标题: | 利用苯硼酸衍生物作为抗体专一位向固定化并应用于前降钙素检测 The Orientation of Antibody Controlled by the Derivatives of Phenylboronic acid and The Application of Procalcitonin for Biosensing |
作者: | 边郁茹 Pien, Yu-Ju 李耀坤 Li, Yaw-Kuen 应用化学系硕博士班 |
关键字: | 石英晶体微天平;清洁金表面;苯硼酸衍生物;前降钙素;QCM;Gold clean;Phenylboronic acid;Procalcitonin |
公开日期: | 2010 |
摘要: | 败血症主要是由全身性细菌感染所引起,传统上的检测方法是酵素连结免疫吸附法(ELISA)或是以血液培养,ELISA大约需要六个小时,而血液培养更是需要两到三天的时间,耗费时间并延误治疗时程,检测血液中CRP的浓度也是常被使用的检测法,但对于鉴别细菌感染与病毒感染仍有部分误差,很容易造成医生误判,延误病情。近年来,用来判别败血症的新生物指标前降钙素 (Procalcitonin)已被研究出来,根据不同的严重程度,血液中的前降钙素浓度也会不同。因此如何检测前降钙素就成为重要的课题。 本研究是以石英晶体微天平为平台感测器,以进行生物感测,此感测器的固体材料为金电极,在尝试过许多清洗金电极的方式之后,发现使用化学方法搭配电化学方法的清洗方式对于金电极有最佳的清洗效果。 目前也有很多文献指出,金的材料与烷基硫醇反应,其表面会形成排列致密的自组装单层薄膜,因此实验设计一端为硫醇一端为苯硼酸的长链烷基化合物,利用此化合物的硫醇与金反应,固定化在金表面上,接着利用另外一端的苯硼酸对前降钙素单株抗体Fc端寡醣区,作有位向性的键结,以增加与前降钙素键结的位置提高实验的再现性,并利用噬菌体与降钙素原键结位置不同,达到增强讯号,使侦测前降钙素灵敏度达到10-11 g/ml的程度。 Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state (called systemic inflammatory response syndrome or SIRS) and the presence of a known or suspected infection. Traditional detection methods used ELISA (Enzyme-linked immunosorbent assay) or blood incubation, it takes 6 hours to screen the blood by ELISA and 2-3 days to incubate bacteria. The method was time consuming, and delay for medical treatment . Detecting CRP concentration in blood is also a method to diagnosis sepsis. Unfortunately, this kind of method also hard to seperate inflammatory and virus infection. In recent years, Procalcitonin (PCT) becomes an innovative and highly specific marker for the diagnosis of clinically relevant bacterial infections and sepsis. Procalcitonin supports early diagnosis and clinical decision making which could direct an effective therapy immediately and save unnecessary spending for critically ill patients. This study is based on Quartz Crystal Microbalance as a biosensor, sensor chip is composed of gold electrode. During different experiments, we found cleaning the gold surface using standard wash (for example: piranha, or standard cleaning method) was inefficient. The result was proved by the fluorescence labeling antibody. Previous study shows that gold substrate and thiol can form densely packed self-assembly monolayer. Base on this idea, the design for our synthesis strategy was that composed of thiol and phenylboronic acid bi-funtional probe immobilized on the gold, and coupling with antibodies via carbohydrate moiety, located on the Fc region. This method offer a gentle oriented immobilization of proteins, maintaining protein’s activity, and increase reproducibility. Furthermore, in order to increase the sensitivity during detection of low concentration, we use the Fab-phage particle to enhance the signal successfully which has different binding site between the procalcitonin monoclonal antibody and Fab-phage. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079825515 http://hdl.handle.net/11536/47603 |
显示于类别: | Thesis |