Acceptor Emission Research Articles

Simultaneous Determination of Two Subdomain Folding Rates Using the “Transfer-Quench” Method

The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring subdomain structures using a single mutant to facilitate resolution of the order and interdependence of such steps. Here, we report the development of the “Transfer-Quench” method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on Förster resonance energy transfer combined with fluorescence quenching. We placed the donor and acceptor at the loop ends, and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple-labeled mutant is monitored by the acceptor emission. The formation of nonlocal contact (loop closure) increases the time-dependent acceptor emission, while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the common model protein, the B domain of staphylococcal protein A. Only natural amino acids were used as probes, and thus possible structural perturbations were minimized. Tyr and Trp residues served as donor and acceptor at the ends of a long loop between helices I and II, and a Cys residue as a quencher for the acceptor. We found that the closure of the loop (segment 14–33) occurs with the same rate constant as the nucleation of helix HII (segment 33–29), in line with the nucleation-condensation model.

Improved Energy Pooling Efficiency through Inhibited Spontaneous Emission

The radiative lifetime of molecules or atoms can be increased by placing them within a tuned conductive cavity that inhibits spontaneous emission. This was examined as a possible means of enhancing three-body, singlet-based upconversion, known as energy pooling. Achieving efficient upconversion of light has potential applications in the fields of photovoltaics, biofuels, and medicine. The affect of the photonically constrained environment on pooling efficiency was quantified using a kinetic model populated with data from molecular quantum electrodynamics, perturbation theory, and ab initio calculations. This model was applied to a system with fluorescein donors and a hexabenzocoronene acceptor. Placing the molecules within a conducting cavity was found to increase the efficiency of energy pooling by increasing both the donor lifetime and the acceptor emission rate--i.e. a combination of inhibited spontaneous emission and the Purcell effect. A model system with a free-space pooling efficiency of 23% was found to have an efficiency of 47% in a rectangular cavity.

Tracking the Energy Flow on Nanoscale via Sample-Transmitted Excitation Photoluminescence Spectroscopy.

Tracking the energy flow in nanoscale materials is an important yet challenging goal. Experimental methods for probing the intermolecular energy transfer (ET) are often burdened by the spectral crosstalk between donor and acceptor species, which complicates unraveling their individual contributions. This issue is particularly prominent in inorganic nanoparticles and biological macromolecules featuring broad absorbing profiles. Here, we demonstrate a general spectroscopic strategy for measuring the ET efficiency between nanostructured or molecular dyes exhibiting a significant donor-acceptor spectral overlap. The reported approach is enabled through spectral shaping of the broadband excitation light with solutions of donor molecules, which inhibits the excitation of respective donor species in the sample. The resulting changes in the acceptor emission induced by the spectral modulation of the excitation beam are then used to determine the quantum efficiency and the rate of ET processes between arbitrary fluorophores (molecules, nanoparticles, polymers) with high accuracy. The feasibility of the reported method was demonstrated using a control donor-acceptor system utilizing a protein-bridged Cy3-Cy5 dye pair and subsequently applied for studying the energy flow in a CdSe560-CdSe600 binary nanocrystal film.

Evaluating Dye-Labeled DNA Dendrimers for Potential Applications in Molecular Biosensing.

DNA nanostructures provide a reliable and predictable scaffold for precisely positioning fluorescent dyes to form energy transfer cascades. Furthermore, these structures and their attendant dye networks can be dynamically manipulated by biochemical inputs, with the changes reflected in the spectral response. However, the complexity of DNA structures that have undergone such types of manipulation for direct biosensing applications is quite limited. Here, we investigate four different modification strategies to effect such dynamic manipulations using a DNA dendrimer scaffold as a testbed, and with applications to biosensing in mind. The dendrimer has a 2:1 branching ratio that organizes the dyes into a FRET-based antenna in which excitonic energy generated on multiple initial Cy3 dyes displayed at the periphery is then transferred inward through Cy3.5 and/or Cy5 relay dyes to a Cy5.5 final acceptor at the focus. Advantages of this design included good transfer efficiency, large spectral separation between the initial donor and final acceptor emissions for signal transduction, and an inherent tolerance to defects. Of the approaches to structural rearrangement, the first two mechanisms we consider employed either toehold-mediated strand displacement or strand replacement and their impact was mainly via direct transfer efficiency, while the other two were more global in their effect using either a belting mechanism or an 8-arm star nanostructure to compress the nanostructure and thereby modulate its spectral response through an enhancement in parallelism. The performance of these mechanisms, their ability to reset, and how they might be utilized in biosensing applications are discussed.

