Origin of simultaneous donor-acceptor emission in single molecules of peryleneimide-terrylenediimide labeled polyphenylene dendrimers

F?rster type resonance energy transfer (FRET) in donor-acceptor peryleneimide-terrylenediimide dendrimers has been examined at the single molecule level. Very efficient energy transfer between the donor and the acceptor prevent the detection of donor emission before photobleaching of the acceptor. Indeed, in solution, on exciting the donor, only acceptor emission is detected. However, at the single molecule level, an important fraction of the investigated individual molecules (about 10-15%) show simultaneous emission from both donor and acceptor chromophores. The effect becomes apparent mostly after photobleaching of the majority of donors. Single molecule photon flux correlation measurements in combination with computer simulations and a variety of excitation conditions were used to determine the contribution of an exciton blockade to this two-color emission. Two-color defocused wide-field imaging showed that the two-color emission goes hand in hand with an unfavorable orientation between one of the donors and the acceptor chromophore.     

Ultrafast fluorescence resonance energy transfer in a reverse micelle: excitation wavelength dependence

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to fluorescein 548 (F548) in a sodium dioctyl sulfosuccinate (AOT) reverse micelle is studied by picosecond and femtosecond emission spectroscopy. In bulk water, at the low concentration of the donor (C480) and the acceptor (F548), no FRET is observed. However, when the donor (C480) and the acceptor (F548) are confined in a AOT reverse micelle very fast FRET is observed. The time constants of FRET were obtained from the rise time of the emission of the acceptor (F548). In a AOT microemulsion, FRET is found to occur in multiple time scales--3, 200, and 2700 ps. The 3 ps component is assigned to FRET in the water pool of the reverse micelle with a donor-acceptor distance, 16 A. The 200 ps component corresponds to a donor-acceptor distance of 30 A and is ascribed to the negatively charged acceptor inside the water pool and the neutral donor inside the alkyl chains of AOT. The very long 2700 ps component may arise due to FRET from a donor outside the micelle to an acceptor inside the water pool and also from diffusion of the donor from bulk heptane to the reverse micelle. With increase in the excitation wavelength from 375 to 405 nm the relative contribution of the FRET due to C480 in the AOT reverse micelle (the 3 and 200 ps components) increases.     

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.     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

G‐Tetraplex‐Induced FRET within Telomeric Repeat Sequences Using PyA‐PerA as Energy Donor–Acceptor Pair

G‐tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside‐tethered FRET pair embedded in the human telomeric G‐quadruplex forming sequence (5′‐A GGG TT ^Py A GGG TT ^Per A GGG TTA GGG‐3′, Py=pyrene, Per=perylene). Conformational change from a single strand to an anti‐parallel G‐quadruplex leads to FRET from energy donor ( ^Py A ) to acceptor ( ^Per A ). The distance between the FRET donor/acceptor partners was controlled by changing the number of G‐quartet spacer units. The FRET efficiency decreases with increase in G‐quartet units. Overall findings indicate that this could be further used for the development of FRET‐based sensing and measurement techniques.     

A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER

Abstract: A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency (ω) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor-acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5- bis (5- tert -butyl-2-benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr₄ was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady-state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and ω were as small as 1.1 × 10⁻⁵ M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.     

A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER

Abstract –A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency ( Ω ) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor-acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5-W.s(5- tert -butyl-2-benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr₄ was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady-state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and Ω were as small as 1.1 times 10^-5 M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.     

A New Robust and Highly Sensitive FRET Donor–Acceptor Pair: Synthesis,Characterization, and Application in a Thrombin Assay

The synthesis of a new, robust fluorescence‐resonance‐energy‐transfer (FRET) system is described. Its donor chromophore is derived from an N‐allyl‐substituted quinolinone attached to 4‐bromophenylalanine via Heck cross‐coupling. The resulting Fmoc‐protected derivative 11 was used as building block in solid‐phase peptide synthesis (SPPS). As FRET acceptor, a sulfonylated ruthenium(II)–bathophenanthroline complex with a peripheral COOH function was prepared for covalent attachment to target molecules. The UV/VIS absorption and emission spectra of peptides bearing only the donor (D) or acceptor (A) dye showed a good overlap of the emission band of the donor with the absorption band of the acceptor. The fluorescence spectra of a peptide bearing both dyes revealed an additional emission after excitation of the donor, which is due to indirect excitation of the acceptor via FRET. The long fluorescence lifetime of the Ru^II complex (0.53 μs) makes it well‐suited for time‐resolved measurements. As a first application of this new FRET system, the peptide 18 , with the recognition sequence for the protease thrombin, flanked by the two dyes, was synthesized and successfully cleaved by the enzyme. The change in the ratio of the fluorescence intensities could be determined.     

