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Aptamers are synthetic oligonucleotides of RNA or single-stranded DNA that can bind to their targets with high affinity and specificity due to their specific secondary or tertiary structures. Thus, they can be used as aptasensor for detection of biological molecules. The aim of this study was to investigate the utility of DNA aptamers against Bungarus multicinctus presynaptic neurotoxin β-Bungarotoxin (β-Bgt) or lysozyme as DNA aptasensors. Four aptamers Bgt1, Bgt2, Bgt3 and Bgt4 against β-Bungarotoxin were synthesized based on previous studies. Electrophoretic mobility shift assay showed that Naja naja (Taiwan cobra) Cardiotoxin 3 (CTX3) reduced the electrophoretic mobility of the four aptamers. Moreover, CTX3 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 CTX3 bound with the aptamers and the folded structure of four aptamers was changed upon binding with CTX3. Moreover, our data showed CTX3 had the highest affinity and specificity for binding with FAM-Bgt2-Dabcyl among the four aptamers. Similarly, Taiwan cobra CTX isotoxins reduced FAM-Bgt2-Dabcyl fluorescence intensity. Compare to Bungarus multicinctus and Protobothrops mucrosquamatus crude venoms, N. atra (Taiwan cobra) crude venom obviously reduced FAM fluorescence intensity of FAM-Bgt2-Dabcyl. Furthermore, a turn-on fluorescent platform was established using coralyne-FAM-Bgt2-Dabcyl complexes. It was found that CTXs and Taiwan cobra crude venom specifically restore FAM fluorescence of coralyne-FAM-Bgt2-Dabcyl complexes. FAM-Bgt2-Dabcyl and lysozyme aptamer (FAM-Apta1-Dabcyl) were also used for construct use to construct turn-on or turn-off aptasensors for detecting lysozyme. Compared to other constructed sensors, FAM-Apta1-Dabcyl sensor showed the best specificity for detecting lysozyme in buffer or in red wine. Taken together, the data of the present study indicate that the sensor systems constructed by Bgt aptamers and lysozyme aptamer are successful for rapidly detecting CTXs, Taiwan cobra crude venoms and lysozyme.
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