【作者】 吕丰；

【导师】 许鑫华；

【作者基本信息】 天津大学 ， 材料学， 2005， 硕士

【摘要】 由于生物素和亲和素间高度的特异性结合力、桥联作用以及多级放大作用,且多数的蛋白质、酶、抗体、DNA等可以较容易地生物素化,生物素化后并不降低其活性,使其在生物学、分子生物学、生物化学、临床医学等领域广泛应用。本文研究了生物素-亲和素自组装膜及基于该自组装膜的生物传感器在葡萄糖检测和端粒酶活性检测的效果。将生物素-亲和素系统和纳米金效应结合起来,以石英晶体微天平(QCM)为手段,实时监测了生物素-亲和素系统固定葡萄糖氧化酶的一系列过程,探讨了不同浓度亲和素、生物素标记酶对吸附的影响,生物素标记前后酶的吸附过程,比较了纳米金修饰前后酶的固定效果。结果表明,通过生物素、亲和素间的特异性结合力能够很好的固定化酶,金片表面修饰纳米金颗粒可使基片表面固定的酶量提高1倍以上。制备了基于生物素-亲和素系统固定化葡萄糖氧化酶的生物传感器,通过循环伏安曲线对电极进行了表征,讨论了酶的层数、溶液pH值、离子强度、搅拌速率、温度等各种实验条件对传感器性能的影响,测试了该传感器检测范围、灵敏度及电极的稳定性。结果表明,应用生物素-亲和素系统固定化酶制备的生物传感器有较大的电流响应及较好的灵敏度,本传感器能检测出0.5mM到40mM的葡萄糖浓度,检测范围较广,传感器的响应时间仅为6s。以Bio-dATP作为聚合酶链式反应(PCR)底物,对端粒酶活性下PCR扩增反应的端粒DNA进行生物素标记,分别用压电石英晶体生物传感器和表面等离子基元共振生物传感器检测端粒酶活性,提出了新的检测端粒酶活性的方法。结果表明,用生物传感器方法检测端粒酶活性快速方便,通过纳米微粒质量放大及增效技术,提高了检测精度和灵敏度,缩短了响应时间,能够较好地分辨端粒酶的阴性和阳性以及不同种类端粒酶的活性,降低了检测下限。更多还原

【Abstract】 The biotin-avidin system (BAS) has the especial appetency, great link and amplificatory function. Meanwhile, most proteins, enzymes, antibodies and DNAs can be linked with biotin easily, which do not decrease their activities. Therefore, BAS can be widely used in the fields of biology, molecular biology, biochemistry and medicine. In this paper, the self-assembly film based on BAS was researched. Furthermore, the biosensor based the technology was applied to test the strength of glucose and the activity of telomerase. BAS and the effect of nano-Au were combined in the experiment. A series of processes of glucose oxidase (GOx) immobility base on BAS were monitored by quartz crystal microbalance (QCM). The impacts such as the strength of avidin and bio-GOx, the effect of biotin and the modification of nano-Au, were discussed. The results show that it is beneficial to immobilize enzyme by BAS and the enzyme of immobility doubled after nano-Au modification. The GOx biosensor was prepared based on BAS. The electrode was charactered by cyclic voltammetry (CV). The conditions such as temperature, pH value, ionic strength and layers of enzyme, which affected the strength of biosensor’s current, were studied. The ranger of detecting, the sensitivity and the stability of electrodes were tested. The results show that the biosensor has a fast current response of 6 s and linear calibration range of 5.0×10-4 to 4.0 × 10-3 M. It has advantages of sensitivity and quick response. Based on bio-dATP, the telomere DNA of Polymerase Chain Reaction (PCR) under the activity of telomerase was labeled by biotin. The activity was examined by the biosensors including QCM and IAsys, which was an innovative technique. The results mean that it is convenient for diagnosing the activity of telomerase to apply the biosensor. The biosensor has a beneficial sensitivity and the fast response time by the modification of nano-Au. It can distinguish the different telomerase with varied activities. Its limit for telomerase activity can be optimized.更多还原