| Summary: | 碩士 === 國立交通大學 === 分子醫學與生物工程研究所 === 98 === 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 ?e 10-11 to 1 ?e 10-7 M with the detection limit of 5 ?e 10-12 M. The established biosensing platform was used to detect serum thrombin with the linear range for thrombin concentration was from 1 ?e 10-10 to 1 ?e 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.
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