Aptamer為單股DNA或RNA片段，能以其形成之特定結構和作用標的物結合，因此可作為偵測結合標的物的Aptasensor，本論文主要探討台灣雨傘節突觸前神經毒素β-Bungarotoxin (β-Bgt)之DNA Aptamers作為生物感測器的應用性。 根據先前文獻報導合成四段β-Bgt aptamers：Bgt1、Bgt2、Bgt3及Bgt4，瓊脂電泳分析顯示β-Bgt可降低四段Aptamers的電泳移動性，此外β-Bgt能使5'端標定Carboxyfluorescein (FAM)及3'端標定4-([4-(dimethylamino)- phenyl]azo)-benzoic acid (Dabcyl)的Aptamers螢光強度下降，顯示β-Bgt能與四段Aptamers結合並改變其結構，由FAM螢光變化分析證實四段 Aptamers中，β-Bgt對Bgt1親和性最高。 另一方面發現Coralyne可明顯降低5'端標定FAM之Bgt1 (FAM-Bgt1)及5'端與3'端分別標定FAM與Dabcyl之Bgt1 (FAM-Bgt1-Dabcyl)的FAM螢光強度，而Palmatine及Berberine僅有些微影響FAM螢光強度，顯示FAM-Bgt1及FAM-Bgt1-Dabcyl可應用於選擇性偵測Coralyne，偵測Coralyne的Limit of detection (LOD)分別為0.021 μM及0.015 μM。 進一步以Coralyne-FAM-Bgt1及Coralyne-FAM-Bgt1-Dabcyl複合物作為感測器以turn-on螢光方式偵測β-Bgt及Heparin，發現β-Bgt及Heparin均可明顯回復Coralyne-FAM-Bgt1及Coralyne-FAM-Bgt1-Dabcyl複合物之FAM螢光，實驗結果顯示偵測β-Bgt的LOD值分別為3.57 nM及2.74 nM，而偵測Heparin的LOD值分別為0.012 μg/ml及0.004 μg/ml，此外偵測血清中的Heparin其LOD值分別為0.027 μg/ml及0.009 μg/ml。 此Coralyne與Bgt1構建的感測系統，針對不同Glycosaminoglycans包括Chondroitin sulfate及Hyaluronic acid僅些許影響，顯示可選擇性偵測Heparin；另一方面，此感測系統雖可有效區分β-Bgt與其他純化蛇毒蛋白，但無法選擇性偵測台灣雨傘節粗蛇毒。 綜合上述結果證實β-Bgt之Aptamers構建之感測器系統可應用於偵測Coralyne及Heparin，但對β-Bgt之偵測無法應用於粗蛇毒之分析。

Aptamers are single-stranded DNA or RNA fragments that bind to targeted molecules via folding into specific structure. Thus, aptamers can be employed as aptasensors for detecting targeted molecules. The aim of this study was to investigate the utility of DNA aptamers against Bungarus multicinctus (Taiwan banded krait) presynaptic neurotoxin β-bungarotoxin (β-Bgt) as biosensors. Four β-Bgt aptamers, Bgt1, Bgt2, Bgt3, and Bgt4 were synthesized according to previously published results. Electrophoretic mobility shift assay showed that β-Bgt reduced the electrophoretic mobility of the aptamers. Moreover, β-Bgt reduced fluorescence intensity of the aptamers labeled with 5'-end carboxyfluorescein (FAM) and 3'-end 4-([4-(dimethylamino)-phenyl]azo)-benzoic acid (Dabcyl). These results showed that the folded structure of aptamers changed upon binding with β-Bgt. Measurement of the changes in FAM fluorescence revealed that β-Bgt had the highest binding affinity for Bgt1 among the four aptamers. In contrast to palmatine and berberine, coralyne drastically reduced FAM fluorescence intensity of 5'-FAM-Bgt1 and 5'-FAM-Bgt1-3'-Dabcyl, suggesting the selectivity of the sensors for detecting coralyne. The limit of detection (LOD) of coralyne determined by 5'-FAM-Bgt1 and 5'-FAM-Bgt1-3'-Dabcyl were 0.034 μM and 0.021 μM, respectively. Furthermore, coralyne-5'-FAM-Bgt1 and coralyne-5'-FAM-Bgt1-3'-Dabcyl complexes were employed as turn-on fluorescent sensors for detecting β-Bgt and heparin. β-Bgt and heparin notably restored FAM fluorescence intensity of coralyne-5'-FAM-Bgt1 and coralyne-5'-FAM-Bgt1-3'-Dabcyl complexes. The LOD of β-Bgt measured by coralyne-5'-FAM-Bgt1 and coralyne-5'-FAM-Bgt1-3'-Dabcyl complexes were 3.57 nM and 2.74 nM, respectively; the LOD of heparin were 0.012 μg/ml and 0.004 μg/ml, respectively; and the LOD for heparin-spiked serum were 0.027 μg/ml and 0.009 μg/ml, respectively. The sensor system constructed by coralyne and Bgt1 showed a high selectivity for heparin detection than for other glycosaminoglycans including chondroitin sulfate and hyaluronic acid. Although the sensor system effectively differentiated β-Bgt from other purified snake venom proteins, it could not specifically detect Taiwan banded krait crude venom. Taken together, our data indicate that aptamer against β-Bgt can be used for constructing biosensors for detecting coralyne and heparin. However, the sensor system is unable to identify the crude venoms of different snake species via β-Bgt detection.

論文審定書+i
致謝+ii
中文摘要+iii
Abstract+iv
英文縮寫表+vi
目錄+viii
第一章 緒論+1
第二章 實驗材料與方法+7
第三章 結果+15
第四章 討論+22
圖表+28
附圖+57
參考文獻+71

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