Comparison of quantum dots and dyes for Förster resonance energy transfer based aptamer sensors

Bio-sensing has enabled the detection and identification of many analytes, including molecules, viruses, diseases, cancers, pollutants, and pesticides. One type of biosensor involves the use of fluorescent dyes capable of Fӧrster Resonance Energy Transfer (FRET) which, although effective in signalling, can be sensitive to environmental changes such as pH, temperature, and ionic concentrations due to their organic nature. In contrast, inorganic quantum dots are resistant to these environmental changes while also producing a controlled and specific signal through its tuneable band gap and narrow emission. Aptamer-based quantum dot biosensors are therefore a promising alternative to organic dye sensors due to the specificity of aptamers and valued properties of the quantum dots. A combination of green and red fluorophores with attached complementary aptamers allows a FRET signal to be observed when there is no target present, and no FRET signal observed when the target is present.

Here, the selectivity and specificity of novel InP/Zns QD-thrombin aptamer conjugates are investigated and compared with Cyanine3 (Cy3) and Cyanine5 (Cy5) in response to a thrombin protein target. Firstly, the fluorophores are synthesised and characterised. Secondly, four systems are tested for their suitability for FRET, QD-QD, QD-dye, dye-QD, and dye-dye. Thirdly, the suitable systems are tested with the thrombin target for detection through FRET interruption, finding that some detection is observed at higher nM concentrations of thrombin within the QD-QD and QD-dye systems, particularly with lower concentrations of fluorophore. Fourthly, interactions between the green donor QD and thrombin protein are investigated as a possible reason behind the lack of detection in some systems. Finally, the QD-dye system is used with a second target, ATP, and a second dye, Texas Red, to demonstrate the applicability of the overall system type with a range of targets. While the biosensor systems tested were not reliable for thrombin detection, knowledge was gained about the differences between QDs and dyes within the systems tested, and the use of non-toxic InP/ZnS QDs within a FRET-based aptamer system shows promise for future diagnostic use.