Fluorescence Resonance Energy Transfer Rate Research Articles

Efficient FRET process between CsPbBr3 quantum dots and RhB dye molecules by pressure regulation

Fluorescence resonance energy transfer (FRET) based on quantum dots (QDs) and dye molecules have great application potential in biochemical fields. How to achieve an efficient energy transfer process has become an important research topic. Pressure can be used to regulate the energy transfer process, but its regulation on metal halide perovskite systems is rarely reported. Herein, the efficient FRET process between CsPbBr3 QDs and Rhodamine B (RhB) molecules under high pressure is investigated. Upon compression to 1.3 GPa, the FRET rate of the CsPbBr3–RhB composite reaches 0.21 ns−1 and the FRET efficiency is improved from 12.4% to 62.4%, due to enhanced spectral overlap and shortened minimum distance between CsPbBr3 QDs and RhB molecules. This study provides a strategy for achieving efficient FRET research and further promotes the development of applications based on halide perovskite molecular systems.

Energy Funneling from Water-Dispersed Perovskites to Chromophores

Cesium lead halide perovskite nanocrystals (PNCs) have enjoyed enormous attention in optoelectronics and photovoltaics. However, instability under polar conditions and limited energy/charge transport due to long-chain capping ligands restrict their large-scale applications. We have engineered a short-chain multidentate bolaamphiphilic ligand (NKE-3), which provides synergistic passivation of the perovskite surface by one multidentate ionic terminal and localizes water molecules by another multidentate ionic terminal, leading to a water-suspended colloidal solution of PNCs. NKE-3 allows efficient long-range dipole-based fluorescence resonance energy transfer (FRET) from perovskites to Rhodamine B isothiocyanate (RITC) in water, with FRET efficiencies ranging from 96% to 98%. We calculated the FRET rate using the acceptor’s rise-time component, as it ensures no contamination from FRET-inactive donors. Moreover, we tuned the emission maxima of PNCs through halide exchange to optimize FRET efficiency. Such energy funneling to a suitable molecular photocatalyst is imperative for PNCs’ potential applications.

Meso- and β-Pyrrole-Linked Chlorin-Bacteriochlorin Dyads for Promoting Far-Red FRET and Singlet Oxygen Production.

A series of chlorin-bacteriochlorin dyads (derived from naturally occurring chlorophyll-a and bacteriochlorophyll-a), covalently connected either through the meso-aryl or β-pyrrole position (position-3) via an ester linkage have been synthesized and characterized as a new class of far-red emitting fluorescence resonance energy transfer (FRET) imaging, and heavy atom-lacking singlet oxygen-producing agents. From systematic absorption, fluorescence, electrochemical, and computational studies, the role of chlorin as an energy donor and bacteriochlorin as an energy acceptor in these wide-band-capturing dyads was established. Efficiency of FRET evaluated from spectral overlap was found to be 95 and 98 % for the meso-linked and β-pyrrole-linked dyads, respectively. Furthermore, evidence for the occurrence of FRET from singlet-excited chlorin to bacteriochlorin was secured from studies involving femtosecond transient absorption studies in toluene. The measured FRET rate constants, kFRET , were in the order of 1011 s-1 , suggesting the occurrence of ultrafast energy transfer in these dyads. Nanosecond transient absorption studies confirmed relaxation of the energy transfer product, 1 BChl*, to its triplet state, 3 Bchl*. The 3 Bchl* thus generated was capable of producing singlet oxygen with quantum yields comparable to their monomeric entities. The occurrence of efficient FRET emitting in the far-red region and the ability to produce singlet oxygen make the present series of dyads useful for photonic, imaging and therapy applications.

Energy-Transfer Phenomena in Thermoresponsive and pH- Switchable Fluorescent Diblock Copolymer Vesicles.

