Development of a novel optical bionanosensor

We have developed a novel optical bionanosensor platform using a supported bilayer lipid membrane (SBLM), which has generic multi-analyte sensing capabilities. The SBLM is produced using a novel combination of ordered nanostructured thin film i.e. Langmuir-Blodgett (LB) and self-assembly deposition methodologies. A heptamer linear RGD (arginine-glycine-aspartate) containing peptide was covalently attached to a BODIPY (donor) lipid dye and utilised as an optical biosensor for integrin alpha(v)beta(3) loaded HUVEC's (sensitivity = 1000 cells ml(-1)). A second BODIPY (acceptor) lipid dye was integrated into the SBLM thus enabling signal amplification via a Forster resonance energy transfer (FRET) mechanism. The fluidity of the bilayer was confirmed via fluorescence recovery after photobleaching (FRAP) techniques and was performed without the need for fusogens.     

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Multiplexed FRET to image multiple signaling events in live cells

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.     

Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy.

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of proteins, lipids, enzymes, DNA, and RNA in vivo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the potential to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, steady-state FRET microscopy measurements can suffer from several sources of distortion, which need to be corrected. These include direct excitation of the acceptor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the analysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detection of acceptor fluorescence with the donor emission filter), and for the dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a recently identified Bcl-2 interacting protein located on chromosome 17q21), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the different filter units.     

Triple-FRET Technique for Energy Transfer Between Conjugated Polymer and TAMRA Dye with Possible Applications in Medical Diagnostics

Three-component Förster resonance energy transfer (FRET) has been used to obtain efficient FRET between the cationic conjugated polymer (CCP) as donor and 5-carboxy tetramethylrhodamine (TAMRA) dye as acceptor, by using an intermediate donor, fluorescein. In spite of the fact that there is enough overlap between the emission spectra of CCP and absorption spectra of TAMRA, the efficiency of FRET between CCP and TAMRA is poor. The reason for this is that while the Förster critical distance is not very sensitive to the overlap, the FRET efficiency is extremely sensitive to it. However, it is observed that the FRET efficiency between CCP and TAMRA improves considerably when fluorescein is introduced in the solution. The triple FRET so obtained can be used for deoxyribonucleic acid sequence detection in medical diagnostics because the fluorescence emission from TAMRA is pH-insensitive.     

Optimizing the cationic conjugated polymer-sensitized fluorescent signal of dye labeled oligonucleotide for biosensor applications

Methods for real time, highly selective and sensitive polynucleotide detection are of vast scientific and economic importance. Fluorescence resonance energy transfer (FRET)-based assays which take advantage of the collective response of water-soluble conjugated polymers (CPs) and the self-assembly characteristic of aqueous polyelectrolytes have been widely used for the detection of DNA, RNA, protein and small molecules. The detection sensitivity of CP-based biosensor is dependent on the signal amplification of dye emission upon excitation of CP relative to that upon direct excitation of the dye. Using cationic polyfluorene derivatives and chromophore (fluorescein or Texas Red) labeled single-stranded DNA molecules (ssDNA-C*) as donor/acceptor pairs, we show that in addition to the spectral overlap, orientation and distance between the donor and the acceptor, the energy levels and fluorescence quenching of the donor/acceptor within the polymer/DNA-C* complexes are also important factors that affect the signal output of dye emission.     

Fluorescent resonance energy transfer (FRET) based detection of a multiplex ligation-dependent probe amplification assay (MLPA) product

A fluorescent resonance energy transfer (FRET)-based hybridization assay for detecting multiplex ligation-dependent probe amplification (MLPA) products has been developed, extending the diagnostic power of the technique and demonstrating the possibility of combining MLPA with microarrays for the detection of multiple mutations. FRET is one of the most commonly used detection techniques for hybridization assays. To investigate the applicability of FRET based detection of MLPA products, a sandwich assay was designed to detect gene copy number by exploiting an immobilized probe labeled with an acceptor dye, Alexa Fluor 555, which hybridises to specific PCR amplicons, followed by hybridization of a second probe labeled with the donor dye, Alexa Fluor 488. Following excitation of the Alexa Fluor 488, a FRET signal was produced only if a DNA sequence specific to the BRCA1 exon 13 was present in the test sample. We have verified this assay on a DNA sample of a patient carrying a heterozygous BRCA1 exon 13 deletion using male genomic DNA as control. Here we demonstrate that the DNA sample containing the heterozygous deletion generated a considerably reduced FRET signal as compared to the control male human DNA. Our results show that the FRET design presented in this study can differentiate between reduced copy numbers any genomic DNA sequence after MLPA analysis, and the reported format is applicable to multiplex detection of MLPA products, using microarrays, or optical biosensor arrays, and future work will focus on the demonstration of this.     

