Fluorescence resonance energy transfer between a quantum dot donor and a dye acceptor attached to DNA (original) (raw)
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Quantum Dot-Based Multiplexed Fluorescence Resonance Energy Transfer
Journal of The American Chemical Society, 2005
We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.
Advanced Materials, 2007
Luminescent quantum dots (QDs), with their large absorption cross sections, superior photo-and chemical stability, broad excitation spectra, and narrow emission bandwidths, are excellent alternatives to traditional organic dyes for fluorescence labeling and emerging nanosensing applications. Using various surface-functionalization techniques (including cap exchange and encapsulation methods), QDs can be dispersed in aqueous media. This has naturally led to their use in biological applications, most notably in cellular labeling, and in the development of sensitive assays that can detect small molecules and oligonucleotides in solution. More recently, we and other groups have shown that QDs are unique donor fluorophores for fluorescence resonance energy transfer (FRET) where multiple acceptor dyes can be positioned around the QD to substantially enhance the overall rate of FRET between the QD and proximal dyes. Because of its exquisite sensitivity to changes in donor-acceptor separation distance (with sixth power dependence), FRET has proven to be a powerful method for detecting molecular-scale interactions, such as binding events and changes in protein conformations. FRET-based QD-biomolecule sensing assemblies that are specific for the detection of target molecules including soluble 2,4,6-trinitrotoluene (TNT), DNA, and the activity of various proteolytic enzymes have been demonstrated. Multiphoton fluorescence microscopy is the preferred highresolution imaging method for thick (ca. 1 mm) tissue samples owing to its intrinsic optical sectioning ability and limited out-of-focus photodamage. It also uses far red and near IR excitation (700-1100 nm), which is ideally located in the tissue optical transparency window. However, FRET performance driven by two-photon excitation has been limited by the photophysical properties of organic dyes and fluorescent proteins. In particular, it is often difficult to devise a donoracceptor pair with substantial spectral overlap for high FRET efficiency and nonoverlapping two-photon absorption spectra for limited acceptor direct excitation. A recent report by Larson et al. showed that water-soluble CdSe-ZnS QDs are superior probes for multiphoton fluorescence imaging where typical QD two-photon action cross sections are about one to two orders of magnitude larger than those of organic molecules designed specifically for such applications. In this report, we demonstrate efficient resonance energy transfer between luminescent QDs and proximal dye acceptors driven by a two-photon process using sub-band excitation energy (far red and near IR photoexcitation). The FRET process between QDs and proximal dyes using this format has two unique features: 1) it exploits the very high two-photon action cross sections of QDs compared to those of conventional dyes, which results in a near-zero background contribution from the dye acceptors due to direct excitation, independent of the excitation wavelength; 2) it provides high signal-to-background ratios in FRET imaging of cells and tissue samples by substantially reducing both autofluorescence and direct excitation contributions to the acceptor photoluminescence (PL) signal. These features can considerably simplify data analysis, in particular when signals of both the QD donor and dye acceptor are required to interpret assay results; they can also improve applications such as intracellular FRET sensing and imaging. Our findings also show that the energy transfer resulting from two-photon excitation is entirely con-
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