Development of smart nanoparticle-aptamer sensing technology

Quantum dots (QDs) are excellent donors in Förster resonance energy transfer (FRET) based sensors because of their broad absorption and narrow symmetric emission. However, the strict requirement of a short donor-acceptor distance to achieve high FRET (hence sensitivity) has posed a significant challenge for QD- FRET based sensors due to challenges associated with the preparation of QD-conjugates that are both compact and highly stable. Consequently, most robust QD-FRET sensors are often too bulky to produce FRET efficiently, especially at low target to QD copy numbers (e.g. 1:1). They have largely relied on increasing the target:QD ratio to achieve high FRET, making them undesirable and inefficient in situations of low target:QD copy numbers. Here we report our recent work in the preparation of stable, compact and water-soluble QDs via ligand exchange and their subsequent conjugation to DNAs to make compact, functional QD-DNA conjugates based smart nanoparticle sensors for labelled and label-free DNA and protein detection. We have developed two strategies to prepare QD-DNA sensors: 1) via QD-thiolated DNA self-assembly, and 2) via covalent coupling between DNA and a QD surface ligand functional group. We show that a thiolated DNA (labelled with a fluorophore) can self-assemble onto a 3-mercaptopropionic acid-capped, CdSe/ZnS core/shell QD to produce highly efficient FRET (~80%) at a low DNA:QD ratio of 1:1 at single molecule level. However, this system suffers from strong, non-specific DNA adsorption and the self-assembled single-stranded (ss) DNA is unable to hybridise to its target complementary DNA. More recently, we found that a dihydrolipoic acid (DHLA)-capped QD-ssDNA self-assembled system can hybridise to its labelled DNA target to produce very efficient FRET that can be exploited for labelled-DNA quantification. By incorporating an anti-thrombin DNA aptamer to the self-assembled QD-DNA system, the resulting QD-DNA aptamer sensor can detect specifically 10 nM unlabelled protein target (thrombin) in standard aqueous buffer. In strategy 2, we show that the non-specific DNA adsorption can be eliminated by introducing a polyethylene glycol (PEG) linker to the QD capping ligands or by capping the QD with a novel chelating dendritic ligand. The resulting QD-DNA sensors can specifically detect 1 nM unlabelled short DNA targets, or 35 pM of a labelled-DNA target using QD sensitised dye FRET signals on a conventional fluorimeter.