Title page for etd-0528118-084541


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URN etd-0528118-084541
Author Yao-De Yin
Author's Email Address No Public.
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Department Institute of Biomedical Sciences
Year 2017
Semester 2
Degree Master
Type of Document
Language zh-TW.Big5 Chinese
Title The Utility of DNA Aptamers against β-Bungarotoxin as Sensors for Detecting β-Bungarotoxin, Coralyne and Heparin
Date of Defense 2018-06-27
Page Count 87
Keyword
  • DNA aptamer
  • Sensors
  • Heparin
  • Coralyne
  • β-Bungarotoxin
  • Abstract 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.
    Advisory Committee
  • Bin-Nan Wu - chair
  • Ying-Jung Chen - co-chair
  • Long-Sen Chang - advisor
  • Files
  • etd-0528118-084541.pdf
  • Indicate in-campus at 5 year and off-campus access at 5 year.
    Date of Submission 2018-06-28

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