戴奧辛生物檢測系統之建立

Abstract

戴奧辛類與類戴奧辛化合物包括多氯二聯苯戴奧辛、多氯二聯苯呋喃及多氯聯苯。戴奧辛類和類戴奧辛化合物為化學合成中的副產物，可經由焚化爐產生的廢氣、汽機車引擎的排放、石化工廠原料製程及一般燃燒行為中產生。在所有的戴奧辛類和類戴奧辛化合物的生物檢測中，2, 3, 7, 8-四氯二聯苯戴奧辛(2, 3, 7, 8-TCDD)被認定是毒性最強。 目前戴奧辛類和類戴奧辛化合物的成分定量檢測主要採用氣象層析-高解析度質譜儀(GC/HRMS)。然而，時間和成本考量使得氣象層析-高解析度質譜儀無法應用於大量日常性的例行檢測分析。本論文的第一部分，我們陳述了高靈敏度與低成本的生物冷光及生物冷光共振能量轉移的替代方案。我們建立了AAPH 細胞株，其穩定表現 AhR 和 Renilla luciferase融合蛋白 (AhR-RL)與Hsp90 和 yellow fluorescent protein 融合蛋白(Hsp90-YFP)。在AAPA細胞株中，則是穩定表現AhR-RL融合蛋白與Arnt 和 yellow fluorescent protein 融合蛋白(Arnt-YFP)。當細胞中兩融合蛋白接近時，Renilla luciferase所產生的冷光可用來激發YFP且應用於偵測檢體中戴奧辛類和類戴奧辛化合物的含量。另外，我們同時發現2, 3, 7, 8-四氯二聯苯戴奧辛會促進AhR-RL融合蛋白降解，因而減少AAPH細胞的冷光值，其偵測靈敏度達到10-17 M。 為減少動物實驗與長時間的細胞培養，我們在論文的第二部分使用AAPA的細胞粹取物來偵測戴奧辛類和類戴奧辛化合物的含量。我們發現AhR 促效藥 3-methylcholanthrene (3MC)會促進AhR 與Arn結合，因而避免遭蛋白酶體降解。 此外。3MC或2, 3, 7, 8-四氯二聯苯戴奧辛會穩定AAPA細胞粹取物的AhR-RL融合蛋白冷光並以偵測戴奧辛類和類戴奧辛化合物的含量，其2, 3, 7, 8-四氯二聯苯戴奧辛偵測靈敏度達到10-18 M。 使用戴奧辛類和類戴奧辛化合物會造成AhR–RL 降解的原理，2, 3, 7, 8-四氯二聯苯戴奧辛在AAPH細胞的偵測靈敏度可達到10 aM.。在AAPA 細胞粹取物中，戴奧辛類和類戴奧辛化合物會穩定AhR–RL融合蛋白，其2, 3, 7, 8-四氯二聯苯戴奧辛偵測靈敏度接近1 aM. 此外，所有的反應可在2到3小時內完成，且費用遠低於其他實驗室所發展的生物檢測系統或氣象層析-高解析度質譜儀方法。總結來說，我們所發展的戴奧辛生物檢測系統將可應用於大量例行性環境汙染物檢測並幫助維護公共衛生。

Dioxins and dioxin-like compounds include polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls, which are byproducts of chemical processes, including municipal waste incineration, automobile engines, fossil-fuels, and backyard barrel burning. 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the most toxic species of all dioxin-like compounds. At present, gas chromatography with high-resolution mass spectrometry (GC/HRMS) is considered the gold standard for detection of dioxins. However, cost-ineffectiveness and excess time consumption limit their routine utilization. In the first part of my thesis, we describe highly sensitive and cost-effective alternative methods, based on bioluminescence and bioluminescence resonance energy transfer. We generated cell lines that stably co-express a fusion protein of AhR and Renilla luciferase (AhR-RL) and either Hsp90 or Arnt tagged with yellow fluorescent protein (Hsp90-YFP in AAPH cells or Arnt-YFP in AAPA cells). The fluorescent signals of YFP are activated by the emission of RL while the interactions between AhR and Hsp90 (or Arnt) were monitored. In addition, TCDD treatment reduced Renilla luminescence in AAPH cells in a concentration-dependent manner, due to degradation of AhR. Intriguingly, the detection limit for dioxin in our AhR degradation assay was as low as 10-17 M. To decrease the use of animals and long-term cell cultures, AAPA cell-free extracts were applied for detection of dioxins and dioxin-like compounds in the second part of my thesis. Treatment with 3-methylcholanthrene (3MC), an AhR agonist, enhanced the interaction between AhR and Arnt and avoided proteosomal degradation. In addition, treatment with 3MC or TCDD stabilized Renilla luciferase from AhR-RL of AAPA cell-free extracts in a concentration-dependent manner. The TCDD detection limit in this cell-free system was as low as 10−18 M. The TCDD detection limit in our cell-based method, which can be used to estimate the rate of AhR–RL degradation, was close to 10 aM. In our cell-free method, which measures the stability of AhR–RL, the detection limit was as low as 1 aM. Moreover, the total reaction time was approximately 2 to 3 h, and the total cost was considerably lower than that of other bioassays or GC technologies. Therefore, our findings may contribute toward the detection of environmental contaminants, thereby helping to protect human health.