"DNA Origami Traffic Lights" with a Split Aptamer Sensor for a Bicolor Fluorescence Readout.

A split aptamer for adenosine triphosphate (ATP) was embedded as a recognition unit into two levers of a nanomechanical DNA origami construct by extension and modification of selected staple strands. An additional optical module in the stem of the split aptamer comprised two different cyanine-styryl dyes that underwent an energy transfer from green (donor) to red (acceptor) emission if two ATP molecules were bound as target molecule to the recognition module and thereby brought the dyes in close proximity. As a result, the ATP as a target triggered the DNA origami shape transition and yielded a fluorescence color change from green to red as readout. Conventional atomic force microscopy (AFM) images confirmed the topology change from the open form of the DNA origami in the absence of ATP into the closed form in the presence of the target molecule. The obtained closed/open ratios in the absence and presence of target molecules tracked well with the fluorescence color ratios and thereby validated the bicolor fluorescence readout. The correct positioning of the split aptamer as the functional unit farthest away from the fulcrum of the DNA origami was crucial for the aptasensing by fluorescence readout. The fluorescence color change allowed additionally to follow the topology change of the DNA origami aptasensor in real time in solution. The concepts of fluorescence energy transfer for bicolor readout in a split aptamer in solution, and AFM on surfaces, were successfully combined in a single DNA origami construct to obtain a bimodal readout. These results are important for future custom DNA devices for chemical-biological and bioanalytical purposes because they are not only working as simple aptamers but are also visible by AFM on the single-molecule level.

Resolution of the Time Sequence of Fast Folding Transition by the “Transfer-Quench” Method

The rates and efficiency of the folding transition of globular protein might be enhanced by early formation of few sub-domain structures. Ultrafast Forster resonance energy transfer (FRET) based methods are ideal for characterization of the transient ensembles of refolding molecules. However, each site specific labeling modification might affect rates of folding of near neighbor structural elements and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring sub-domain structures using a single mutant in order to facilitate resolution of the order and interdependence of such steps. Here we report the development of the “Transfer-Quench” method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on FRET combined with fluorescence quenching. We placed the donor and acceptor at the loop ends’ and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple labeled mutant is monitored at the acceptor emission. The formation of non-local contact (loop closure) increases the time dependent acceptor emission while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the B domain of staphylococcal protein A. Only natural amino acids were used as probes and thus possible structural perturbations were minimized (Tyr and Trp residues as donor and acceptor at the ends of a long loop between helices I and II, and Cys residue as a quencher for the acceptor). We found that the closure of the loop is formed with the same rate constant as the nucleation of helix II in line with the nucleation-condensation model.

