Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

Quantum dots as simultaneous acceptors and donors in time-gated Förster resonance energy transfer relays: characterization and biosensing

The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in F?rster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.     

Quantum dot-based multiplexed fluorescence resonance energy transfer

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.     

Solution-phase single quantum dot fluorescence resonance energy transfer

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.     

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.     

Joint statistical analysis of multichannel time series from single quantum dot-(Cy5)n constructs

One of the major challenges in single-molecule studies is how to extract reliable information from the inevitably noisy data. Here, we demonstrate the unique capabilities of multichannel joint statistical analysis of multispectral time series using F?ster resonance energy transfer (FRET) in single quantum dot (QD)-organic dye hybrids as a model system. The multispectral photon-by-photon registration allows model-free determination of intensity change points of the donor and acceptor channels independently. The subsequent joint analysis of these change points gives high-confidence assignments of acceptor photobleaching events despite the interference from background noise and from intermittent blinking of the QD donors and acceptors themselves. Finally, the excited-state lifetimes of donors and acceptors are calculated using the joint maximum likelihood estimation (MLE) method on the donor and acceptor decay profiles, guided by a four-state kinetics model.     

High‐Performance Förster Resonance Energy Transfer (FRET)‐Based Dye‐Sensitized Solar Cells: Rational Design of Quantum Dots for Wide Solar‐Spectrum Utilization

High‐performance Förster resonance energy transfer (FRET)‐based dye‐sensitized solar cells (DSSCs) have been successfully fabricated through the optimized design of a CdSe/CdS quantum‐dot (QD) donor and a dye acceptor. This simple approach enables quantum dots and dyes to simultaneously utilize the wide solar spectrum, thereby resulting in high conversion efficiency over a wide wavelength range. In addition, major parameters that affect the FRET interaction between donor and acceptor have been investigated including the fluorescent emission spectrum of QD, and the content of deposited QDs into the TiO₂ matrix. By judicious control of these parameters, the FRET interaction can be readily optimized for high photovoltaic performance. In addition, the as‐synthesized water‐soluble quantum dots were highly dispersed in a nanoporous TiO₂ matrix, thereby resulting in excellent contact between donors and acceptors. Importantly, high‐performance FRET‐based DSSCs can be prepared without any infrared (IR) dye synthetic procedures. This novel strategy offers great potential for applications of dye‐sensitized solar cells.     

High-Efficiency FRET Processes in BODIPY-Functionalized Quantum Dot Architectures

Efficient FRET systems are developed combining colloidal CdSe quantum dots (QDs) donors and BODIPY acceptors. To promote effective energy transfer in FRET architectures, the distance between the organic fluorophore and the QDs needs to be optimized by a careful system engineering. In this context, BODIPY dyes bearing amino-terminated functionalities are used in virtue of the high affinity of amine groups in coordinating the QD surface. A preliminary QD surface treatment with a short amine ligand is performed to favor the interaction with the organic fluorophores in solution. The successful coordination of the dye to the QD surface, accomplishing a short donor–acceptor distance, provides effective energy transfer already in solution, with efficiency of 76 %. The efficiency further increases in the solid state where the QDs and the dye are deposited as single coordinated units from solution, with a distance between the fluorophores down to 2.2 nm, demonstrating the effectiveness of the coupling strategy.     

Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

Comparative efficiency of energy transfer from CdSe-ZnS quantum dots or nanorods to organic dye molecules

Fluorescence resonance energy transfer (FRET) in conjugates of CdSe-ZnS semiconductor nanocrystals of different shapes (FRET donors) and an Alexa Fluor organic dye (FRET acceptors) is examined. The dye molecules are chemically conjugated with quantum dots (QDs) or nanorods (NRs) in dimethyl sulfoxide colloidal solutions, and FRET efficiency in the purified conjugates is measured. The FRET from NR to a single dye molecule is less efficient than that of the QD-dye conjugates and this effect is explained in terms of distance-limited energy-transfer rate in the case of a point-like acceptor and extended donor dipoles. However, the larger surface area of NRs allows for many more dye acceptors to be bound, and the total FRET efficiency in NR-dye conjugates approaches those of QD-dye conjugates.     

