Steady State and Time Resolved Spectroscopic Study of CdSe and CdSe/ZnS QDs:FRET Approach

This article highlights some physical studies on the relaxation dynamics and Förster resonance energy transfer (FRET) of semiconductor quantum dots (QDs) to proximal dye molecule and the way these phenomena change with core to core-shell QD is discussed. Efforts to understand the optical and carrier relaxation dynamics of CdSe and CdSe/ZnS QDs are made by using absorption, steady-state fluorescence and time-resolved fluorescence (TCSPC) techniques. Steady-state as well as time-resolved fluorescence measurements were employed to evaluate the QD PL quenching induced by the proximal Rhodamine 101 dye molecule and to examine the influence of deep trap states on energy transfer efficiency. The FRET parameters such as spectral overlap, Förster distance, intermolecular distance for each donor-acceptor pair are determined and variation of these parameters from core to core-shell QD is discussed.     

Quantitative analysis of caspase-3 activation by fitting fluorescence emission spectra in living cells

Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death.     

CdTe量子点与罗丹明B水溶液体系下的双光子激发荧光共振能量转移

采用时间分辨荧光光谱技术研究了在双光子激发下不同尺寸的量子点与罗丹明B 之间的荧光共振能量转移. 研究结果表明, 在800 nm的双光子激发条件下, 体系间能量转移效率随着供体吸收光谱与受体荧光光谱的光谱重叠程度增加而增加; 理论分析表明, 供体和受体间的Förster半径增加是导致其双光子能量转移效率增大的物理原因. 同时, 研究了罗丹明B浓度对荧光共振能量转移效率的影响. 研究结果表明, 量子点的荧光寿命随着罗丹明B浓度的增加而减小; 量子点与罗丹明B之间的荧光共振能量转移效率随着罗丹明B浓度的增加而增加; 当罗丹明B浓度为3.0×10^-5 mol·L^-1时, 双光子荧光共振能量转移效率为40.1%.     

Optimization of FRET algorithms for sensitized-emission FRET detection

Sensitized-emission fluorescence resonance energy transfer（FRET） detection method based on threechannel fluorescence microscopy is widely used.Several FRET algorithms,such as N_FRET,FRETn,FR, FRET_R,and F^C/Df,are developed recently to quantitatively gauge and compare FRET signals between different experimental groups.However,the algorithms are difficult to choose and interpret.In this letter, we optimize the suitable yellow fluorescent protein（YFP） to cyan fluorescent protein（CFP） concentration ratio range for the above FRET algorithms.We also test the effect of YFP-to-CFP concentration ratio on the calculated energy transfer efficiency E and use the optimized FRET algorithms in the analysis of fas-associated protein with death domain（FADD） self-association directly in living cells.     

Detection of the homo- and hetero-interaction of proteins using fluorescence resonance energy transfer spectrum method

Fluorescence spectrometry based on fluorescence resonance energy transfer (FRET) principle is a simple but effective tool for investigating protein-protein interactions. In this paper, we report a spectrometry to quantify FRET efficiency based on our home-designed spectral probe system and spectral data-processing procedure. In our method, the fluorescence spectrum from each specimen is recorded at two wavelengths 454 and 502 nm. Least-squares linear fitting algorithm is applied directly to decompose the spectra of donor and acceptor under these two wavelengths to obtain FRET efficiency, which takes both spectral intensity and spectral profile into account compared with traditional three-step analysis. This system and the data-processing procedure enabled us to detect the homo-interaction and hetero-interaction of proteins in living cell.     

Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.     

FRET Studies of the Interaction of Dimeric Cyanine Dyes with DNA

Fluorescence Resonance Energy Transfer (FRET) is a powerful tool to determine distances between chromophores bound to macromolecules, since the efficiency of the energy transfer from an initially excited donor to an acceptor strongly depends on the distance between the two dye molecules. The structure of the noncovalent complex of double-strand DNA (dsDNA) with thiazol orange dimers (TOTO) allows FRET analysis of two intercalated chromophores. By intercalation of two different TOTO dyes we observe an energy transfer from TOTO-1 as donor and TOTO-3 as acceptor. In this manner we are able to determine the mean distance between two proximate TOTO molecules bound to dsDNA. Thus the maximum number of binding positions for this type of intercalation dyes in the dsDNA can be obtained. Furthermore the dependency of the acceptor emission on the donor concentration is analysed. The emission of TOTO-3 reaches a maximum when the acceptor-to-donor ratio is 1:10.     

Fluorescence resonance energy transfer between perylene and riboflavin in micellar solution and analytical application on determination of vitamin B2

Fluorescence resonance energy transfer (FRET) between perylene and riboflavin is studied in micellar solution of sodium dodecyl sulfate. The fluorescence of perylene is quenched by riboflavin and quenching is in accordance with Stern-Volmer relation. The efficiency of energy transfer is found to depend on the concentration of riboflavin. The value of critical energy transfer distance (R₀) calculated by using Foster relation is 32.13 Å, and as it is less than 50 Å, it indicates efficient energy transfer in the present system. The analytical relation was established between extent of sensitization and concentration of riboflavin, which helped to estimate vitamin B₂ directly from pharmaceutical tablets.     