The Long Road to Micro-Dynamic Presynaptic FRET Measurements

I will review the history and lead-up to our attempt to measure changes in FRET in assembled SNARE complexes in a very small space and a very short time, where only a tiny fraction of each of the labeled SNARE proteins is part of a SNARE complex. After recounting the saga of grant support and technical development, I will summarize our main findings: Using donor dequenching by receptor bleach as well as FLIM, we find a resting FRET indicating a number of assembled SNARE complexes exceeding the expected fraction of synaptobrevins (or VAMPs) that are in assembled SNAREs of docked and primed vesicles, likely reflecting the existence of still-assembled “orphan SNAREs” left over from prior bouts of secretion. We can detect the dispersion of all three SNARE proteins – VAMP, SNAP-25, and syntaxin – as well as the FRETting complex of assembled SNAREs itself, from the center of the presynaptic active zone to the periphery during and after a train of action potentials. Using sensitized acceptor emission on donor excitation, we also detect the disassembly of N-terminally labeled SNAREs prior to endocytosis of vesicle membrane and proteins, and the assembly of new SNAREs as replacement vesicles dock and prime, and we occasionally see a transient FRET increase while vesicles fuse during the train. By donor quenching, we also detect the rearrangement of C-terminally labeled SNAREs when vesicles fuse with the plasma membrane, and their subsequent dispersion, disassembly, and re-assembly. A variety of control experiments and statistical tests rule out most sources of artefact and alternative interpretations. I will also speculate on why SNARES disperse and are disassembled in the periphery, rather than in the central active zone where exocytosis occurs, as we had originally expected.

Efficient Förster Resonance Energy Transfer Donors of In(Zn)P/ZnS Quantum Dots

We demonstrate a detailed investigation of the effect of the type and concentration of zinc precursor on the optical properties of In(Zn)P/ZnS quantum dots. We achieved up to 87% quantum yield along with 54 nm emission bandwidth for the green emitters with changing the concentration of the Zn precursors. Employing efficient green emitters as the donor species, we demonstrated an efficient Förster resonance energy transfer (FRET) couple of green and red emitting InP-based quantum dots. With a FRET efficiency level of 70.3% achieved (analyzed from the donor lifetime with and without an acceptor), we further demonstrated the enhancement of the acceptor emission nearly twofold due to the energy transfer. Our results provide new insights toward the understanding of the excitonic interactions among donor and acceptor quantum dots of the III–V family for light harvesting applications.

Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors.

Quantum dots (QDs) and upconverting nanoparticles (UCNPs) are luminescent nanoparticles (NPs) commonly used in bioassays and biosensors as resonance energy transfer (RET) donors. The narrow and tunable emissions of both QDs and UCNPs make them versatile RET donors that can be paired with a wide range of acceptors. Ratiometric signal processing that compares donor and acceptor emission in RET-based transduction offers improved precision, as it accounts for fluctuations in the absolute photoluminescence (PL) intensities of the donor and acceptor that can result from experimental and instrumental variations. Immobilizing NPs on a solid support avoids problems such as those that can arise with their aggregation in solution, and allows for facile layer-by-layer assembly of the interfacial chemistry. Paper is an attractive solid support for the development of point-of-care diagnostic assays given its ubiquity, low-cost, and intrinsic fluid transport by capillary action. Integration of nanomaterials with paper-based analytical devices (PADs) provides avenues to augment the analytical performance of PADs, given the unique optoelectronic properties of nanomaterials. Herein, we describe methodology for the development of PADs using QDs and UCNPs as RET donors for optical transduction of nucleic acid hybridization. Immobilization of green-emitting QDs (gQDs) on imidazole functionalized cellulose paper is described for use as RET donors with Cy3 molecular dye as acceptors for the detection of SMN1 gene fragment. We also describe the covalent immobilization of blue-emitting UCNPs on aldehyde modified cellulose paper for use as RET donors with orange-emitting QDs (oQDs) as acceptors for the detection of HPRT1 gene fragment. The data described herein is acquired using an epifluorescence microscope, and can also be collected using technology such as a typical electronic camera.

Growth, structural and optical properties of orderly SnO2 microhemispheres on patterned sapphire substrates

