Quantum pathways for resonance energy transfer

A quantum electrodynamical calculation is presented that focuses individually on the two quantum pathways or time orderings for resonance energy transfer. Conventional mathematical procedures necessitate summing the quantum pathway amplitudes at an early stage in the calculations. Here it is shown, by the adoption of a different strategy that allows deferral of the amplitude summation, that it is possible to elicit key information regarding the relative significance of the two pathways and their distinct distance dependences. A special function integration method delivers equations that also afford new insights into the behavior of virtual photons. It is explicitly demonstrated that both time-ordered pathways are effective at short distances, while in the far field the dissipation of virtual traits favors one pathway. Hitherto unknown features are exhibited in the oblique asymptotic behavior of the time-ordered contributions and their quantum interference. Consistency with the rate equations of resonance energy transfer is demonstrated and results are presented graphically.     

Classical model for electronically non-adiabatic collision processes resonance effects in electronic-vibrational energy transfer

A classical model for electronically non-adiabatic collision processes is applied to E → V energy transfer in a collinear system, A + BC (v = 1) → A¹ + BC (v = 0), resembling Br-H₂.The model, which treats electronic as well as translational, rotational, and vibrational degrees of freedom by classical mechanics, describes the resonance features in this process reasonably well.     

Fluorescence resonance energy transfer in dye-labeled DNA

We present the results of molecular modeling of dye-labeled, double-stranded DNA. The structural information obtained from the simulations are used as input to an analysis of energy transfer in this system. The simulations reveal the nature of the interaction between a pair of fluorophores and DNA. The donor, tetramethylrhodamine, TMR, attached to the 5′-end of DNA with a six-carbon tether, interacts primarily with DNA's minor groove, but occasionally stacks against the DNA base pairs. The acceptor, Cy5, attached to the opposite strand at positions n (n = 7, 12, 14, 16, 19, 24, 27), binds in the major groove in two distinct locations on the upper and lower part of the groove. We analyzed in detail the dye-to-dye distances, dipole orientation factors and fluorescence resonance energy transfer (FRET) rates. Tests of the validity of the Förster model were conducted using the transition density cube (TDC) method, which provides the exact Coulombic interaction within a certain model chemistry. Our studies show that the use of long tethers does not guarantee rotational freedom of the dyes, as intended in the experiments. Instead, the tethers allow Cy5 to bind in two different geometries, which causes a large uncertainty in the dye-to-dye distances. Our results also show significant fluctuation in the orientation factor, κ², which, together with uncertainty in dye-to-dye distances, cause considerable uncertainty in interpreting FRET measurements. We suggest that molecular modeling, combined with the TDC method, provides a useful tool in designing and interpreting FRET experiments.     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

Fluorescence resonance energy transfer in polydiacetylene liposomes

Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.     

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.     

Ultrafast dynamics of resonance energy transfer in cryptochrome

In this communication, we report the ultrafast dynamics of resonance energy transfer in a blue-light photoreceptor, Vibrio cholerae cryptochrome. The transfer was observed to occur in 60 ps. We also studied the local rigidity and solvation around the binding site of the photoantenna molecule. The results for the first time show energy transfer in cryptochrome suggesting some mechanistic similarities between photolyase that repairs damaged DNA and cryptochrome that mediates blue-light signaling.     

Modulating fluorescence resonance energy transfer in conjugated liposomes

We report here a novel system where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism.     

Ultrafast fluorescence resonance energy transfer in a micelle

Ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) to rhodamine 6G (R6G) is studied in a neutral PEO(20)-PPO(70)-PEO(20) triblock copolymer (P123) micelle and an anionic micelle (sodium dodecyl sulfate, SDS) using a femtosecond up-conversion setup. Time constants of FRET were determined from the rise time of the acceptor emission. It is shown that a micelle increases efficiency of FRET by holding the donor and the acceptor at a close distance (intramicellar FRET) and also by tuning the donor and acceptor energies. It is demonstrated that in the P123 micelle, intramicellar FRET (i.e., donor and acceptor in same micelle) occurs in 1.2 and 24 ps. In SDS micelle, there are two ultrafast components (0.7 and 13 ps) corresponding to intramicellar FRET. The role of diffusion is found to be minor in the ultrafast components of FRET. We also detected a much longer component (1000 ps) for intramicellar FRET in the larger P123 micelle.     

Simple chemiluminescence aptasensors based on resonance energy transfer

In the present work, two aptamer-based probes and related sensor systems were developed with chemiluminescence signaling. The detection was based on "turning-on" chemiluminescence with switching "off" of the resonance energy transfer after the aptamer's recognition of the target molecule. In this design, a DNA/aptamer duplex linked a chemiluminescence group and a gold nanoparticle together. Only low-intensity chemiluminescence was obtained due to the highly efficient resonance energy transfer. After introducting the target molecule, structure-switching took place with turning off the energy transfer; thus, a restoration and turning on of the chemiluminescence was obtained. The two designs differed in the chemiluminescence groups, since one was a covalently linked luminol molecule, while the other was a conjugated horseradish peroxidase for the catalysis of further chemiluminescence reactions. These schemes provided simple and effective sensing toward a model analyte, adenosine.     

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.     

The smallest resonance energy transfer acceptor for tryptophan

The utility of diazirine ligands as acceptors in resonance energy transfer (RET) distance measurements with tryptophan or tryptophan analogues as donor is reported. The principle is demonstrated for a diazirine derivative of d-mannitol, 2-azi-2-deoxy-d-arabino-hexitol, and single-tryptophan-containing mutants of the mannitol transporter, EIImtl, from E. coli. The F?rster distance is 10 A for the tryptophan-diazirine donor-acceptor couple, allowing the measurement of distances up to 17 A. The versatility of tryptophan as an intrinsic spectroscopic probe in protein chemistry and the small size of the diazirine group makes this a very attractive donor-acceptor couple for accurate RET distance information in protein chemistry.     

Mediation of resonance energy transfer by a third molecule

The influence of a third molecule on the rate of resonance energy transfer is studied using diagrammatic perturbation theory within the framework of molecular quantum electrodynamics. Two distinct mechanisms are identified. One corresponds to direct transfer between donor and acceptor while the other involves relay of energy by the third species. Fermi Golden rule transition rates valid for all separation distances beyond wave function overlap are evaluated for these two processes as well as for the interference term between direct and indirect exchange, thereby extending previous work which was limited to the near-zone only. Short- and long-range limits are also obtained in each case. It is found that in the near-zone the indirect rate contribution exhibits inverse sixth power dependence on relative distances of emitter and absorber relative to the third body, in contrast to its far-zone counterpart, which exhibits inverse square behavior. The interference term, however, displays inverse cubic dependence on all three distance vectors at short-range and inverse behavior in the far-zone. Interestingly, for a collinear arrangement of the three molecules in the near-zone, the interference term is negative, reducing the overall rate of energy transfer. The results obtained are interpreted in terms of microscopic and macroscopic pictures of transfer occurring within a surrounding medium.     

Control of emission by intermolecular fluorescence resonance energy transfer and intermolecular charge transfer

