标题: | 建构功能性奈米金球之核酸配体电化学生物感测试片运用于凝血酶的检测 Fabrication of The Aptamer-based Electrochemical Biosensing Strips with Functionalized Au Nanoparticles for Thrombin Detection |
作者: | 刘庭妤 Liu, Ting-Yu 林志生 Lin, Chih-Sheng 分子医学与生物工程研究所 |
关键字: | 网版印刷碳电极;核酸配体;凝血酶;奈米金球;电化学;Screen-printed carbon electrode (SPCE);Aptamer;Thrombin;Gold nanoparticles;Electrochemistry |
公开日期: | 2009 |
摘要: | 本研究系以网版印刷碳电极(screen-printed carbon electrode, SPCE)建构一高灵敏度,且具放大效应之电化学三明治式生物感测平台,并针对凝血酶(thrombin)进行检测。Thrombin为一多功能的丝氨酸蛋白酶,其在凝血机制中为不可或缺之要角,在心血管疾病中亦扮演重要角色,并为发炎反应之重要调控因子。人体内thrombin浓度的改变为某些病理学上的征状,其包括血栓栓塞疾病、癌症、神经退化性疾病等等。为建构一电化学生物感测SPCE试片,我们首先利用13 nm之奈米金球(Au nanoparticles, AuNPs)与二茂铁(ferrocenedicarboxylic acid, FeDc;做为介电子)进行工作电极之表面修饰,藉以改善其导电度和感测表现。接着将卵白素(streptavidin)固定于电极表面,再以5’端修饰生物素(biontin)之核酸配体(aptamer)(序列为5’-biotin-(T)25 GGT TGG TGT GGT TGG-3’)经由streptavidin-biotin亲和力键结方式将aptamer修饰于SPEC电极表面,此aptamer形成SPEC试片的第一层thrombin专一性的侦测探针。在本SPEC试片检测策略中,当thrombin被aptamer辨识捕获后,再运用第二层thrombin专一性探针进行反应,此探针为anti-thrombin抗体(Antibody, Ab)与辣根过氧化氢酶(horseradish peroxidase, HRP)共同修饰在AuNPs表面所组成的AuNPs/Ab-HRP(即功能性AuNPs),AuNPs/Ab-HRP可辨识被SPCE试片捕获之thrombin。上述所建构的SPCE试片以H2O2作为HRP的受质,并藉由侦测反应电流讯号强度即可做为所检测样品中thrombin的浓度计量。在本研究中,利用循环伏安法(cyclic voltammetry)可测得修饰之SPCE电极表面的电化学特性,在样品检测上则运用安培检测法于相对参考电极300 mV的电压下进行检测。当所侦测thrombin的浓度范围在1 □ 10-11至1 □ 10-7 M时,可得到一thrombin浓度与检测电流强度间的直线正相关性,而thrombin的侦测低限为5 □ 10-12 M。所建构的SPCE试片也被用来检测血清中thrombin浓度,其可侦测之thrombin浓度范围为1 □ 10-10至1 □ 10-7 M。另外,为了测试本SPCE试片运用于临床检测的可行性,血浆中thrombin的生成量也被检测,其结果显示血浆中的thrombin生成量可随着prothrombin之转换率增加而增加。总结,在本研究中所建构之aptamer生物感测SPCE试片与功能性AuNPs(即AuNPs/Ab-HRP)结合运用,其具潜力发展成一临床上快速医疗诊断工具。 The goal of this work was to fabricate a sensitive and amplified electrochemical sandwich assay by screen-printed carbon electrode (SPCE) strips for thrombin detection. Thrombin, a multifunctional serine protease of coagulation cascade, plays the role in cardiovascular diseases and regulates many processes in inflammation. Alteration in thrombin concentration has been demonstrated to be a common feature of many pathological conditions, including the thromboembolic disease, cancer and neurodegenerative diseases. In the present study, the surface of working electrode of SPCE was firstly modified with 13 nm Au nanoparticles (AuNPs) and ferrocenedicarboxylic acid (FeDC, as mediators) to enhance the conductivity and sensing performance. Furthermore, the streptavidin was immobilized onto the electrode surface of SPCE. Then, the biotinylated aptamer (5’-biotin-(T) 25 GGT TGG TGT GGT TGG-3’) was immobilized onto the SPCE via streptavidin-biotin affinity binding. The applied aptamer could be as primary probe to capture thrombin in the detected sample. The secondary probe applied in the SPCE strips was anti-thrombin antibody (Ab) and horseradish peroxidase (HRP) co-modified AuNPs (AuNPs/Ab-HRP) that were used for recognizing thrombin captured on the SPCE. Hydrogen peroxide was used as the substrate for HRP and the response signal of current can be detected. The electrochemical property of the modified SPCE was characterized by cyclic voltammetry. Amperometric detection was performed to produce the response signal at a potential +300 mV vs. counter/reference electrode. Under optimal conditions, the detection sensitivity showed a linear response for thrombin in the range of 1 □ 10-11 to 1 □ 10-7 M with the detection limit of 5 □ 10-12 M. The established biosensing platform was used to detect serum thrombin with the linear range for thrombin concentration was from 1 □ 10-10 to 1 □ 10-7 M. To test the reliability of our developing method used in clinical detection, the generation assay of plasma thrombin was also investigated and the result shows that plasma thrombin was increasing dependent on the conversion efficiency of prothrombin. In conclusion, combination of the aptamer-based biosensing SPCE strip and the use of AuNPs/Ab-HRP developed in this study provide a potential tool for clinical applications in medical diagnosis. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079750504 http://hdl.handle.net/11536/45805 |
显示于类别: | Thesis |