One-dimensional nanoscaled materials, such as nanotubes, nanowires and nanobelts, have attracted a great deal of attention in recent years because of their unique electronic, optical, and mechanical properties. Their potential applications are found in next generation devices, functional materials, and sensors. A material of particular interest is stannic oxide (SnO2), which is a novel oxide semiconductor material for ultraviolet and blue luminescence devices due to its wide band gap of 3.6 eV at room temperature. SnO2 can also be widely used in many fields, such as gas sensors, optoelectronic devices, and transparent conductive glass, because of its high optical transparency in the visible range, low resistivity, and higher chemical and physical stability. In recent years, one-dimensional nanostructures of SnO2 materials, such as nanobelts, nanotubes, and nanowires, have been reported. However, the preparations of orderly SnO2 micro/nanostructures have been rarely reported. In this paper, orderly SnO2 microhemispheres with different sizes are grown on patterned sapphire substrates by a traditional chemical vapor deposition method without using any catalyst. The patterned sapphire substrates are cleaned by using a standard sapphire wafer cleaning procedure. High-purity metallic Sn powders (99.99%) and oxygen gas are used as Sn and oxygen sources, respectively. The flow rate of high-purity Ar carrier gas is controlled at 200 sccm, and the oxygen reactant gas with a flow rate of 100 sccm is introduced into the system. In the growth process, the whole system is kept at 1000℃ for 30 min. The surface morphologies, structural and optical properties of the SnO2 microhemispheres are investigated by the field emission scanning electron microscope (HITACHI S4800), the X-ray diffraction with a Cu Kup radiation (0.15418 nm), the optical absorption spectroscope (UV-3600 UV-VIS-NIR, Shimadzu), and the photoluminescence spectroscope with an excitation source of He-Cd laser (=325 nm) to identify the As related acceptor emission, respectively. These results show that the diameters of SnO2 microhemispheres become larger, and the crystal quality is degraded with the increase of Sn powder mass. The special selective growth of SnO2 microhemisphere on a patterned sapphire substrate is found. In addition, we also find that the optical band gaps of the samples A-D are all redshifted with the increase of Sn powder mass. The shrinkage of Eg in the absorption spectrum should be partly attributed to the degradation of crystal quality because of excess Sn sources. This growth method of SnO2 microhemisphere provides a feasible and effective way of preparing the high density, orderly arrangement of SnO2 micro/nanostructures.

Preparation and properties of Ag doped ZnO nanorods with N plasmon treatment

ABSTRACTIn this paper, Ag doped ZnO nanorods with N plasmon treatment were synthesized on Si substrate based on diffusion doping principle. At the same time, pure ZnO, Ag doped ZnO and ZnO nanorods with N plasmon treatment were also prepared. The experimental results showed that the surface of ZnO/Ag/N is the roughest, but there is no redundant Ag on the surface. The red shift of the absorption edge is the largest. The most noteworthy is that ZnO/Ag/N has a good acceptor emission behavior, and the calculated acceptor binding energy is about 128 meV.

Enabling Förster Resonance Energy Transfer from Large Nanocrystals through Energy Migration

The stringent distance dependence of Förster resonance energy transfer (FRET) has limited the ability of an energy donor to donate excitation energy to an acceptor over a Förster critical distance (R0) of 2-6 nm. This poses a fundamental size constraint (<8 nm or ∼4R0) for experimentation requiring particle-based energy donors. Here, we describe a spatial distribution function model and theoretically validate that the particle size constraint can be mitigated through coupling FRET with a resonant energy migration process. By combining excitation energy migration and surface trapping, we demonstrate experimentally an over 600-fold enhancement over acceptor emission for large nanocrystals (30 nm or ∼15R0) with surface-anchored molecular acceptors. Our work shows that the migration-coupled approach can dramatically improve sensitivity in FRET-limited measurement, with potential applications ranging from facile photochemical synthesis to biological sensing and imaging at the single-molecule level.

Fluorescence Resonance Energy Transfer of Fluorescent Molecules in Joint‐Linker Type Organic‐Inorganic Hybrid Gels