Control of emission by intermolecular fluorescence resonant energy transfer (IFRET) and intermolecular charge transfer (ICT) is investigated with the quantum-chemistry method using two-dimensional (2D) and three-dimensional (3D) real space analysis methods. The work is based on the experiment of tunable emission from doped 1,3,5-triphenyl-2-pyrazoline (TPP) organic nanoparticles (Peng, A. D.; et al. Adv. Mater. 2005, 17, 2070). First, the excited-state properties of the molecules, which are studied (TPP and DCM) in that experiment, are investigated theoretically. The results of the 2D site representation reveal the electron-hole coherence and delocalization size on the excitation. The results of 3D cube representation analysis reveal the orientation and strength of the transition dipole moments and intramolecular or intermolecular charge transfer. Second, the photochemical quenching mechanism via IFRET is studied (here "resonance" means that the absorption spectrum of TPP overlaps with the fluorescence emission spectrum of DCM in the doping system) by comparing the orbital energies of the HOMO (highest occupied molecular orbital) and the LUMO (lowest unoccupied molecular orbital) of DCM and TPP in absorption and fluorescence. Third, for the DCM-TPP complex, the nonphotochemical quenching mechanism via ICT is investigated. The theoretical results show that the energetically lowest ICT state corresponds to a pure HOMO-LUMO transition, where the densities of the HOMO and LUMO are strictly located on the DCM and TPP moieties, respectively. Thus, the lowest ICT state corresponds to an excitation of an electron from the HOMO of DCM to the LUMO of TPP.     

Vibrational energy transfer in ozone by infrared-ultraviolet double resonance

Absorption transients at 254 nm have been observed in O₃-O₂ mixtures following laser irradiation at 9.64 μm. From analysis of these transients, we are able to determine vibrational relaxation rate constants (O₃-O₂ λ₁^?1/[O₂] = (2560±370) Torr^?1 S^?1, λ₂^?1/[O₂] = (640±50) Torr^?1 S^?1, and also a v₁-v₃ equilibration rate constant (O₃-O₃) of (1.5±1.0) × 10⁶ Torr^?1 S^?1.     

Vibrational dephasing and resonance energy transfer in molecular liquids

The influence of vibrational band-broadening mechanisms (pure dephasing, resonance transfer, depopulation processes) on the vibrational correlation functions of dense fluids is discussed. The coupling between the vibrational and the rotational-translational subsystems is assumed to be weak. It is found that even for weak coupling the homogeneously broadened linewidth (the inverse dephasing time τ^?1) cannot be represented as a sum of widths related to the individual mechanisms. Using an exponential repulsive interaction potential, we obtain numerical estimates for the dephasing time of the 1 ← 0 transition of liquid nitrogen, which agree very well with experimental observations. It turns out that the most significant contribution for τ arises from the weak anharmonicities of the molecular oscillator.     

Continuous-flow protease assay based on fluorescence resonance energy transfer

A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence of inhibitors. In the continuous-flow assay, an inhibitor solution and an enzyme solution were first eluted into the system and allowed to react with each other in a reaction coil. Subsequently, the substrate solution was added to an enzyme-inhibitor mixture in a second reaction coil and incubated for 1 min. Finally, the fluorescence intensity was monitored.The system was also utilized to measure the inhibition of the protease by two weak acidity inhibitors which are 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylenediaminetetraacetic acid (EDTA). Using the obtained optimum conditions for AEBSF, a detection limit of 0.3 mmol/l was achieved and the relative standard deviation was below 3.7% in the 2.5-7.5 mmol/l range. For EDTA, which required a 20 times higher substrate concentration than AEBSF, a detection limit of 0.2 mmol/l was obtained and the relative standard deviation was below 9.6% in the 0.5-7.5 mmol/l range.The optimization of pH, substrate concentration, enzyme concentration, reaction time and temperature are described. Organic modifier effects were also investigated. Methanol, acetonitrile and DMSO could be tolerated up to 30%.     

利用荧光共振能量转移体系测定食品中NO2-的研究

在λcx/λem=450／580nm,0．1mol／L的HCl溶液中,番红花红T和吖啶橙能够发生有效的共振能量转移,使得番红花红T荧光增强,同时吖啶橙的荧光猝灭,而NO2^-的加入使得两者的荧光强度同时减弱。由此建立了一种新的测定痕量NO2^-的方法。结果表明,NO2^-在0．02～10μg／mL范围内与染料的荧光强度减弱程度呈良好的线性关系,方法检出限为1．73ng／mL;该法用于食品中NO2^-的测定,回收率为105．0％～112．4％。