Förster resonant energy transfer in quantum dot layers

Förster resonant energy transfer (FRET) in quantum dot (QD) layer structures has been analyzed. Small and large colloidal CdTe QDs were used as donors and acceptors, respectively. A FRET theory for random donor/acceptor distributions in two dimensions, taking into account exclusion zones around the donors, was applied to characterize FRET in a mixed monolayer. The exclusion zones provide a possibility to include the QD size in the FRET analysis and to determine its impact on the FRET efficiency. The acceptor concentration dependence of the FRET efficiency can also be described within this theory. In a separate donor/acceptor layer structure the distance dependence of the FRET efficiency as well as the acceptor enhancement was investigated. Both were found to agree well with the model of FRET between donor and acceptor layers.     

Theoretical study of fluorescence resonant energy transfer dynamics in individual semiconductor nanocrystal–DNA–dye conjugates

Motivated by recent experimental studies of fluorescence resonant energy transfer (FRET), we consider the influence of the temperature-dependent microscopic spectral overlap and relative orientation of the transition dipoles of fluorophores on the nanosecond dynamics of photon statistics and energy transfer efficiency in semiconductor nanocrystal–DNA–organic dye conjugates using Monte-Carlo simulations. Our calculated mean energy transfer efficiencies are found to be well consistent with those measured in experiment at low temperatures. For the higher temperatures, our results demonstrate that the use of Förster radius estimated from the isotropic dynamic average value of 2/3 for the orientation parameter term may lead to overestimation of energy transfer efficiency for the cases of the rigid arrangement of fluorophore transition moments, and thereby deteriorate the precision of the analysis of donor–acceptor distances. Our theoretical results here underline the importance of a detailed understanding of the microscopic picture of FRET for exploiting this spectroscopic technique in various nano- and bio-applications.     

Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.     

Fluorescence resonance energy transfer: FRET studies of ligand binding to cell surface receptors

We describe a simple optical system employing fluorescence resonance energy transfer (FRET) to identify potential binding domains on the macrophage scavenger receptor for the ligand maleylated bovine serum albumin (mal-BSA). Using a plasma membrane vesicle system, we placed donor probes on the ligand and acceptor probes in the membrane to determine the distance of bound ligand from the cell surface. Two donors and three acceptors were employed. Transfer between ligand covalently modified with multiple dansyl molecules and hexadecanoylaminoeosin in the membrane yielded a distance of 46.5 ± 7.5 å; transfer from the same type of donors to octadecylrhodamine B in the membrane gave a distance of 58.5 ± 3.0 å. No transfer was observed between ligand mono-labeled with fluorescein and l,l′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate in the membrane. This suggests that the orientation of mal-BSA bound to the receptor places the fluorescein probe too far from the lipid surface to experience energy transfer. The distance information identifies a potential location for the binding site, which can be compared to structural information about the receptor and used to extract a binding sequence.     

聚二甲基硅氧烷微流道中光流控荧光共振能量转移激光

将单一折射率的石英裸光纤植入由聚二甲基硅氧烷构成的基片微流道中,以低折射率的罗丹明B(RhB)和吡啶821(LDS821)乙醇溶液构成的供体和受体对作为激光增益介质.采用沿光纤轴向消逝波抽运方式,首先以波长为532nm的连续波激光器作为激励光,对荧光共振能量转移特性参数进行了研究.然后以波长为532nm的脉冲激光器作为抽运光,通过直接激励供体分子RhB,并将其能量转移给临近的受体分子LDS821,在不改变抽运光波长的条件下,实现了较低阈值(1.26μJ/mm~2)的受体LDS821激光辐射.     

Resonant transfer of one- and two-photon excitations in quantum dot–bacteriorhodopsin complexes

Light-sensitive protein bacteriorhodopsin (BR), which is capable of electrical response upon exposure to light, is a promising material for photovoltaics and optoelectronics. However, the rather narrow absorption spectrum of BR does not allow achieving efficient conversion of the light energy in the blue and infrared spectral regions. This paper summarizes the results of studies showing the possibility of extending the spectral region of the BR function by means of the Förster resonance energy transfer (FRET) from CdSe/ZnS quantum dots (QDs), which have a broad spectrum of one-photon absorption and a large twophoton absorption cross section (TPACS), to BR upon one- and two-photon excitation. In particular, it is shown that, on the basis of QDs and BR-containing purple membranes, it is possible to create electrostatically associated bio-nano hybrid systems in which FRET is implemented. In addition, the large TPACS of QDs, which is two orders of magnitude larger than those of BR and organic dyes, opens up a means for selective two-photon excitation of synthesized bio-nano hybrid complexes. On the basis of the results of this work, the spectral region in which BR converts the light energy into electrical energy can be extended from the UV to near-IR region, creating new opportunities for the use of this material in photovoltaics and optoelectronics.     

Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ₁ ) to selectively detect the fluorescence from donor, and a FRET channel (CH ₂ ) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.     

