Förster Resonance Energy Transfer System Research Articles

Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT).

Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.

A ratiometric fluorescent sensor based on g-CNQDs@Zn-MOF for the sensitive detection of riboflavin via FRET.

A novel ratiometric fluorescent sensor based on Förster resonance energy transfer (FRET) platform was designed for riboflavin (RF) detection. The graphitic carbon nitrides quantum dots - Zn-MOF composite (g-CNQDs@Zn-MOF) was used as the fluorescent probe. In the FRET system, g-CNQDs@Zn-MOF and RF acted as donor and acceptor, respectively. The probe exhibited high sensitivity and good selectivity to RF, and had been successfully used for the detection of RF in milk and vitamin B2 tablets. The detection limit of the sensor was 15nM. The strategy expanded the application of MOF in sensing filed and provided a new method for the detection of RF.

Construction of a FRET System in a Double-Stranded DNA Using Fluorescent Thymidine and Cytidine Analogs.

Förster resonance energy transfer (FRET) has been used as a molecular ruler to monitor the distance between or within biomolecules. A distance- and orientation-dependent FRET system can be a more powerful tool to investigate biomolecules because of its higher spatial sensitivity. For this purpose, distance- and orientation-dependent FRET pairs have been developed using a DNA scaffold; however, none of them use an isomorphic thymidine analog. Herein, we report the construction of a FRET pair using a fluorescent thymidine analog, dioxT, as the donor and tC as the acceptor. The experimental FRET efficiency was calculated based on steady-state fluorescence spectra and time-resolved fluorescence (lifetime). The calculated values showed a periodical change, which suggests the sensitivity of FRET efficiency to the orientation of fluorophores. For further evaluation of our FRET pair, we compared the theoretical FRET efficiency with the experimental value and observed a relatively high consistency. This is the first color variable FRET system that used a fluorescent thymidine analog, which is expected to expand the applicability of nucleobase-analog FRET pairs.

Förster Resonance Energy Transfer: An Efficient Way to Develop Stimulus-Responsive Room-Temperature Phosphorescence Materials and Their Applications

Summary In this work, Forster resonance energy transfer (FRET) is utilized to construct a stimulus-responsive room-temperature phosphorescence (RTP) system. Owing to the specificity of the FRET system and its sensitivity to distance, the resultant material shows turn-on persistent RTP emission upon force or heating stimulus. Furthermore, a near-perfect compatibility with existing printing technology is demonstrated for this material, especially for thermal printing. This finding could provide a simple, efficient, and truly commercially viable construction strategy for RTP material.

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Ionic Liquid-Based Dye for Highly Sensitive Optical Sensing Based on FRET and Ion Extraction System Using Plasticized PVC Membrane

Efficient Cascade Resonance Energy Transfer in Dynamic Nanoassembly for Intensive and Long-Lasting Multicolor Chemiluminescence.

Light emission induced by chemical reactions, known as chemiluminescence (CL), has been widely used for bioassays, biosensors, imaging, and illumination applications. Most known CL systems exhibit flash-type single-color light emissions, which limit their applications. Long-lasting multicolor CL in aqueous solutions is highly desirable, especially for biological applications, but remains a challenge. Herein, we report a simple strategy of achieving highly efficient cascade Förster resonance energy transfer (FRET) in the dynamic nanoassembly of β-cyclodextrin (β-CD), CL reagents, and fluorophores in aqueous solution, which emits intensive multicolor CL with adjustable wavelength within 410-610 nm. β-CD can bind CL reagents and fluorophores to form a dynamic nanoassembly. These nanoassemblies can bring the included luminescent intermediate and fluorophores into close proximity and proper alignment, which should greatly enhance the FRET efficiency between luminescent intermediate and fluorophores. Indeed, the cascade FRET efficiency in this supramolecular nanoassembly reaches up to 92%, which is comparable with the cascade FRET systems based on covalently linked donors and acceptors. By using hydroxypropyl methylcellulose as the thickener to slow the diffusion (to elongate the CL emission), and using Ca(OH)2 solid (a low solubility strong base) as buffer to maintain the pH in the optimal range for the CL reaction, this nanoassembly system has been further developed to achieve slow-diffusion-controlled catalytic CL reactions, which enables long-lasting multicolor CL in aqueous solution that is visible to naked eyes and lasts for more than 20 h. The multicolor CL systems can be used to prepare transformable two-dimensional multicolor codes for encryption application.

