title: A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: application to label-free thrombin detection.
abstract: A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg(2+), coupling time, and aptamer type on the beacon's performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well.
authors.affiliation: Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan.
authors.forename: Chun-Wei
authors.initials: CW
authors.lastname: Chi
authors.forename: Yeh-Hsing
authors.initials: YH
authors.lastname: Lao
authors.forename: Yi-Shan
authors.initials: YS
authors.lastname: Li
authors.forename: Lin-Chi
authors.initials: LC
authors.lastname: Chen
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D002232:Carbocyanines / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007364:Intercalating Agents / Q000737:chemistry*; D045663:Quantum Dots; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis*
chemical_list: D052157:Aptamers, Nucleotide; C098829:BOBO 3; D002232:Carbocyanines; D007364:Intercalating Agents; C081676:thrombin aptamer; D013917:Thrombin