Submicron structure in L-alpha-dipalmitoylphosphatidylcholine monolayers and bilayers probed with confocal, atomic force, and near-field microscopy.

Langmuir-Blodgett (LB) monolayers and bilayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC), fluorescently doped with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diIC18), are studied by confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM). Beyond the resolution limit of confocal microscopy, both AFM and NSOM measurements of mica-supported lipid monolayers reveal small domains on the submicron scale. In the NSOM studies, simultaneous high-resolution fluorescence and topography measurements of these structures confirm that they arise from coexisting liquid condensed (LC) and liquid expanded (LE) lipid phases, and not defects in the monolayer. AFM studies of bilayers formed by a combination of LB dipping and Langmuir-Schaefer monolayer transfer exhibit complex surface topographies that reflect a convolution of the phase structure present in each of the individual monolayers. NSOM fluorescence measurements, however, are able to resolve the underlying lipid domains from each side of the bilayer and show that they are qualitatively similar to those observed in the monolayers. The observation of the small lipid domains in these bilayers is beyond the spatial resolving power of confocal microscopy and is complicated in the topography measurements taken with AFM, illustrating the utility of NSOM for these types of studies. The data suggest that the small LC and LE lipid domains are formed after lipid transfer to the substrate through a dewetting mechanism. The possible extension of these measurements to probing for lipid phase domains in natural biomembranes is discussed.     

Homogeneous genotyping assays for single nucleotide polymorphisms with fluorescence resonance energy transfer detection

A homogeneous detection mechanism based on fluorescence resonance energy transfer (FRET) has been developed for two DNA diagnostic tests. In the template-directed dye-terminator incorporation (TDI) assay, a donor dye-labeled primer is extended by DNA polymerase using allele-specific, acceptor dye-labeled ddNTPs. In the dye-labeled oligonucleotide ligation (DOL) assay, a donor dye-labeled common probe is joined to an allele-specific, acceptor dye-labeled probe by DNA ligase. Once the donor and acceptor dyes become part of a new molecule, intramolecular FRET is observed over background intermolecular FRET. The rise in FRET, therefore, can be used as an index for allele-specific ddNTP incorporation or probe ligation. Real time monitoring of FRET greatly increases the sensitivity and reliability of these assays. Change in FRET can also be measured by end-point reading when appropriate controls are included in the experiment. FRET detection proves to be a robust method in homogeneous DNA diagnostic assays.     

CTAB enhancement of FRET in DNA structures

The effect of cetyl‐trimethylammonium bromide (CTAB) on enhancing the fluorescence resonance energy transfer (FRET) between two dye‐conjugated DNA strands was studied using fluorescence emission spectroscopy and dynamic light scattering (DLS). For hybridized DNA where one strand is conjugated with a TAMRA donor and the other with a TexasRed acceptor, increasing the concentration of CTAB changes the fluorescence emission properties and improves the FRET transfer efficiency through changes in the polarity of the solvent, neutralization of the DNA backbone and micelle formation. For the DNA FRET system without CTAB, the DNA hybridization leads to contact quenching between TAMRA donor and TexasRed acceptor producing reduced donor emission and only a small increase in acceptor emission. At 50 µM CTAB, however, the sheathing and neutralization of the dye‐conjugated dsDNA structure significantly reduces quenching by DNA bases and dye interactions, producing a large increase in FRET efficiency, which is almost four fold higher than without CTAB.

