negative probe spot were used to detect the hybridization performance of each probe by using enzyme-labeled chemiluminescence method. 6. The performance of the chip is tested by SPR detection system. 7. The detection characteristics of the chip were compared with the conventional clinical detection method. Results: 1. Seven common clinical pathogens and rare but important clinical pathogens have been identified. 2.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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