Energy Transfer-Based Multiplex Analysis Using Quantum Dots

Abstract

Semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as a significant new class of materials over the past decade. The capabilities of QDs – high quantum yield, improved sensitivity, high photostability, and size-tunable colors have paved the way for numerous studies including imaging, sensing and targeting biomolecules. Thus QDs are now rapidly replacing traditional fluorophores in almost all fluorescence-based applications. Unlike organic dyes and fluorescent proteins, QDs are sizetunable with non-overlapping emission band profiles due to their narrow and symmetric emission bands (full width at half maximum of 25–40 nm) that can span the light spectrum from the ultraviolet even to the infrared. As illustrated in Figure 1, this property enables the QDs to be useful for multiplexing assay in a single run (Chan et al., 2002; Jaiswal et al., 2003; Wu et al., 2003). Moreover, QDs typically have very broad absorption spectra with very large molar extinction coefficients (0.5–5×106 M−1 cm−1) (Hawrylak et al., 2000; Moreels and Hens, 2008). This makes QDs absorb 10–50 times more photons than organic dyes at the same excitation photon flux, providing a sufficient brightness for the sensing system (Gao et al., 2004). Owing to high photostability, QD-based sensing and imaging are favorable for continuous tracking studies over a long period of time. Most importantly, when the QD is harnessed in fluorescence resonance energy transfer (FRET), several advantages over organic dye-based probes have been acquired. Multiple binding of an energy acceptor per a single QD is expected to increase the overall energy transfer efficiency (Clapp et al., 2004; Zhang et al., 2005). Additionally, large Stokes shift of the QD can avoid the crosstalk between the donor QD and the acceptor counterpart because its broadband absorption allows excitation at a short wavelength that does not directly excite the acceptor. The continuously tunable emissions that can be matched to any desired acceptor, makes it possible to use many fluorophores for multiplexed assay (Medintz et al., 2003). The quencher (orgarnic (Mauro et al., 2003) or metal (Kim et al., 2008a; Oh et al., 2005; Oh et al., 2006; Wargnier et al., 2004) substances) or emissive fluorescent molecules (fluorophores, proteins, or other QDs) (Wang et al., 2002) can be promising acceptors. While QDs are frequently used as donors in FRET, they may also play a critical role as energy acceptors either in bioluminescence resonance energy transfer (BRET)(Rao et al., 2006a; Rao et al., 2006b) or chemiluminescene resonance energy transfer (CRET)(Huang et al., 2006) as the energy donor (Figure 2). To this end, energy transfer system allows QDs to be suitable for many biological applications, such as the analyses of enzyme activity, protein-protein