A Fluorescent Indicator for Imaging Lysosomal Zinc(II) with Förster Resonance Energy Transfer (FRET)‐Enhanced Photostability and a Narrow Band of Emission

We demonstrate a strategy to transfer the zinc(II) sensitivity of a fluoroionophore with low photostability and a broad emission band to a bright and photostable fluorophore with a narrow emission band. The two fluorophores are covalently connected to afford an intramolecular Förster resonance energy transfer (FRET) conjugate. The FRET donor in the conjugate is a zinc(II)‐sensitive arylvinylbipyridyl fluoroionophore, the absorption and emission of which undergo bathochromic shifts upon zinc(II) coordination. When the FRET donor is excited, efficient intramolecular energy transfer occurs to result in the emission of the acceptor boron dipyrromethene (4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene or BODIPY) as a function of zinc(II) concentration. The broad emission band of the donor/zinc(II) complex is transformed into the strong, narrow emission band of the BODIPY acceptor in the FRET conjugates, which can be captured within the narrow emission window that is preferred for multicolor imaging experiments. In addition to competing with other nonradiative decay processes of the FRET donor, the rapid intramolecular FRET of the excited FRET‐conjugate molecule protects the donor fluorophore from photobleaching, thus enhancing the photostability of the indicator. FRET conjugates 3 and 4 contain aliphatic amino groups, which selectively target lysosomes in mammalian cells. This subcellular localization preference was verified by using confocal fluorescence microscopy, which also shows the zinc(II)‐enhanced emission of 3 and 4 in lysosomes. It was further shown using two‐color structured illumination microscopy (SIM), which is capable of extending the lateral resolution over the Abbe diffraction limit by a factor of two, that the morpholino‐functionalized compound 4 localizes in the interior of lysosomes, rather than anchoring on the lysosomal membranes, of live HeLa cells.     

Structural Control of Cell Permeability with Highly Emissive Europium(III) Complexes Permits Different Microscopy Applications

Four anionic europium complexes are described based on triazacyclononane tris‐carboxylate or phosphinate ligands. In each case, the three sensitising chromophores comprise a substituted aryl–alkynyl pyridine group, with complex brightness in water falling in the range 4 to 23 mM ^?1 cm^?1. para‐Substitution of the aryl ring with carboxymethyl groups gives complexes that are taken into cells, stain the lysosomes selectively and unexpectedly permit lifetime measurements of lysosomal pH. In contrast, the introduction of sulfonate groups inhibits cell uptake enabling the Eu complex to be used as an extracellular donor for FRET applications at the membrane surface. Using time‐gated FRET microscopy, the cell membrane structure was highlighted, in which Cell Mask Deep Red was used as a membrane‐ localized FRET acceptor.     

Exceptional Blueshifted and Enhanced Aggregation‐Induced Emission of Conjugated Asymmetric Triazines and Their Applications in Superamplified Detection of Explosives

A novel conjugated asymmetric donor–acceptor (CADA) strategy for preventing the redshift in photoluminescence, as well as preserving the merits of donor–acceptor architectures, was proposed and demonstrated for two triazine derivatives, which showed highly efficient, narrow, and blueshifted ultraviolet light emission in solid films along with special aggregation‐induced emission behavior. A mechanism of aggregation‐induced locally excited‐state emission by suppressing the twisted intramolecular charge‐transfer emission for the spectacular optoelectronic phenomena of these CADA molecules was suggested on the basis of both experimental measurements and theoretical calculations. By taking advantage of this special CADA architecture, fluorescent probes based on aggregates of conjugated asymmetric triazines in THF/water for the detection of explosives show superamplified detection of picric acid with high quenching constants (>1.0×10⁷ M ^?1) and a low detection limit of 15 ppb.     

From Dark to Light to Fluorescence Resonance Energy Transfer (FRET): Polarity‐Sensitive Aggregation‐Induced Emission (AIE)‐Active Tetraphenylethene‐Fused BODIPY Dyes with a Very Large Pseudo‐Stokes Shift

The work presented herein is devoted to the fabrication of large Stokes shift dyes in both organic and aqueous media by combining dark resonance energy transfer (DRET) and fluorescence resonance energy transfer (FRET) in one donor–acceptor system. In this respect, a series of donor–acceptor architectures of 4,4‐difluoro‐4‐bora‐3a,4a‐diaza‐s‐indacene (BODIPY) dyes substituted by one, two, or three tetraphenylethene (TPE) luminogens were designed and synthesised. The photophysical properties of these three chromophore systems were studied to provide insight into the nature of donor–acceptor interactions in both THF and aqueous media. Because the generation of emissive TPE donor(s) is strongly polarity dependent, due to its aggregation‐induced emission (AIE) feature, one might expect the formation of appreciable fluorescence emission intensity with a very large pseudo‐Stokes shift in aqueous media when considering FRET process. Interestingly, similar results were also recorded in THF for the chromophore systems, although the TPE fragment(s) of the dyes are non‐emissive. The explanation for this photophysical behaviour lies in the DRET. This is the first report on combining two energy‐transfer processes, namely, FRET and DRET, in one polarity‐sensitive donor–acceptor pair system. The accuracy of the dark‐emissive donor property of the TPE luminogen is also presented for the first time as a new feature for AIE phenomena.     

Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors

We used luminescent CdSe-ZnS core-shell quantum dots (QDs) as energy donors in fluorescent resonance energy transfer (FRET) assays. Engineered maltose binding protein (MBP) appended with an oligohistidine tail and labeled with an acceptor dye (Cy3) was immobilized on the nanocrystals via a noncovalent self-assembly scheme. This configuration allowed accurate control of the donor-acceptor separation distance to a range smaller than 100 A and provided a good model system to explore FRET phenomena in QD-protein-dye conjugates. This QD-MBP conjugate presents two advantages: (1) it permits one to tune the degree of spectral overlap between donor and acceptor and (2) provides a unique configuration where a single donor can interact with several acceptors simultaneously. The FRET signal was measured for these complexes as a function of both degree of spectral overlap and fraction of dye-labeled proteins in the QD conjugate. Data showed that substantial acceptor signals were measured upon conjugate formation, indicating efficient nonradiative exciton transfer between QD donors and dye-labeled protein acceptors. FRET efficiency can be controlled either by tuning the QD photoemission or by adjusting the number of dye-labeled proteins immobilized on the QD center. Results showed a clear dependence of the efficiency on the spectral overlap between the QD donor and dye acceptor. Apparent donor-acceptor distances were determined from efficiency measurements and corresponding F?rster distances, and these results agreed with QD bioconjugate dimensions extracted from structural data and core size variations among QD populations.     

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).     

Metal ion induced FRET On-Off in naphthyl-pyrenyl pendent tetrahomodioxacalix[4]arene

A novel tetrahomodioxacalix[4]arene (7) bearing both naphthyl- and pyrenyl-amide pendants was synthesized as FRET-based fluorometric sensor for Cu²⁺ ion. Intramolecular FRET from the naphthalene emission to the pyrene absorption affords Cu²⁺ ion selectivity over other metal ions. Upon addition of Cu²⁺ ion, the complex solution of 7 gave a significantly decreased pyrene acceptor emission along with an enhancement of naphthalene donor emission via FRET On-Off event.     

Nonradiative energy transfer from Tb3+ → Er3+ in calibo glass

Energy transfer studies have been made in a terbium-erbium coactivated calibo-glass system at room temperature and at liquid-air temperature. A study of the emission and decay of ⁵D₄ level of Tb³⁺ has been made by varying the acceptor (Er³⁺) concentration. Probabilities and efficiencies of energy transfer as well as donor-acceptor distances have been calculated. At low acceptor concentration the decay of the donor (Tb³⁺) emission has been found to be diffusion limited. At high acceptor concentration the mechanism governing the transfer is found to be dipole-dipole.     

Flexibility of the aldolase molecule measured using quenching-induced variations of the Forster distance for fluorescence energy transfer

The range of flexibility of the rabbit muscle aldolase molecule was studied using fluorescent labelled aldolase. The protein molecule was specifically labelled on the opposite sites of the enzyme subunit with the fluorescence energy donor and acceptor residues. Labelled aldolase with full enzymatic activity was used as a tool in the FRET studies between IAEDANS — donor or Cys-289 and IAF — acceptor on Cys-239. A range of Forster distance (R) was obtained by collisional quenching of the donor emission. The experiments of donor fluorescence quenching with wide range of acrylamide concentrations have shown the changes of donor-acceptor distances. In the absence of quencher the D-A distance distribution in characterized by an average value of 40.4 ?, and a half-width of 0.13 ?. A dramatic increase in half-width to 17.7? is observed after expositions of the enzyme to high acrylamide concentrations (0.13 M-0.68 M).     

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

Ultrafast fluorescence resonance energy transfer in a bile salt aggregate: Excitation wavelength dependence

Fluorescence resonance energy transfer (FRET) from Coumarin 153 (C153) to Rhodamine 6G (R6G) in a secondary aggregate of a bile salt (sodium deoxycholate, NaDC) is studied by femtosecond up-conversion. The emission spectrum of C153 in NaDC is analysed in terms of two spectra-one with emission maximum at 480 nm which corresponds to a non-polar and hydrophobic site and another with maximum at ∼530 nm which arises from a polar hydrophilic site. The time constants of FRET were obtained from the rise time of the emission of the acceptor (R6G). In the NaDC aggregate, FRET occurs in multiple time scales — 4 ps and 3700 ps. The 4 ps component is assigned to FRET from a donor (D) to an acceptor (A) held at a close distance (R _DA ∼ 17 ?) inside the bile salt aggregate. The 3700 ps component corresponds to a donor-acceptor distance ∼48 ?. The long (3700 ps) component may involve diffusion of the donor. With increase in the excitation wavelength (λ _ex) from 375 to 435 nm, the relative contribution of the ultrafast component of FRET (∼4 ps) increases from 3 to 40% with a concomitant decrease in the contribution of the ultraslow component (∼3700 ps) from 97 to 60%. The λ _ex dependence is attributed to the presence of donors at different locations. At a long λ _ex (435 nm) donors in the highly polar peripheral region are excited. A short λ _ex (375 nm) ‘selects’ donor at a hydrophobic location.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.