Fluorescence resonance energy transfer (FRET) sensitized emission of perylene by pyrene in a joint‐linker type gel is observed for the first time. The joint‐linker type organic‐inorganic hybrid gels containing pyrene and perylene synthesized from multi‐functional cyclic siloxane, 1,3,5,7‐tetramethylcyclotetrasiloxane (TMCTS), or cubic silsesquioxane, 1,3,5,7,9,11,13,15‐octakis(dimethylsilyloxy)pentacyclo‐[9,5,1,1,1,1]octasilsesquioxane (POSS), as joint molecules, and α,ω‐nonconjugated dienes, 1,5‐hexadiene (HD), or 1,9‐decadiene (DD), as linker molecules, are involved in a photo hydrosilylation reaction with a bis(acetylacetonato)platinum catalyst in a toluene solution of pyrene and perylene. FRET from pyrene (donor) to perylene (acceptor) in the gels is studied quantitatively. The FRET efficiencies (E) of gels containing 300 or 30 x 10–6 m of pyrene and perylene are about 0.6–0.8 or 0.5, respectively, independent of the monomer concentration. Sensitized acceptor emission is observed in the gels, especially in the gels containing 300 x 10–6 m of pyrene and perylene molecules. The ratio of the increase of the sensitized acceptor emission to the loss of donor fluorescence, the G factor, is determined to evaluate the efficiency of the sensitized emission of the acceptor in the gels. The G factors of the gels containing 300 or 30 x 10–6 m of pyrene and perylene molecules are 0.4–0.75 or 0.07‐0.1, repectively, and the gels with TMCTS or DD have a slightly higher G factor than gels with POSS or HD. image

FRET on Surface of Silica Nanoparticle: Effect of Chromophore Concentration on Dynamics and Efficiency

Forster resonance energy transfer (FRET) has been studied between fluorescein (donor, D) and rhodamine B (acceptor, A) bound covalently to the surface of silica nanoparticle (SNP-dye). This is a part of an ongoing effort toward development of light harvesting nanoantennae. The role of the D:A ratio and the total chromophore content on the efficiency of FRET has been investigated. At low number density (ca. 75 ± 15 dye molecules/particle) efficiency is dependent on the D:A ratio. It appears that the distribution of the dyes on the surface is inhomogeneous and that FRET occurs only between D and A molecules in very close proximity. The effect of self-quenching of the donor is not very significant for this number density. At higher number densities (ca. 700–1100 dye molecules/particle), a prominent rise in the acceptor emission is observed, perhaps indicating a more homogeneous distribution of the dye molecules, and D emission is quenched completely. However, D–D quenching is an issue here. So, it appears th...

Application of strain and calibration of Förster Resonance Energy Transfer (FRET) emission for in vitro live cell response to cytoskeletal deformation

Cellular mechanotransduction is an integral part of many crucial physiological processes, but non-invasive tools for quantifying intracellular strain in vivo are not available for complex tissues such as bone. As a first step to address this gap, we have utilized a novel, non-invasive approach to quantify cellular strain in vitro by employing a transfected alpha-actinin Förster Resonance Energy Transfer (FRET) sensor. Following validation experiments, mouse fibroblasts transfected to express FRET sensors were seeded to a silicone membrane and subjected to up to 10% tensile strain mounted on a multi-photon microscope. During tensile strain, fluorescent emission of acceptor (YFP) and donor (CFP) proteins was quantified. YFP/CFP ratio was normalized to the initial baseline (unstretched) ratio for each cell which demonstrates a negative linear correlation between the relative proximity ratio of emission spectra and cell strain, with a mean decrease of 1.017% normalized ratio for every percent strain experienced by the cell. The exciting implications of our findings are that the discovery of the stable correlation between loss of FRET and experimentally applied strain opens intriguing possibilities for future use of this technology with in vivo research, leading to discoveries improving disease treatments in mechanically sensitive tissues such as bone.

Wide-field microscopic FRET imaging using simultaneous spectral unmixing of excitation and emission spectra.

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has the inherent ability to resolve donor emission, fluorescence resonance energy transfer (FRET)-sensitized acceptor emission and directly excited acceptor emission. We here develop an ExEm unmixing-based quantitative FRET measurement method (EES-FRET) independent of excitation intensity and detector parameter setting. The ratio factor (rK), predetermined using a donor-acceptor tandem construct, of total acceptor absorption to total donor absorption in excitation wavelengths used is introduced for determining the concentration ratio of acceptor to donor. We implemented EES-FRET method on a wide-field microscope to image living cells expressing tandem FRET constructs with different donor-acceptor stoichiometry.

