建構功能性奈米金球之核酸配體電化學生物感測試片運用於凝血酶的檢測

Abstract

本研究係以網版印刷碳電極（screen-printed carbon electrode, SPCE）建構一高靈敏度，且具放大效應之電化學三明治式生物感測平台，並針對凝血酶（thrombin）進行檢測。Thrombin為一多功能的絲氨酸蛋白酶，其在凝血機制中為不可或缺之要角，在心血管疾病中亦扮演重要角色，並為發炎反應之重要調控因子。人體內thrombin濃度的改變為某些病理學上的徵狀，其包括血栓栓塞疾病、癌症、神經退化性疾病等等。為建構一電化學生物感測SPCE試片，我們首先利用13 nm之奈米金球（Au nanoparticles, AuNPs）與二茂鐵（ferrocenedicarboxylic acid, FeDc；做為介電子）進行工作電極之表面修飾，藉以改善其導電度和感測表現。接著將卵白素（streptavidin）固定於電極表面，再以5’端修飾生物素（biontin）之核酸配體（aptamer）（序列為5’-biotin-(T)25 GGT TGG TGT GGT TGG-3’）經由streptavidin-biotin親和力鍵結方式將aptamer修飾於SPEC電極表面，此aptamer形成SPEC試片的第一層thrombin專一性的偵測探針。在本SPEC試片檢測策略中，當thrombin被aptamer辨識捕獲後，再運用第二層thrombin專一性探針進行反應，此探針為anti-thrombin抗體（Antibody, Ab）與辣根過氧化氫酶（horseradish peroxidase, HRP）共同修飾在AuNPs表面所組成的AuNPs/Ab-HRP（即功能性AuNPs），AuNPs/Ab-HRP可辨識被SPCE試片捕獲之thrombin。上述所建構的SPCE試片以H2O2作為HRP的受質，並藉由偵測反應電流訊號強度即可做為所檢測樣品中thrombin的濃度計量。在本研究中，利用循環伏安法（cyclic voltammetry）可測得修飾之SPCE電極表面的電化學特性，在樣品檢測上則運用安培檢測法於相對參考電極300 mV的電壓下進行檢測。當所偵測thrombin的濃度範圍在1 □ 10-11至1 □ 10-7 M時，可得到一thrombin濃度與檢測電流強度間的直線正相關性，而thrombin的偵測低限為5 □ 10-12 M。所建構的SPCE試片也被用來檢測血清中thrombin濃度，其可偵測之thrombin濃度範圍為1 □ 10-10至1 □ 10-7 M。另外，為了測試本SPCE試片運用於臨床檢測的可行性，血漿中thrombin的生成量也被檢測，其結果顯示血漿中的thrombin生成量可隨著prothrombin之轉換率增加而增加。總結，在本研究中所建構之aptamer生物感測SPCE試片與功能性AuNPs（即AuNPs/Ab-HRP）結合運用，其具潛力發展成一臨床上快速醫療診斷工具。

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.