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.     

Quantitative genotyping of single nucleotide polymorphism by single-molecule multi-color fluorescence resonance energy transfer

We developed a new single nucleotide polymorphism (SNP) genotyping method based on single-molecule multi-color fluorescence resonance energy transfer (FRET). We demonstrated that this new method uses less than 1 fmol of sample and is also highly quantitative with a detection level of 1% or lower in the minor allele fraction.     

吖啶红-罗丹明B荧光共振能量转移法测定汞

研究了吖啶红(供体)和罗丹明B(受体)之间荧光共振能量转移的最佳条件,建立了荧光共振能量转移猝灭法测定污水和废旧电池中痕量汞的方法。室温中,采用十六烷基三甲基溴化胺(CTMAB),在pH=7.0条件下,吖啶红与罗丹明B之间能产生有效的共振能量转移,汞离子的加入能使体系中罗丹明B荧光峰强猝灭从而测定汞的含量。汞离子浓度在0.05～2.5μg/mL范围内与罗丹明B荧光强度变化ΔF呈现良好线性关系(r=0.9997),检出限(3σ/K)为0.95ng/mL,加标回收率98.0%～104.5%。该方法可用于污水和废旧电池中痕量汞的测定。     

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％。     

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.     

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.     

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.     

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.     

荧光共振能量转移测定牛血清白蛋白的研究

随着生物分析技术进入了后基因组时代,生命科学领域里的研究课题不断深入,DNA、RNA、蛋白质和其他生物大分子的检测技术发展十分迅速,生命科学中单分子分析技术不断揭示出生命活动的客观规律.相关的新的分析方法和仪器不断取得进展,成为生命科学的前沿领域.     

荧光共振能量转移测定牛血清白蛋白的研究

随着生物分析技术进入了后基因组时代,生命科学领域里的研究课题不断深入,DNA、RNA、蛋白质和其他生物大分子的检测技术发展十分迅速,生命科学中单分子分析技术不断揭示出生命活动的客观规律.相关的新的分析方法和仪器不断取得进展,成为生命科学的前沿领域.     

Self-assembly dynamics of a cylindrical capsule monitored by fluorescence resonance energy transfer

The constituent cavitands of a cylindrical capsule were labeled with donor and acceptor fluorophores, and fluorescence resonance energy transfer (FRET) was employed as a tool to study the dynamics of self-assembly. When donor and acceptor dyes are present in the same capsular assembly, they are brought within 25 A of each other, a distance suitable for efficient energy transfer to occur between them. This allowed for the study of interacting species at nanomolar concentrations providing information unattainable from NMR experiments. The kinetic stability of the capsule in the presence of various guest molecules was investigated which revealed a range of more than 4 orders of magnitude in the rates of cylindrical capsule exchange. While the thermodynamic stability of the capsule generally dictates the self-assembly dynamics, it was discovered that longer rigid guests can impart a significant kinetic barrier to monomer exchange.     

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%.     

Synthesis and assembly of monofunctionalized pyrogallolarene capsules monitored by fluorescence resonance energy transfer

Pyrogallol[4]arenes were monofunctionalized with fluorophores and fluorescence resonance energy transfer (FRET) was used to follow the self-assembly and exchange of the hexameric capsules at micromolar concentrations.     

Clear evidence of fluorescence resonance energy transfer in gas-phase ions

Fluorescence resonance energy transfer (FRET) is a distance-sensitive method that correlates changes in fluorescence intensity with conformational changes, for example, of biomolecules in the cellular environment. Applied to the gas phase in combination with Fourier transform ion cyclotron resonance mass spectrometry, it opens up possibilities to define structural/conformational properties of molecular ions, in the absence of solvent, and without the need for purification of the sample. For successfully observing FRET in the gas phase it is important to find suitable fluorophores. In this study several fluorescent dyes were examined, and the correlation between solution-phase and gas-phase fluorescence data were studied. For the first time, FRET in the gas phase is demonstrated unambiguously.     

Electron transfer reaction in a single protein molecule observed by total internal reflection fluorescence microscopy

To observe an electron transfer (ET) process in a single protein molecule, we constructed a model system, Alexa-HCytb5, in which cytochrome b5 (Cytb5) is modified with a fluorescent probe, Alexa Fluor 647 dye. In this model system, intramolecular transfer of an electron from the Alexa dye to heme in Cytb5 is supposed to oxidize the probe and quench its fluorescence, and the ET reaction at the single-molecule level can be monitored as the intermittent change in the fluorescence intensity. Alexa-HCytb5 was fixed on the glass surface, and illumination of laser light by the total internal reflection resulted in blinking of the fluorescence from the single Alexa-HCytb5 molecule in the time scale of several hundred milliseconds. Each Alexa-HCytb5 molecule is characterized by its own rate constant of the blinking, corresponding to the ET rate constant at the single-molecule level, and its variation ranges between 1 and 10 s(-1). The current system thus enables us to visualize the ET reaction in the single protein molecule, and the protein ET reaction was found to be explained by the distribution of the rate constants. On the basis of the Marcus theory, we suggest that the origin of this rate distribution is the distance change associated with the structural fluctuation in the protein molecule.     

RNA degradation in cell extracts: real-time monitoring by fluorescence resonance energy transfer

Short noncoding RNAs are increasingly recognized as key regulators of essential cellular processes such as RNA interference. A better understanding of the processes by which such RNAs are degraded is necessary to expand our knowledge of these processes and our ability to harness them. To this end we have developed a novel fluorescence resonance energy transfer (FRET) assay to monitor in real-time the degradation kinetics of short RNAs by a purified RNase and S100 cytosolic HeLa cell extract. An unstructured RNA is found to be degraded more rapidly than a stem-loop RNA under all conditions tested except for low concentrations of cell extract, showing that secondary structure confers protection against RNase activity. The assay also allows for the quantitative comparison of inhibitors such as Contrad70 and aurin tricarboxylic acid (ATA). Finally, gel electrophoretic FRET analysis confirms that HeLa cell extract is dominated by 5' to 3' exonucleolytic activity.     

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin-quencher is bound to Eu-streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher-biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2-2 nmol L⁻¹. In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay.