整合導電連接分子與電化學阻抗分析儀以檢測丙型干擾素之研究

Abstract

結核病是一全球性傳染病，在未開發及開發中國家尤其盛行，據世界衛生組織統計，全世界約有三分之一的人受到結核分枝桿菌感染，每秒約有一個人受到結核病的感染。丙型干擾素 (IFN-γ) 為肺結核的指標蛋白質，全血丙型干擾素檢驗 (IGRA) 被認為是取代結核菌素皮膚試驗 (TST) 成為潛伏結核感染 (LTBI) 最佳診斷方法，雖然透過酶免疫測定法來檢測IFN-γ已有了很大的進步，但此法仍有耗費人力和費時等缺點。為了改善上述缺點，本團隊採用IFN-γ抗體來檢測不同濃度的IFN-γ抗原，以檢測丙型干擾素為目標，期望能夠發展出一套具有免標定、高精準度、快速檢測的檢驗方法。本研究選擇以電化學方法作為生物感測器的開發基礎，並利用導電連結分子使蛋白質分子與金電極鍵結在一起以量測IFN-γ抗體抗原間相互反應。 在本論文中，我們利用電化學循環伏安法以及阻抗分析法來量測數種導電連結分子的導電特性。從實驗結果可以發現導電連結分子的導電度與碳鏈長短具有相當大的關係，當碳鏈越短時，導電的效果越好，致使訊雜比提升進而改善檢測極限。為了確保連結分子能夠鍵結在金電極上面，我們採用螢光顯微術來分析其鍵結效果。最後透過循環伏安法與電化學阻抗分析法來量測IFN-γ抗體抗原間的交互作用，實驗結果顯示此感測器在10 pM~50 nM的量測範圍內，阻抗隨著濃度上升而上升，但當濃度高於此範圍後，阻抗隨著濃度上升而逐漸下降，我們推測造成此現象的原因，主要為此電化學系統中空間障礙與靜電作用力相互競爭所造成的結果。

Tuberculosis (TB) is an ancient disease constituted a long-term menace to public health. According to World Health Organization (WHO), mycobacterium tuberculosis (MTB) infected nearly a third of people of the world. There is about one new TB occurrence every second worldwide today. Interferon-gamma (IFN-γ) is associated with susceptibility to TB, and interferon-gamma release assays (IGRA) is considered to be the best alternative of tuberculin skin test (TST) for diagnosis of latent tuberculosis infection (LTBI). Although significant progress has been made with regard to the design of enzyme immunoassays for IFN-γ, adopting this assay is still labor-intensive and time-consuming. To alleviate these drawbacks, we used IFN-γ antibody to facilitate the detection of IFN-γ. Our team chose IFN-γ as the target protein and expected to pursue a label-free, high-precision, and rapid detection technique. We chose electrochemical method as the development foundation of the biosensor and adopted conductive linker to form a self-assembled monolayer (SAM). Through the impedance measurement, we can understand the interaction of IFN-γ antibody and IFN-γantigen. In the thesis, cyclic voltammetry and electrochemical impedance spectroscopy are used to measure the conductive characteristics of three kinds of conductive linkers. From the experimental results, it can be found that conductivity is associated with the number of methylene chain. As the number of methylene chain decreased, the conductivity increased. Through the conductivity enhancement, we can obtain a higher signal/noise ratio such that the detection limit is improved. Fluorescence microscopy was used to verify that the conductive linkers have capability to bind with gold. Finally, we used the electrochemical methods to observe the interaction of IFN-γ antibody and IFN-γ antigen. The results show that for the range from 10 pM to 50 nM, the impedance would increase along with the rising of IFN-γ concentration. When the concentration exceeded this range, the impedance would decrease along with the rising of IFN-γ concentration. It is our conjecture that these two trends can be attributed to the competition between steric hindrance and electrostatic force.