Detection of FRET efficiency in imaging systems by photo-bleaching acceptors

Fluorescence resonance energy transfer (FRET) is widely used to obtain the distance between a donor and an acceptor in biological research. However, the detection of FRET efficiencies with fluorescence microscopy imaging systems remains a great challenge due to the difficulties of transferring gray scales of the images into fluorescence intensities, and the absence of exact quantum yields of donors and acceptors. Herein, we presented a new method to detect the FRET efficiency in imaging systems by analyzing the photo-bleaching-induced changes in fluorescent intensities of quantum dots (QDs, donors) and Cy5 dyes (acceptors). Our method is different from the previous acceptor-photo-bleaching studies in imaging systems by theoretically analyzing the bleaching process, and bringing forward a new parameter which is universal for samples of the same kind. It is convenient for calculating FRET efficiencies. There is hardly any spectral crosstalk between 605QD and Cy5, thus the FRET result is more accurate than that of many other common FRET pairs. The lengths of single-stranded and double-stranded DNA fragments in solution were determined via the analysis of FRET efficiency values. This technique provides a reliable approach to study biomacromolecules in living cells through fluorescent imaging and in situ measurements.     

Förster Resonance Energy Transfer Investigations Using Quantum‐Dot Fluorophores

F?rster resonance energy transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited donor fluorophore to a proximal ground-state acceptor fluorophore, is a well-characterized photophysical tool. It is very sensitive to nanometer-scale changes in donor-acceptor separation distance and their relative dipole orientations. It has found a wide range of applications in analytical chemistry, protein conformation studies, and biological assays. Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophores with unique optical and spectroscopic properties that could enhance FRET as an analytical tool, due to broad excitation spectra and tunable narrow and symmetric photoemission. Recently, there have been several FRET investigations using luminescent QDs that focused on addressing basic fundamental questions, as well as developing targeted applications with potential use in biology, including sensor design and protein conformation studies. Herein, we provide a critical review of those developments. We discuss some of the basic aspects of FRET applied to QDs as both donors and acceptors, and highlight some of the advantages offered (and limitations encountered) by QDs as energy donors and acceptors compared to conventional dyes. We also review the recent developments made in using QD bioreceptor conjugates to design FRET-based assays.     

Lanthanides to quantum dots resonance energy transfer in time-resolved fluoro-immunoassays and luminescence microscopy

A time-resolved fluoro-immunoassay (TR-FIA) format is presented based on resonance energy transfer from visible emitting lanthanide complexes of europium and terbium, as energy donors, to semiconductor CdSe/ZnS core/shell nanocrystals (quantum dots, QD), as energy acceptors. The spatial proximity of the donor-acceptor pairs is obtained through the biological recognition process of biotin, coated at the surface of the dots (Biot-QD), and streptavidin labeled with the lanthanide markers (Ln-strep). The energy transfer phenomenon is evident from simultaneous lanthanide emission quenching and QD emission sensitization with a 1000-fold increase of the QD luminescence decay time reaching the hundred mus regime. Delayed emission detection allows for quantification of the recognition process and demonstrated a nearly quantitative association of the biotins to streptavidin with sensitivity limits reaching 1.2 pM of QD. Spectral characterization permits calculation of the energy transfer parameters. Extremely large F?rster radii (R(0)) values were obtained for Tb (104 A) and Eu (96 A) as a result of the relevant spectral overlap of donor emission and acceptor absorption. Special attention was paid to interactions with the varying constituents of the buffer for sensitivity and transfer efficiency optimization. The energy transfer phenomenon was also monitored by time-resolved luminescence microscopy experiments. At elevated concentration (>10(-)(5) M), Tb-strep precipitated in the form of pellets with long-lived green luminescence, whereas addition of Biot-QD led to red emitting pellets, with long excited-state decay times. The Ln-QD donor-acceptor hybrids appear as highly sensitive analytical tools both for TR-FIA and time-resolved luminescence microscopy experiments.     