We describe the development of a polymeric vesicle that not only selectively fluoresces at low pH, a condition prevailing in cancer cells, but also can potentially monitor the thermoresponsive release of a drug even if the drug is nonfluorescent. The developed fluorescence resonance energy transfer (FRET)-based thermoresponsive vesicular nanocarriers are composed of a new poly(PEGMA)-b-poly(NIPA-r-R6GMED) block copolymer, which undergoes pH-switchable superior turn on-off fluorescence characteristics. The block copolymer was synthesized using the RAFT technique, and its solution properties and self-assembly behavior were investigated by turbidity measurements, fluorescence spectroscopy, 1H NMR, dynamic light scattering, and transmission electron microscopy. The block copolymer self-assembled to form nanostructured vesicles above the critical aggregation temperature under physiologically relevant conditions. Steady-state and time-resolved fluorescence spectroscopy were utilized to study the FRET process between encapsulated hydrophobic guest C-153 (donor) and polymer-bound R6GMED units (acceptor) in the thermoresponsive vesicles. The FRET rate and efficiency were found to vary as a result of the pH-dependent changes in the quantum yield of the acceptor molecules. The occurrence of a highly efficient FRET in this polymeric vesicular nanocarrier at acidic pH, a condition similar to the cytoplasm and cell nucleus in leukemic tissues, and the ability to encapsulate hydrophilic and hydrophobic molecules and their temperature-controlled release make it potentially useful in imaging guided real-time monitoring of drug-delivery vehicles.

Synaptotagmin 1 and Ca2+ drive trans SNARE zippering

Synaptotagmin 1 (Syt1) is a major Ca2+-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca2+, indicating C2AB does not interfere with the initial nucleation of SNARE assembly. However, C2AB-Ca2+ accelerated the FRET rate significantly at membrane proximal region, indicating C2AB-Ca2+ promotes the transition from a partially assembled SNARE complex to the fusion-competent SNAREpin. Similar enhancement was also observed at the end of the transmembrane domain of SNARE proteins. The stimulatory effect disappeared if there was no membrane or only neutral membrane present.

Study of Fluorescence Resonance Energy Transfer in Zwitterionic Micelle: Ionic-Liquid-Induced Changes in FRET Parameters

The fluorescence resonance energy transfer (FRET) using Coumarin-153 (C-153) as the donor and Rhodamine 6G (R6G) as the acceptor is studied in an aqueous solution of N-hexadecyl-N,N-dimethylammonio-1-propanesulfonate (SB-16) micelles by steady-state and picosecond time-resolved fluorescence spectroscopy. We have determined the rate of FRET (k(FRET)) from the rise of the acceptor (R6G) emission. In the absence of donor (C-153), the acceptor (R6G) displays a single-exponential decay with average lifetime of 4.77 ns, whereas in presence of donor (C-153), the acceptor (R6G) exhibits a biexponential fluorescence transient having a distinct rise component of 0.94 ns and decay component of 5.16 ns. We have carried out a comparative study of changes in FRET parameters upon addition of three different ionic liquids (ILs), 1-ethyl-3-methylimidazolium ethylsulfate [C(2)mim][C(2)SO(4)], 1-ethyl-3-methylimidazolium n-butylsulfate [C(2)mim][C(4)SO(4)], and 1-ethyl-3-methylimidazolium n-hexylsulfate [C(2)mim][C(6)SO(4)], where each ionic liquid bears the same cationic part and the anionic parts differ in the alkyl chain length only. It has been observed that with gradual addition of the ILs [C(2)mim][C(2)SO(4)], [C(2)mim][C(4)SO(4)], and [C(2)mim][C(6)SO(4)], the rise component gradually decreases and the rate of FRET (k(FRET)) gradually increases. The k(FRET) was found to be 1.06 × 10(9) s(-1) in 28 mM aqueous SB-16 micelles. With the addition of 100 mM [C(2)mim][C(2)SO(4)], the k(FRET) increases by a factor of 1.33 (1.41 × 10(9) s(-1)), whereas with the addition of 100 mM [C(2)mim][C(6)SO(4)] it increases by a factor of 3.25 (3.45 × 10(9) s(-1)). This rapid increase in k(FRET) in the case of [C(2)mim][C(6)SO(4)] can be explained by our earlier observation ( Rao, V. G.; Ghatak, C.; Ghosh, S.; Mandal, S.; Sarkar, N. J. Phys. Chem. B2012, 116, 3690-3698 ), where we have shown that with the addition of [C(2)mim][C(6)SO(4)], C-153 moves toward the outer surface of the micelle. This movement of C-153 causes reduction in donor-acceptor distance and enhancement in FRET rate (k(FRET)). This is well-supported by the reduced donor-acceptor distance (R(DA)) observed with the addition of [C(2)mim][C(6)SO(4)]. The R(DA) was found to be 29.1 Å in 28 mM aqueous SB-16 micelles. With the addition of 100 mM [C(2)mim][C(6)SO(4)], the R(DA) decreases to 24.8 Å. With further increase in the concentration of [C(2)mim][C(6)SO(4)], the R(DA) decreases, but the time constant for the rise of acceptor emission decreases to such an extent that we are unable to observe it by our instrumental setup.