Cavity-enhanced energy transfer between nano-emitters and monolayer graphene

Photonic cavities have been shown to efficiently control and enhance the Förster resonance energy transfer (FRET) between point-like donors and acceptors. In this work, we experimentally implemented photonic cavity control of the energy transfer between point-like donors and two-dimensional acceptors, using an integrated system combining an Al/SiO2 cavity with nano-emitters and graphene. We demonstrate that the Al/SiO2 cavity can control energy transfer between nanodiamond nitrogen-vacancy (NV) centers and monolayer graphene by tuning the thickness of the SiO2 layer, providing a two-fold enhancement of both the FRET efficiency and rate. Our results of point-to-layer FRET systems pave the way for a graphene-integrated dielectric cavity platform for application in enhanced light–matter interaction and photodetection devices.

Poly-tryptophan/carbazole based FRET-system for sensitive detection of nitroaromatic explosives

Sensitive detection of nitroaromatic explosives, a hot topic of worldwide concern, is greatly asked for, due to the adverse effect on ecosystems and human health originating from the excessive use and inadequate management. Here, we report the development of a Förster Resonance Energy Transfer (FRET) based sensor system for highly sensitive detection of nitro-aromatic compounds (NACs). The sensor system is composed of a copolymer poly[(N,N-dimethylacrylamide)-co-(Boc-Trp-EMA)] (RP) bearing tryptophan derivative in the side chain as donor and carbazole (CZ) as an acceptor. Primarily, the fluorescence of RP copolymer is quenched by non-radiative energy transfer to CZ which only happens once the two molecules are within Förster critical distance. The efficiency and rate of energy transfer process were calculated for this deliberated FRET system. This FRET pair exhibited a specific fluorescence response to NACs such as 2,4,6-trinitrophenol (TNP), 2,4-dinitrotoluene (DNT) and 2,4,6-trinitrotoluene (TNT) with 6.4, 5.9 and 1.4 μM limit of detections (LODs), respectively. Interestingly, the order of sensitivities of the FRET pair to NACs was very high. The sensing process occurs through a static and dynamic quenching pathway, confirmed by Stern−Volmer analysis of steady-state and time-resolved fluorescence as well as UV–Vis Spectroscopy.

Exploring dynamics of resonance energy transfer in hybrid Quantum Dot Sensitized Solar Cells (QDSSC)

Graphene based nanomaterials are known to provide new avenues to improve semiconductor based light harvesting devices. This work makes use of graphene quantum dots (GQD) to improve the efficiency of a CdSe Quantum Dot Sensitized Solar Cell (QDSSC) by Förster Resonance Energy Transfer (FRET) mechanism. FRET describes non-radiative energy transfer between two adjacent molecules typically in range from 1 to 10 nm with one molecule as donor and other molecule as acceptor. If the acceptor is in close proximity of the excited donor, then their dipoles align resulting in transfer of excitation energy from donor to acceptor. Here graphene quantum dot acts as the energy donor to enhance light harvesting of CdSe quantum dot which acts as an acceptor in the hybrid solar cell. The introduction of GQD increases the efficiency of CdSe sensitized QDSSC from 0.18 to 0.28% showing an efficiency enhancement of 55%. The improved efficiency is mainly attributed to the 46% increase in current density of the GQD-CdSe solar cell compared to the CdSe QDSSC. The increased performance of the QDSSC owes to the existence of non-radiative energy transfer (FRET) between GQD and CdSe evident from photoluminescence (PL) quenching and lifetime measurements. This FRET system of GQD (donor)-CdSe (acceptor) shows an energy transfer of 48.7% providing new insights for selective light harvesting of the solar spectrum which can be utilised for various potential applications in future.

Genetically Encoded Fluorescent Amino Acid for Monitoring Protein Interactions through FRET.

Förster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer β-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of β-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 μM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

Abstract The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.

NIR-Emitting Alloyed CdTeSe QDs and Organic Dye Assemblies: A Nontoxic, Stable, and Efficient FRET System.