Optofluidic FRET Lasers and Their Applications in Novel Photonic Devices and Biochemical Sensing

Incorporating fluorescence resonance energy transfer (FRET) into a laser cavity can increase the sensitivity of FRET-based biochemical sensors due to the nonlinear dependence of the lasing output on the FRET parameters. Here, we carry out a comprehensive theoretical analysis of optofluidic FRET lasers based on a Fabry–Perot microcavity using a rate equation model. We compare conceptually distinct cases of donor and acceptor molecules diffusing freely in a bulk solution versus molecules connected by a fixed-length linker and show that the latter arrangement is especially well suited for sensing of low-concentration analytes. By comparing FRET lasing-based sensors with conventional FRET sensors, we show that for optimal pump fluence and FRET-pair concentration, FRET lasing can lead to more than 100-fold enhancement in detection sensitivities of conformational changes in the Forster radius range. We also show that for optimal experimental conditions, donor and acceptor emission intensities become over 20-fold more sensitive to FRET-pair concentration changes in the presence of FRET lasing. We study the dependence of the sensitivity enhancement on the cavity Q-factor. We show that the highest enhancements can be obtained for Q-factors between $10^4\text{--}10^6$ , and enhancement values decrease for Q-factors above $10^6$ due to the radiative energy transfer in the cavity.

Competition between Förster Resonance Energy Transfer and Donor Photodynamics in Plasmonic Dimer Nanoantennas

Plasmonic optical antennas enhance and control the emission of quantum sources in the far-field. Interestingly, the antenna concept can also be applied to enhance the electric field produced by a quantum emitter in the near-field and increase the rate of Forster resonance energy transfer (FRET) between two nearby donor and acceptor dipole emitters. However, plasmonic antennas also influence numerous other photophysical processes such as the donor excitation intensity and decay dynamics and the acceptor emission yield, which compete with the observation of FRET. Understanding the balance between FRET and these processes and monitoring FRET under intense resonant optical confinement in plasmonic nanoantennas have remained challenging open questions. Here, we use DNA-driven self-assembly to accurately produce 40 and 60 nm gold nanoparticle dimer antennas containing a single FRET pair located in the center of a 14 nm gap. The spontaneous donor decay rate constants are increased by 2 orders of magnitude, creat...

Simple Method for Proper Analysis of FRET Sensor Titration Data and Intracellular Imaging Experiments Based on Isosbestic Points

Forster resonance energy transfer (FRET) sensors and other ratiometric probes are increasingly used in life sciences to obtain quantitative information in complex environments such as the cell interior or blood plasma. When using FRET sensors, either to determine the affinity of the sensor for its analyte in vitro, or to apply the sensor to measure an unknown concentration in situ, the ratio of donor and acceptor emission is commonly used as a measure of the sensor occupancy. However, it has been recently demonstrated that the underlying assumption of a linear relationship between the emission ratio and the relative sensor occupancy is not correct. Here we present a simple solution to this problem by using the fluorescence intensity at the isosbestic wavelength as an internal standard. The isosbestic wavelength of FRET-sensors based on the widely used CFP/YFP pair was determined to be at 513 nm, independent of the specific sensor architecture and pH. We show that using the ratio of either the donor or the...

Excitation energy transfer in partly ordered polymer films differing in donor and acceptor transition moments orientation

Based on spectroscopic measurements selected properties of nonradiative Förster energy transport are studied in uniaxially stretched polyvinyl alcohol thin films for three systems differing in donor and acceptor transition moments orientation relative to the axis of stretching. In particular, donor – acceptor emission anisotropy spectra yield completely different regularities for these systems in uniaxially stretched films, whereas they are similar in unstretched films. In particular it is shown that acceptor fluorescence can be either strongly polarized after nonradiative energy transfer in stretched films or depolarized depending on the angular distribution of acceptor transition moments in the matrix. Donor and acceptor emission anisotropy decays exhibit similar regularities to those of steady-state measurements. The obtained results are analyzed with the help of Monte Carlo simulations.