FRET based ultra sensor for detection of Hg (II) in water: A comparative study using citrate and marcapto propanoic acid as stabilizer of AuNPs

Rhodamin 6G (Rh6G) functionalized Au nanoparticles (AuNPs) are being used as efficient fluorescence probe for selective sensing of heavy metal ions. It has been observed that the quenching of fluorescence of Rh6G in presence of AuNPs takes place through fluorescence resonance energy transfer (FRET). Recovery of fluorescence of Rh6G emission in presence of Hg (II) in AuNPs–Rh6G coupled system has been explained as the increasing affinity of heavy metal ions to form shell and thereby reduced the FRET rate. In presence of heavy metal ions such as Hg (II), Rh6G molecules are released from the AuNPs surface and the fluorescence of Rh6G is restored. The modulation of photoluminescence quenching efficiency of Rh6G–AuNPs in the presence of Hg (II) ions can achieve a large turn on fluorescence enhancement. Our experiment using marcapto propanoic acid (MPA) caped AuNPs show much less fluorescence enhancement in presence of Hg 2+ compares to tri sodium citrate (TSC) stabilized AuNPs.

Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G(s) and G(i) proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G(i) inhibitor pertussis toxin (PTX). Further studies revealed that activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/G(i) coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (> or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR blocked receptor internalization and coupling to G(i) proteins, probably by preventing the receptor's transportation to access G(i) proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G(i)-specific inhibitor, G(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.

Molecular dynamics study of chemically engineered green fluorescent protein mutants: Comparison of intramolecular fluorescence resonance energy transfer rate

Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N-terminal or C-terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, kappa(2). In agreement with experiment, the construct with the C-terminally attached dye was predicted to have higher energy transfer rate than observed for the N-terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, kappa(2), deviates from the commonly assumed value, the implications of which are also considered.

The fluorescence resonance energy transfer between dye compounds in micellar media

The fluorescence resonance energy transfer from fluorescein to merocyanine 540 in aqueous sodium dodecyl sulfate, cetyltrimethylammonium bromide and Triton X-100 micellar solutions as well as deionized water was investigated at room temperature using steady-state and time-resolved fluorescence spectroscopy techniques. Fluorescence resonance energy transfer rate constants ( k T), obtained using both Stern–Volmer and Förster theories, were in good agreement. Moreover, energy transfer efficiency values of 0.14, 0.38, 0.77 and 0.85 for deionized water, sodium dodecyl sulfate, TX-100 and cetyltrimethylammonium bromide micellar solution were obtained at the highest acceptor concentration at which fluorescence resonance energy transfer was achieved. The data obtained from steady-state absorption, fluorescence spectral studies and time-resolved lifetime measurements indicated that the fluorescence resonance energy transfer from fluorescein to merocyanine 540 occurred most effectively in aqueous cetyltrimethylammonium bromide micellar solutions.

