We provide an overview of the progress made in the past few years in investigating fluorescence resonance energy transfer (FRET) using semiconductor quantum dots (QDs) and the application of QD-based FRET to probe specific biological processes. We start by providing some of the pertinent conceptual elements involved in resonance energy transfer, and then discuss why the Förster dipole-dipole mechanism applies to QD fluorophores. We then describe the unique QD photophysical properties of direct relevance to FRET and summarize the main advantages offered, along with some of the limitations encountered by QDs as exciton donors and/or acceptors. Next we describe the overall progress made and discuss a few representative examples where QD-based FRET sensing of specific biological processes has been demonstrated. We also detail some of the advances of single molecule FRET using QD-conjugates and highlight the unique information that can be extracted. We conclude by providing an assessment of where QD-based FRET investigations may be evolving in the near future.
"In general, the application of FRET technique to quantification of a binding event can be carried out by either the emission-shift method or the donor-quenching method . The emission-shift strategy can exhibit two-dimensional signals (decrease in QD emission and increase in dye emission) and was used in this work. "
[Show abstract][Hide abstract] ABSTRACT: This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian influenza viruses were used as the targets in this work. Two oligonucleotides (16 mers and 18 mers) that specifically recognize two separate but neighboring regions of the H5 sequences were served as the capturing and reporter probes, respectively. The capturing probe was conjugated to QD655 (donor) in a molar ratio of 10:1 (probe-to-QD), and the reporter probe was labeled with Alexa Fluor 660 dye (acceptor) during synthesis. The sandwich hybridization assay was done in a 20 μL transparent, adhesive frame-confined microchamber on a disposable, temperature-adjustable indium tin oxide (ITO) glass slide. The FRET signal in response to the sandwich hybridization was monitored by a homemade optical sensor comprising a single 400 nm UV light-emitting diode (LED), optical fibers, and a miniature 16-bit spectrophotometer. The target with a concentration ranging from 0.5 nM to 1 μM was successfully correlated with both QD emission decrease at 653 nm and dye emission increase at 690 nm. To sum up, this work is beneficial for developing a portable QD-based nucleic acid sensor for on-site pathogen detection.
"Therefore, an exciton is trapped in the CdSe core of the QDs. The overall exciton wave function is confined within the physical dimension of the nanocrystal and essentially localized at the center of the QDs vanishing at its edge . EET occurs between the confined lowest excited state in the CdSe quantum well and the absorbing S 0 → S 1 transition of the PCB chromophores. "
[Show abstract][Hide abstract] ABSTRACT: The present work describes results obtained on hybrid systems formed in aqueous buffer solution by self-assembly of different CdSe quantum dots (QDs) surrounded by a ZnS shell and functionalized by covering the surface with anionic and cationic groups and various isolated pigment-protein complexes from the light-harvesting antennae of photosynthetic organisms (light-harvesting complexes 1 and 2 (LH1 and LH2, respectively) from purple bacteria, phycobiliproteins (PBPs) from cyanobacteria and the rod-shaped PBP from the cyanobacterium Acaryochloris marina). Excitation energy transfer (EET) from QDs to PBP rods was found to take place with varying and highly temperature-dependent efficiencies of up to 90%. Experiments performed at room temperature on hybrid systems with different QDs show that no straightforward correlation exists between the efficiency of EET and the parameter J/(R(12)(6)) given by the theory of Förster resonance energy transfer (FRET), where J is the overlap integral of the normalized QD emission and PBP absorption and R(12) the distance between the transition dipole moments of donor and acceptor. The results show that the hybrid systems cannot be described as randomly orientated aggregates consisting of QDs and photosynthetic pigment-protein complexes. Specific structural parameters are inferred to play an essential role. The mode of binding and coupling seems to change with the size of QDs and with temperature. Efficient EET and fluorescence enhancement of the acceptor was observed at particular stoichiometric ratios between QDs and trimeric phycoerythrin (PE). At higher concentrations of PE, a quenching of its fluorescence is observed in the presence of QDs. This effect is explained by the existence of additional quenching channels in aggregates formed within hybrid systems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
"The binding between polyhistidine and the zinc on QD surfaces is known to be extremely stable with a dissociation constant of ∼1 × 10−9 M . Using the Zn2+-rich surface of QDs and hexahistidine-tagged peptide assembly we have successfully applied this mechanism to create a number of different FRET and charge transfer based biosensors that were capable of monitoring DNA hybridization, enzymatic activity and pH changes [2,38,40]. "
[Show abstract][Hide abstract] ABSTRACT: Förster resonance energy transfer (FRET) from luminescent terbium complexes (LTC) as donors to semiconductor quantum dots (QDs) as acceptors allows extraordinary large FRET efficiencies due to the long Förster distances afforded. Moreover, time-gated detection permits an efficient suppression of autofluorescent background leading to sub-picomolar detection limits even within multiplexed detection formats. These characteristics make FRET-systems with LTC and QDs excellent candidates for clinical diagnostics. So far, such proofs of principle for highly sensitive multiplexed biosensing have only been performed under optimized buffer conditions and interactions between real-life clinical media such as human serum or plasma and LTC-QD-FRET-systems have not yet been taken into account. Here we present an extensive spectroscopic analysis of absorption, excitation and emission spectra along with the luminescence decay times of both the single components as well as the assembled FRET-systems in TRIS-buffer, TRIS-buffer with 2% bovine serum albumin, and fresh human plasma. Moreover, we evaluated homogeneous LTC-QD FRET assays in QD conjugates assembled with either the well-known, specific biotin-streptavidin biological interaction or, alternatively, the metal-affinity coordination of histidine to zinc. In the case of conjugates assembled with biotin-streptavidin no significant interference with the optical and binding properties occurs whereas the histidine-zinc system appears to be affected by human plasma.
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