Location of Novel Benzanthrone Dyes in Model Membranes as Revealed by Resonance Energy Transfer

Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) as a donor and newly synthesized benzanthrones (referred to here as A8, A6, AM12, AM15 and AM18) as acceptors has been examined to gain insight into molecular level details of the interactions between benzanthrone dyes and model lipid membranes composed of zwitterionic lipid phosphatidylcholine and its mixtures with anionic lipids cardiolipin (CL) and phosphatidylglycerol (PG). FRET data were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for A8 location in phospholipid headgroup region has been obtained. Inclusion of CL and PG into PC bilayer has been found to induce substantial relocation of A6, AM12, AM15 and AM18 from hydrophobic membrane core to lipid-water interface.     

Fluorescent polystyrene microspheres with large Stokes shift by fluorescence resonance energy transfer

We prepared fluorescent microspheres with notably large Stokes shift and long-wavelength fluorescence by applying fluorescence resonance energy transfer (FRET) between two common julolidine dyes. Short distance between dye molecules caused by high dye concentration results in efficient FRET in microspheres. However, adequate dye concentration and moderate molar ratio of the donor and acceptor should be chosen to avoid aggregation of dye molecules, which leads to the decrease of fluorescent intensity. Microrspheres with average distance between dye molecules of 1.94 nm and molar ratio of 3.08:1 realize highly efficient FRET with no fluorescence of donor and intense long-wavelength emission of acceptor. In addition, the applied solvent evaporation method for preparing microspheres provided better protection of dyes from ambient medium than traditional surface-labeled method. These results demonstrate the feasibility of applying FRET in microspheres to expand useful fluorescent probes, and reveal their potential application in bioassays field.     

Spectroscopic studies on the molecular interaction between salicylic acid and riboflavin (B2) in micellar solution

The interaction between salicylic acid (SA) and riboflavin (RF) was studied by Fluorescence Resonance Energy Transfer (FRET) in micellar solution. The riboflavin strongly quenches the intrinsic fluorescence of SA by radiative energy transfer. The extent of energy transfer in sodium dodecyl sulphate (SDS) micellar solution of different concentration is quantified from the energy transfer efficiency data. It is seen that the energy transfer is more efficient in the micellar solution. The critical energy transfer distance (R₀) was determined from which the mean distance between SA and RF molecules was calculated. The quenching was found to fit into Stern-Volmer relation. The results on variation of Stern-Volmer constant (K_sv) with quencher concentration obtained at different temperatures suggested the formation of complex between SA and RF. The association constant of complex formation was estimated and found to decrease with temperature. The values of thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were estimated and the results indicated that the molecular interaction between SA and RF is electrostatic in nature.     

Detection of <Emphasis Type="Italic">Bifidobacterium</Emphasis> Species-specific 16S rDNA Based on QD FRET Bioprobe

Fluorescence resonance energy transfer (FRET) that consists of quantum dot as donors and organic fluorophore dyes as acceptors has been a very important method to detect biomolecules such as nucleic acids. In this work, we established a new FRET detection system of Bifidobacterium species-specific 16S rDNA using QD—ROX FRET bioprobe, in which 525 nm QD-DNA conjugation consisted of the carboxyl-modified QD and the amino-modified DNA in the presence of EDC. Both ROX-DNA and the conjugation above could hybridize with the target DNA after forming the QD—ROX bioprobe. When the hybridization made the distance between the QD and ROX to meet FRET effect needed, 525 nm QD fluorescence intensity decreased and ROX fluorescence intensity increased. In the control, there was no notable change of fluorescence intensities without target DNA. It is very clear that the change of the QD and ROX fluorescence intensities provide the good base and guaranty for this rapid and simple detection system.     

庆大霉素与水溶性CdTe量子点的荧光共振能量转移作用

以巯基丙酸(mercaptopropionic acid,MPA)为稳定剂合成水溶性CdTe最子点(quantum dots,QDs),以CdTe QDs作为能量供体.庆大霉素(Gentamycin,GT)作为能垦受体,建立了荧光共振能量转移(fluorescence resonance energy transfer,FRET)体系.在690 nm处可见发射峰,半峰宽约10 nm,在一定范围内荧光强度与GT的含量旱线性关系,线性范围为2～20 mg·L-1,相关系数r=0.986 7.优化了不同的激发波长、pH、离子强度、时间和温度等凼素对反应的影响,并应用傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)和高效液相色谱(high-performance liquid chromatography,HPLC)分别表征了化学结构和相对专一性.结果表明巯基丙酸的巯基中S原子和羧基中氧原子与纳米微粒表面的富Cd离子发生了配位作用,CdTe QDs与GT的耦合主要是通过量子点周围巯基丙酸羧基(-COOH)中的氧原子与GT的胺基(-NH2)形成分子问氧键实现的;GT与CdTe QDs的结合率为0.35:1.研究表明GT可以作为检测CdTe QDs标记牛血清白蛋白(bovine serum albumin,BSA)的荧光增敏剂,荧光强度值增强6倍,应用前景广阔.