In the present work, we synthesize Near Infrared (NIR)-emitting alloyed mercaptopropionic acid (MPA)-capped CdTeSe quantum dots (QDs) in a single-step one-hour process, without the use of an inert atmosphere or any pyrophoric ligands. The quantum dots are water soluble, non-toxic, and highly photostable and have high quantum yields (QYs) up to 84%. The alloyed MPA-capped CdTeSe QDs exhibit a red-shifted emission, whose color can be tuned between visible and NIR regions (608–750 nm) by controlling the Te:Se molar ratio in the precursor mixtures and/or changing the time reaction. The MPA-capped QDs were characterized by UV-visible absorption spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), and zeta potential measurements. Photostability studies were performed by irradiating the QDs with a high-power xenon lamp. The ternary MPA-CdTeSe QDs showed greater photostability than the corresponding binary MPA-CdTe QDs. We report the Förster resonance energy transfer (FRET) from the MPA-capped CdTeSe QDs as energy donors and Cyanine5 NHS-ester (Cy5) dye as an energy acceptor with efficiency (E) up to 95%. The distance between the QDs and dye (r), the Förster distance (R0), and the binding constant (K) are reported. Additionally, cytocompatibility and cell internalization experiments conducted on human cancer cells (HeLa) cells revealed that alloyed MPA-capped CdTeSe QDs are more cytocompatible than MPA-capped CdTe QDs and are capable of ordering homogeneously all over the cytoplasm, which allows their use as potential safe, green donors for biological FRET applications.

Quantitative recognition and ratiometric cell imaging of HSO3− inspired of confined-space based FRET system within human serum albumin

A ratiometric fluorescent probe for HSO3− was assembled using a pair of Förster resonance energy transfer (FRET)-matchable fluorescent dyes within a single human serum albumin (HSA). In this work, the micro-environment-sensitive dyes B1 and HE were designed and synthesized, wherein B1 located in site II as the FRET donor while HE located in site I as the FRET acceptor and receptor for HSO3−. This B1−HE FRET system exhibited a good efficiency in the sensitive, selective, and ratiometric recognition of HSO3− with a detection limit of 99 nM in aqueous media. Furthermore, this B1−HE system could realize the ratiometric imaging of HSO3− in human breast cancer cells.

Fluorescence method for quickly detecting ochratoxin A in flour and beer using nitrogen doped carbon dots and silver nanoparticles

In this paper, a FRET (Forster resonance energy transfer) based fluorescence method was developed for the quickly detection of ochratoxin A (OTA) in agricultural products (e.g., flour and beer). A highly fluorescent nitrogen doped carbon dots (CD) were served as energy donor, the DNA and MCH (6-mercapto-1-hexanol) modified Ag nanoparticles were served as energy acceptor in the FRET system. OTA can be detected in a concentration range between 10 and 5000 nM, the limit of detection is 8.7 nM. This method has three advantages: (1) an enhanced fluorescent intensity can be acquired by utilizing the nitrogen doped CD synthesized by one-step approach without sophisticated modification of nanoparticles; (2) OTA detection was accomplished quickly (less than 30 min) by using MCH as assistant molecule; (3) an extended OTA detection linear range was acquired, which may facilitate the OTA detection in real agricultural samples, and is helpful for solving food safety problems.

Development of a genetically encodable FRET system using fluorescent RNA aptamers

Fluorescent RNA aptamers are useful as markers for tracking RNA molecules inside cells and for creating biosensor devices. Förster resonance energy transfer (FRET) based on fluorescent proteins has been used to detect conformational changes, however, such FRET devices have not yet been produced using fluorescent RNA aptamers. Here we develop an RNA aptamer-based FRET (apta-FRET) system using single-stranded RNA origami scaffolds. To obtain FRET, the fluorescent aptamers Spinach and Mango are placed in close proximity on the RNA scaffolds and a new fluorophore is synthesized to increase spectral overlap. RNA devices that respond to conformational changes are developed, and finally, apta-FRET constructs are expressed in E. coli where FRET is observed, demonstrating that the apta-FRET system is genetically encodable and that the RNA nanostructures fold correctly in bacteria. We anticipate that the RNA apta-FRET system could have applications as ratiometric sensors for real-time studies in cell and synthetic biology.

