Semiconductor quantum dots are light-emitting nanocrystals (2-10 nm) that straddle the border between condensed matter and atomic physics. In a quantum dot, all three spatial dimensions of the crystal are limited to less than the exciton radius of the material such that discrete energy levels arise due to quantum confinement effects and the spacing of which can be controlled by manipulation of crystal size. This effect leads to several superior spectroscopic properties of quantum dots such as high quantum yields, photostability and wide color availability [1, 2], making them ideal for applications where organic fluorophores fail.

Quantum dots have therefore been used as a simple label to substitute organic dyes for fluorescently enhanced detection and imaging [3]. Recently, quantum dots have been employed as an active participant in molecular self-assembly and fluorescence resonant energy transfer (FRET) processes, facilitating ultrasensitive biosensing. This report highlights the new advances of quantum dot based genomic detection assays for analyzing cancer markers including point mutations and DNA methylation.

Quantum Dots-mediated Fluorescence Resonance Energy Transfer

Early applications used quantum dots to substitute organic fluorescent dyes to improve imaging and assay performance. In more recent advances, quantum dots act not only as luminescent tags but also as scaffolds upon which more complex hybrid inorganic/organic biosensors are built [4, 5]. In these applications, the high surface area to volume ratio, and well-documented conjugation chemistries for quantum dots allow attachment of biomolecular probes, thus transforming the nanocrsytals into scaffolds for molecular sensing. Signal transduction in these biosensors is most often accomplished through fluorescence resonance energy transfer (FRET). FRET is a non-radioactive energy transfer process in which energy is transferred via dipole-dipole interactions between donor-acceptor chromophore pairs. Since the efficiency of FRET phenomena is highly dependent on the intermolecular distance of the energy transfer pair, it can be used as an effective signal transduction method in biosensing.

Quantum dots make excellent FRET donors that overcome pitfalls associated with conventional molecular FRET.

The property of size-tunable narrow emission wavelengths minimizes spectral crosstalk, while the broad absorption allows the choice of an excitation wavelength in order to minimize the direct excitation of the acceptor. These extraordinary features have benefited the development of DNA nanosensors that possess an extremely low background fluorescence and high sensitivity, necessary for detecting rare DNA markers in clinical samples [4, 6]. In the initial design of the quantum dots-FRET based DNA nanosensors [4], each comprised a quantum dot and a pair of oligonucleotide probes, which include a reporter probe labeled with Cy5 and a capture probe labeled with biotin (Fig. 1). If the sample being tested contains target DNA, the probe and the target form a sandwich hybrid that is subsequently captured by the quantum dot. The resulting nanoassembly brings the quantum dot (donor) and Cy5 (acceptor) to a close proximity, leading to acceptor emissions via FRET upon selectively exciting the donor. In addition to being a FRET donor, quantum dots function as a concentrator that amplifies target signal by confining several targets in a nanoscale domain through the multiple binding sites present on the streptavidin that the quantum dot is encapsulated with. It is demonstrated that the nanosensor is >100 folds more sensitive than molecular FRET sensors and is capable of detecting DNA targets even at the femtomolar level.

Quantum Dots for Point Mutation Detection in Cancer

Cancer may be caused by the accumulation of multiple mutations in the genes that regulate cell growth, death and other cellular behaviors. Since the majority of mutations are associated with sequence variations such as single nucleotide substitutions, deletions, and insertions, point mutations can serve as generic markers for cancer diagnostics. In combination with oligonucleotide ligation reaction assay (OLA), quantum dot nanosensors can be extended to point mutation detection [4, 7]. Briefly, a biotinylated common capture and Cy5-labeled discrimination report probes are enzymatically ligated together in the presence of a perfect match DNA target. Streptavidin-functionalized quantum dots are then used to capture successful ligation products, leading to energy transfer between quantum dots and Cy5. In contrast, mismatched targets prevent ligation of capture and reporter probes, and hence energy transfer does not occur. Consequently, the polymorphisms can be differentiated according to the emission of Cy5. The quantum dot-based mutation detection assay has been successfully applied to detect Kras and Braf point mutation in clinical samples from patients with ovarian serous borderline tumors [4, 7].