Evolution of Energy Transfer Process between Quantum Dots of Two Different Sizes during the Evaporation of Solvent

CdSe quantum dots (QDs) of two different sizes were dispersed in toluene solvent. We observed that the photoluminescence (PL) intensity of smaller QDs was gradually depressed by larger QDs during the evaporation of solvent. This is attributed to fluorescence resonance energy transfer (FRET) between two different sized QDs, when the distance between QDs is enough small to ensure close proximity. From PL intensity ratio of smaller to larger QDs, three stages can be distinguished during the evaporation and FRET rate begin to increase in the gel like stage. We estimated the mean distance variation between smaller and larger QDs during the evaporation from PL measurement and Dexter theory. In the solid resulted from the evaporation, FRET rate from smaller to larger dots and FRET rate within smaller QDs were quantitatively estimated to be 0.13 and 0.08 ns-1, respectively. The change in PL properties due to the aggregation of QDs by evaporation is discussed.

Dissecting interacting molecular populations by FRET

COMMENTARY Fluorescence resonance energy transfer (FRET) (1), which used to be a biophysical technique strictly for specialists a few decades ago, has become a standard tool to study molecular conformation, interactions, and colocalization at the nanometer scale in cell biology and related fields (2–9). This penetration is exemplified by new editions of cell biology textbooks covering FRET among classical methods such as coimmunoprecipitation or magnetic resonance (MR) (10). The increased popularity of FRET is due to several factors including the availability of brighter, more photostable dyes, quantum dots, and color variants of fluorescent proteins, and the development of new features, add-ons and dedicated hardware and software that are available for commercial microscopes (11) and flow cytometers (12), and facilitate the smooth application of FRET techniques. In addition to the fact that FRETmeasurements can be applied to detect molecular associations (2–9,13), the FRET phenomenon can be used to improve spectral characteristics of luminescent probes to be utilized in cytometry (14,15). In this commentary we will address two concerns frequently encountered in FRET imaging: the need for being able to dissect molecular populations displaying different FRET efficiencies (16), and the surmounting of artifacts that may arise due to molecular diffusion during the FRETexperiment (17,18). FRET is the nonradiative transfer of excitation energy from an excited, fluorescent donor dye to a (not even necessarily fluorescent) acceptor in the 2–10 nm vicinity of the donor. The probability of the process is characterized by the FRET efficiency (E), which is a measure of the fraction of excitation energy quanta transferred from the donor to the acceptor. Because of the dependence of the rate of FRET on the negative 6th power of the donor–acceptor distance, FRET is also called a spectroscopic ruler (19) allowing sensitive determination of interand intramolecular distances under ideal conditions. Because of FRET, the fluorescence intensity and lifetime of the donor dye decrease, whereas its anisotropy increases (provided that the donor and the acceptor are two different dyes and there is no significant back transfer of energy from the acceptor to the donor) (20). At the same time, the fluorescence intensity of the acceptor increases (sensitized emission) and its anisotropy decreases. Different methods to evaluate the FRET efficiency take advantage of one or more of these spectroscopic changes (3). We can classify them, somewhat arbitrarily, into (a) intensitybased methods measuring donor quenching and/or sensitized emission, and (b) lifetime-based methods determining the fluorescence lifetime components directly or instead, a spectroscopic quantity related to the lifetime (such as the characteristic donor photobleaching rate in a donor photobleaching experiment). Subcellular inhomogeneity of protein–protein interactions necessitates the resolution of high-FRET and low-FRET molecular populations within the same cell. However, by using classical intensity based methods no distinction can be made between the situation of a single population of donor–acceptor pairs characterized by a single intermediate FRETefficiency value, and a mixture of high-FRET and low-FRET donor– acceptor pairs producing the same average FRET efficiency as in the first case. The reason is that in these measurements