A Potential Carcinogenic Pyrene Derivative under Förster Resonance Energy Transfer to Various Energy Acceptors in Nanoscopic Environments

Picosecond‐resolved Förster resonance energy transfer (FRET) from various vibronic bands in benzo[a]pyrene (BP) shows a strong dependency on the spectral overlap of an energy acceptor in a confined environment. Our study on the dipolar interactions between BP and different acceptors, including ethidium (Et), acridine orange (AO), and crystal violet (CV), at the surface of a model anionic micelle revealed that the Förster distance (R₀) and the rate of energy transfer is dependent on the individual spectral overlap of the vibronic bands of BP with the absorption spectra of the different energy acceptors. The differential behavior of the vibronic bands is compared with that of different dyes [quantum dots (QDs)] in a “dye‐blend” (mixture) under FRET to an energy acceptor. Comparison of the FRET of the QDs with that of BP confirmed the independent nature of the dipolar interaction of the vibronic bands with other organic molecules, and the use of deconvolution techniques in the interpretation of the donor–acceptor (D –A) distance was also justified. We also showed that the consideration of differential FRET from the vibronic bands of BP and from the QDs in the dye‐blend is equally acceptable in theoretical frameworks including the Infelta–Tachiya model and D –A distribution analysis in nanoenvironments.     

The photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of Cy3.5-graphene oxide

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.     

Energy Relay from an Unconventional Yellow Dye to CdS/CdSe Quantum Dots for Enhanced Solar Cell Performance

A new design for a quasi‐solid‐state Forster resonance energy transfer (FRET) enabled solar cell with unattached Lucifer yellow (LY) dye molecules as donors and CdS/CdSe quantum dots (QDs) tethered to titania (TiO₂) as acceptors is presented. The Forster radius is experimentally determined to be 5.29 nm. Sequential energy transfer from the LY dye to the QDs and electron transfer from the QDs to TiO₂ is followed by fluorescence quenching and electron lifetime studies. Cells with a donor–acceptor architecture (TiO₂/CdS/CdSe/ZnS‐LY/S^2?‐multi‐walled carbon nanotubes) show a maximum incident photon‐to‐current conversion efficiency of 53 % at 530 nm. This is the highest efficiency among Ru‐dye free FRET‐enabled quantum dot solar cells (QDSCs), and is much higher than the donor or acceptor‐only cells. The FRET‐enhanced solar cell performance over the majority of the visible spectrum paves the way to harnessing the untapped potential of the LY dye as an energy relay fluorophore for the entire gamut of dye sensitized, organic, or hybrid solar cells.     

Fluorescence resonance energy transfer between a quantum dot donor and a dye acceptor attached to DNA

We show that direct coupling of a dye-labelled DNA (acceptor) to a quantum dot (QD) donor significantly reduces the donor-acceptor distance and improves the FRET efficiency: a highly efficient FRET (approximately 88%) at a low acceptor-to-donor ratio of 2 has been achieved at the single-molecule level.     

Surface effects on quantum dot-based energy transfer

CdSe quantum dot (QD)-phthalocyanine (Pc) conjugates were prepared as energy transfer donor-acceptor pairs, and the efficiency of the energy transfer process in this system was investigated as a function of QD size and under different surface chemistry conditions. The kinetics and efficiency of the energy transfer process were studied by femtosecond time-resolved laser spectroscopy. We observed that the energy transfer efficiency does not follow a linear dependence on spectral overlap integrals as predicted by the F?rster theory for molecules. This observation is found to be due to the involvement of QD surface states in the energy transfer process from the photoexcited QDs to the molecular energy acceptor.     

Thermal profiles of förster energy transfer preliminary studies of luminescent probes of protein dynamics in transferrin and calmodulin

Fluorescence energy transfer, the transfer of energy from a donor to an acceptor via a dipole/induced dipole mechanism, has long been used to measure distances between donors and acceptors in proteins and other macromolecules. Because the transfer can occur over time scales larger than protein bending and breathing modes, multiple conformational states can be sampled. The analysis of these states is weighted by the donor-acceptor distance; shorter distances carry more weight, because the energy transfer depends on the inverse sixth power of the distance. The usefulness of fluorecence energy transfer in probing these large amplitude protein motions is studied here. The method involves measuring the nergy transfer efficiency while perturbing the protein conformation with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in flexible region of the protein. This hypothesis was tested in two different protein systems; calmodulin, a calcium- activated regulatory protein, and transferrin, a blood serum iron shuttle. The preliminary studies show a differential sensitivity of the transfer efficiency to heat for the systems. Normalized energy transfer over 10 Å in calmodiulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 297 to 322 K. Normalized energy transfer over 42 Å in transferrin from a Tb(III) donor to an Fe(III) acceptor increase 35% over the same temperature range. In marked contrast to these systems, energy transfer from tyrosine to a chelated Tb(III) shows anomalously high temperature- dependence.