Probing the Dynamic Nature of Self‐Assembling Cyclic Peptide–Polymer Nanotubes in Solution and in Mammalian Cells

AbstractSelf‐assembling cyclic peptide–polymer nanotubes have emerged as a fascinating supramolecular system, well suited for a diverse range of biomedical applications. Due to their well‐defined diameter, tunable peptide anatomy, and ability to disassemble in situ, they have been investigated as promising materials for numerous applications including biosensors, antimicrobials, and drug delivery. Despite this continuous effort, the underlying mechanisms of assembly and disassembly are still not fully understood. In particular, the exchange of units between individual assembled nanotubes has been overlooked so far, despite its knowledge being essential for understanding their behavior in different environments. To investigate the dynamic nature of these systems, cyclic peptide–polymer nanotubes are synthesized, conjugated with complementary dyes, which undergo a Förster resonance energy transfer (FRET) in close proximity. Model conjugates enable to demonstrate not only that their self‐assembly is highly dynamic and not kinetically trapped, but also that the self‐assembly of the conjugates is strongly influenced by both solvent and concentration. Additionally, the versatility of the FRET system allows studying the dynamic exchange of these systems in mammalian cells in vitro using confocal microscopy, demonstrating the exchange of subunits between assembled nanotubes in the highly complex environment of a cell.

Confined-Space Mechanism Inspired by the Ingenious Fabrication of a Förster Resonance Energy Transfer System as a Ratiometric Probe for Ag+ Recognition

In this work, a facile and interesting fabrication of the Forster resonance energy transfer (FRET) system is presented for the first time by introducing an independent donor and acceptor into a confined space, which is different from the typical “donor–linker–acceptor” FRET system. Human serum albumin (HSA), as a proof-of-concept, is selected to load a pair of spectra-matchable but independent fluorescent dyes (ES1 and R1) in its different binding sites. Furthermore, this space-confined FRET system could act as a ratiometric sensor for the quantitative recognition of Ag+ in the water samples with good selectivity and high sensitivity.

Development of a Vivid FRET System Based on a Highly Emissive dG-dC Analogue Pair.

A new type of Förster Resonance Energy Transfer (FRET) system using highly emissive isomorphic nucleobase analogues is reported. The FRET pair consists of 2-aminothieno[3,4-d]pyrimidine G-mimic deoxyribonucleoside (th dG) as an energy donor and 1,3-diaza-2-oxophenothiazine (tC) as an energy acceptor. The distance and orientation between donor and acceptor was controlled by systematic incorporation of th dG and tC into DNA sequences to investigate the FRET efficiencies. This is the first Watson-Crick base-pairable FRET pair to produce vivid colors. In addition, this nucleic acid-based FRET pair was used to monitor DNA conformation and achieved visualization of the B-Z transition.

Orientation-dependent FRET system reveals differences in structures and flexibilities of nicked and gapped DNA duplexes.

Differences in structures and flexibilities of DNA duplexes play important roles on recognition by DNA-binding proteins. We herein describe a novel method for structural analyses of DNA duplexes by using orientation dependence of Förster resonance energy transfer (FRET). We first analyzed canonical B-form duplex and correct structural parameters were obtained. The experimental FRET efficiencies were in excellent agreement with values theoretically calculated by using determined parameters. We then investigated DNA duplexes with nick and gaps, which are key intermediates in DNA repair systems. Effects of gap size on structures and flexibilities were successfully revealed. Since our method is facile and sensitive, it could be widely used to analyze DNA structures containing damages and non-natural molecules.

Multifunctional Polymer Ligand Interface CdZnSeS/ZnS Quantum Dot/Cy3-Labeled Protein Pairs as Sensitive FRET Sensors.

High-quality CdZnSeS/ZnS alloyed core/thick-shell quantum dots (QDs) as energy donors were first exploited in Förster resonance energy transfer (FRET) applications. A highly efficient ligand-exchange method was used to prepare low toxicity, high quantum yield, stabile, and biocompatible CdZnSeS/ZnS QDs densely capped with multifunctional polymer ligands containing dihydrolipoic acid (DHLA). The resulting QDs can be applied to construct QDs-based Förster resonance energy transfer (FRET) systems by their high affinity interaction with dye cyanine 3 (Cy3)-labeled human serum albumin (HSA). This QD-based FRET protein complex can serve as a sensitive sensor for probing the interaction of clofazimine with proteins using fluorescence spectroscopic techniques. The ability of FRET imaging both in vitro and in vivo not only reveals that the current FRET system can remain intact for 2 h but also confirms the potential of the FRET system to act as a nanocarrier for intracellular protein delivery or to serve as an imaging probe for cancer diagnosis.