A quantum dot-aptamer beacon using a DNA intercalating dye as the FRET reporter: Application to label-free thrombin detection

• Published in: Biosensors & bioelectronics Vol. 26; no. 7; pp. 3346 - 3352
• Main Authors: Chi, Chun-Wei, Lao, Yeh-Hsing, Li, Yi-Shan, Chen, Lin-Chi
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 15.03.2011; Elsevier
• Subjects: Aptamer beacon; Aptamers, Nucleotide - chemistry; Beacons; Biological and medical sciences; Biotechnology; BOBO-3; Carbocyanines - chemistry; Deoxyribonucleic acid; Dyes; Electrophoresis; Fluorescence Resonance Energy Transfer - methods; Folding; Fretting; Fundamental and applied biological sciences. Psychology; Humans; Intercalating Agents - chemistry; Joining; Quadruplex; Quantum dot; Quantum Dots; Sensitivity and Specificity; Thrombin; Thrombin - analysis; BOBO-3; Thrombin; Quantum dot; Quadruplex; Aptamer beacon; Dyes; Serine endopeptidases; Enzyme; Peptidases; DNA; Hydrolases; Aptamer; Detection; Application
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2011.01.015

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.