Theory of resonance energy transfer involving nanocrystals: The role of high multipoles

A theory for the fluorescence resonance energy transfer (FRET) between a pair of semiconducting nanocrystal quantum dots is developed. Two types of donor-acceptor couplings for the FRET rate are described: dipole-dipole (d-d) and the dipole-quadrupole (d-q) couplings. The theory builds on a simple effective mass model that is used to relate the FRET rate to measureable quantities such as the nanocrystal size, fundamental gap, effective mass, exciton radius, and optical permittivity. We discuss the relative contribution to the FRET rate of the different multipole terms, the role of strong to weak confinement limits, and the effects of nanocrystal sizes.

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe ( BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system ( BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.

Simulating FRET from Tryptophan: Is the Rotamer Model Correct?

We present a computational model study designed to simulate the results of time-resolved fluorescence spectra of tryptophan in proteins. In such measurements, the occurrence of more than one fluorescence lifetime is generally attributed to the existence of several tryptophan rotamers and/or structural conformations of the protein structure. The protein system we chose for this initial study is the tetracycline repressor (TetR), an interesting model system for the investigation of the mechanisms of transcriptional regulation. Fluorescence resonance energy transfer (FRET) from tryptophan to tetracycline is frequently observed in complexes of the TetR with the antibiotic tetracycline. We use a combined classical/quantum mechanical approach to model the structure and the spectroscopic properties of the TetR-tetracycline complex. A classical molecular dynamics simulation provides input geometries for semiempirical quantum mechanical/molecular mechanical (QM/MM) single-point configuration interaction (CI) calculations, which are used to calculate tryptophan vertical absorption and fluorescence energies and intensities as well as relative FRET rate constants. These rate constants together with the Einstein coefficients for spontaneous emission and an assumed rate for nonradiative deactivation allow us to simulate fluorescence decay curves with and without FRET and for the entire ensemble as well as for individual rotamers. Our results indicate that the classical "rotamer model", used to explain the multiexponential fluorescence-decay curves of time-resolved tryptophan emission spectra, can be extended to systems with FRET acceptors present in the protein matrix but that the interpretation of the fitted lifetimes is different to that usually used.

Fluorescence resonance energy transfer (FRET) in chemistry and biology: Non-Förster distance dependence of the FRET rate

Fluorescence resonance energy transfer (FRET) is a popular tool to study equilibrium and dynamical properties of polymers and biopolymers in condensed phases and is now widely used in conjunction with single molecule spectroscopy. In the data analysis, one usually employs the Förster expression which predicts (l/R 6) distance dependence of the energy transfer rate. However, critical analysis shows that this expression can be of rather limited validity in many cases. We demonstrate this by explicitly considering a donor-acceptor system, polyfluorene (PF6)-tetraphenylporphyrin (TPP), where the size of both donor and acceptor is comparable to the distance separating them. In such cases, one may expect much weaker distance (as l/R 2 or even weaker) dependence. We have also considered the case of energy transfer from a dye to a nanoparticle. Here we find l/R 4 distance dependence at large separations, completely different from Förster. We also discuss recent application of FRET to study polymer conformational dynamics.

A Mechanical Force Accompanies Fluorescence Resonance Energy Transfer (FRET)

We show that the coupling responsible for fluorescence resonance energy transfer (FRET) generates a mechanical force that is distinct from the van der Waals force between ground-state molecules and can be either attractive or repulsive. The underlying potential has the same 1/r6 distance dependence as the rate of FRET, and the two are connected by a Kramers−Kronig relation. Just as the rate of FRET can be derived either classically or quantum mechanically, so too can the interaction potential. Because of the FRET force, time-resolved FRET measurements contain information on the mechanical stiffness of the matrix containing donor and acceptor.