     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

High-contrast imaging of fluorescent protein FRET by fluorescence polarization microscopy

Detection of Forster resonance energy transfer (FRET) between fluorescent protein labeled targets is a valuable strategy for measurement of protein-protein interactions and other intracellular processes. Despite the utility of FRET, widespread application of this technique to biological problems and high-throughput screening has been limited by low-contrast measurement strategies that rely on the detection of sensitized emission or photodestruction of the sample. Here we report a FRET detection strategy based on detecting depolarized sensitized emission. In the absence of FRET, we show that fluorescence emission from a donor fluorescent protein is highly polarized. Depolarization of fluorescence emission is observed only in the presence of energy transfer. A simple detection strategy was adapted for fluorescence microscopy using both laser scanning and wide-field approaches. This approach is able to distinguish FRET between linked and unlinked Cerulean and Venus fluorescent proteins in living cells with a larger dynamic range than other approaches.     

Competitive binding assay using fluorescence resonance energy transfer for the identification of calmodulin antagonists

The ubiquitous calcium regulating protein calmodulin (CaM) has been utilized as a model drug target in the design of a competitive binding fluorescence resonance energy transfer assay for pharmacological screening. The protein was labeled by covalently attaching the thiol-reactive fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) to an engineered C-terminal cysteine residue. Binding of the environmentally sensitive hydrophobic probe 2,6-anilinonaphthalene sulfonate (2,6-ANS) to CaM could be monitored by an increase in the fluorescence emission intensity of the 2,6-ANS. Evidence of fluorescence resonance energy transfer (FRET) from 2,6-ANS (acting as a donor) to MDCC (the acceptor in this system) was also observed; fluorescence emission representative of MDCC could be seen after samples were excited at a wavelength specific for 2,6-ANS. The FRET signal was monitored as a function of the concentration of calmodulin antagonists in solution. Calibration curves for both a selection of small molecules and a series of peptides based upon known CaM-binding domains were obtained using this system. The assay demonstrated dose-dependent antagonism by analytes known to hinder the biological activity of CaM. These data indicate that the presence of molecules known to bind CaM interfere with the ability of FRET to occur, thus leading to a concentration-dependent decrease of the ratio of acceptor:donor fluorescence emission. This assay can serve as a general model for the development of other protein binding assays intended to screen for molecules with preferred binding activity.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.     

Two-photon excitation fluorescence resonance energy transfer with small organic molecule as energy donor for bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.     

Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies.     

Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions

Fluorescence resonance energy transfer (FRET) in association with a time-resolved fluorescence mode of detection was used to design a new homogeneous technology suitable to monitor biomolecular interactions. A lanthanide cryptate characterised by a long lived fluorescence emission was used as donor and a cross-linked allophycocyanine was used as acceptor. This new donor/acceptor pair displayed an exceptionally large Forster radius of 9 nm. This allowed to build up a set of labelling strategies to probe the interactions between biomolecules with an emphasis on fully indirect cassette formats particularly suitable for high throughput screening applications. Herein we describe the basics of the technology, review the latest applications to the study of molecular interactions involved in cells and new oligonucleotides based assays.     

荧光共振能量转移效率的实时定量测量

荧光共振能量转移（FRET）广泛用于研究分子间的距离及其相互作用,与荧光显微镜结合,可定量获取有关生物活体内蛋白质、脂类、DNA和RNA的时空信息。随着绿色荧光蛋白（GFP）的发展,FRET荧光显微镜有可能实时测量活体细胞内分子的动态性质。提出了一种定量测量FRET效率以及供体与受体间距离的简单方法,仅需使用一组滤光片和测量一个比值,利用供体和受体的发射谱肖除光谱间的串扰。该方法简单快速,可实时定量测量FRET的效率和供体与受体间的距离,尤其适用于基于GFP的供体－受体对。     

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies – one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength – have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ²). An increase in range for κ² with increasing FRET efficiency has been observed, with average κ² values different from the dynamic random average of 2/3. These observations call for the need of κ² determination in proximity measurements, where the donor and acceptor orientations are not predictable.An increasing range of κ² with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a “fluidity gradient” exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy.Two new quantities – the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the “dissymmetry index” of the polarized FRET efficiency components – have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.