開發偵測柯楠因之生物感測器

Abstract

本論文研究包含兩個部分，利用抗癌藥物柯楠因可以與複數腺嘌呤分子具有高度專一性的結合並形成錯合物的特性，設計出柯楠因之生化感測器並用以定量柯楠因。在第一部分的實驗中，首先分別合成出金奈米粒子 (Au NPs) 及包覆金之四氧化三鐵奈米粒子 (Au@Fe3O4 NPs)，並於奈米粒子表面修飾三磷酸腺苷 (ATP) 作為偵測柯楠因之探針。當溶液中存在分析物柯楠因時，柯楠因會與粒子表面的三磷酸腺苷有專一性結合，使得兩種奈米粒子會透過柯楠因彼此連接而聚集。透過外加磁場，使溶液中的ATP-Au NPs透過分析物柯楠因與 ATP-Au@Fe3O4 NPs 相互連接後，被磁場吸引而吸附在電極的表面，由於金奈米粒子具有良好的導電性，可以有效地增加電化學活性物質的吸引與電極表面的電子傳遞效率，可減小電化學阻抗頻譜量測所得阻抗值。當分析物柯楠因的濃度增加時，使得更多的 ATP-Au NPs 透過柯楠因與 ATP-Au@Fe3O4 NPs 被磁場吸引一起聚集到電極表面，使得偵測到的阻抗下降。透過偵測電極表面阻抗值的改變，即可偵測柯楠因的濃度。在第二部分的實驗中，透過人工設計之一段單股 DNA，其序列兩端為連續的腺嘌呤來做為偵測柯楠因之探針分子。當加入分析物柯楠因，柯楠因與腺嘌呤透過氫鍵的作用，使得 DNA 髮夾結構的末段形成雙股結構。此時，核酸外切酶 III對雙股結構才能產生催化反應，催化反應由DNA的3端羥基末端往5端作用，將單核苷酸切除。此時分析物柯楠因將再被釋放出來，繼續與尚未被切除的探針 DNA 作用，反覆循環並促使 DNA 被大量切除。接著利用溶液中的 DNA 合成銀奈米團簇。由於文獻指出，環型結構且富含胞嘧啶的DNA序列所合成出的銀奈米團簇之螢光強度會比直鏈單股結構的胞嘧啶大，由於堆積效應的影響，利用此螢光差異作為偵測柯楠因的訊號來源。當加入分析物柯楠因後，由於核酸外切酶 III 將單核苷酸切除，使得切除後剩下單股的 DNA，其螢光強度相較於原本的髮夾型結構會明顯下降。利用此螢光變化可以用來偵測分析物柯楠因的濃度。本論文所呈現之兩種偵測方法均具有簡單、方便、快速與靈敏度高等優點，可準確定量柯楠因於樣品中之濃度。

Coralyne is one of isoquinoline anti-cancer medicine, which can inhibit the growth of leukemia cells and have significant anti-tumor activity. According to the previous reports, one coralyne molecule could specifically interact with four adenine molecules and forming A2-coralyne-A2 complex. In the first part, a highly sensitive electrochemical impedance sensor based on Au@Fe3O4 magnetic nanoparticles (Au@Fe3O4 NPs) for the detection of coralyne was presented. Based on this specific interaction, we designed an electrochemical method that use the adenosine triphosphate (ATP) stabilized Au@Fe3O4 magnetic nanoparticles and gold nanoparticles (Au NPs) as probe to detect coralyne. In the presence of coralyne, both ATP- Au@Fe3O4 NPs and ATP-Au NPs were aggregated via linkage of coralyne. After applying external magnetic field, aggregated ATP-Au NPs were gathered with ATP- Au@Fe3O4 NPs from sample solution to the surface of screen-printed electrode. Because of the high conductivity of ATP-Au NPs, the obtained impedance was decrease with the increase of coralyne concentration. Using magnetic nanoparticles can enhance the sensitivity with high isolation efficiency. In the second part, label-free hairpin DNA-scaffolded silver nanoclusters for fluorescent detection of coralyne using exonuclease III-assisted target recycling amplification was presented. The assay was carried out through target-induced conformational change of hairpin DNA, while the signal derived from the formed silver nanoclusters on hairpin DNA probes. Initially, target coralyne was specifically coordinated with adenine–adenine (A–A) mismatches to form an intact hairpin DNA. The newly formed hairpin DNA was digested through exonuclease III from blunt 3’ termini and restrained at 3’ protruding terminus, thus resulting in the release of target coralyne from probe DNA. The liberated target coralyne initiated the next cycling, thereby causing the conformational change of numerous hairpin probes from the stem-loop DNA structure to single-stranded DNA. Under the optimal conditions, the fluorescent intensity of the as-produced DNA-Ag NCs decreased with the increasing coralyne concentration. With both new strategies, coralyne can be analyzed and quantitated easily with high sensitivity and accuracy.