Homogeneous assay based on anti-Stokes' shift time-resolved fluorescence resonance energy-transfer measurement

We report here a novel, time-resolved, lanthanide-based energy-transfer assay utilizing nonoverlapping acceptor fluorophores, which have their absorption energetically at a higher level than the emittive transitions of the donor. The technique was studied by comparing a series of nonoverlapping acceptors in a homogeneous DNA model assay utilizing Eu3+ chelate as a donor. The assay provides strong energy-transfer enhanced acceptor emission and enables the anti-Stokes' shift FRET measurement, in which the induced acceptor emission is at shorter wavelength than the donor emission. This results in high sensitivity, and 0.8 pM detection limit was measured for the DNA target. The acceptor signal of the assay is characterized by exceptional lifetime properties and is not strictly following the Forster's theory. The mechanism of nonoverlapping energy transfer is considered, and we propose that when nonoverlapping acceptors are utilized, the energy transfer arises from the upper 5D2 and 5D1 excited states of europium. The assumption was studied using a simplified energy level scheme of the Eu3+ donor and the acceptors, and a correlation between the acceptor emission behavior and the energy level scheme was found.     

Multimode laser emission from dye doped polymer optical fiber

Multimode laser emission is observed in a polymer optical fiber doped with a mixture of Rhodamine 6G (Rh 6G) and Rhodamine B (Rh B) dyes. Tuning of laser emission is achieved by using the mixture of dyes due to the energy transfer occurring from donor molecule (Rh 6G) to acceptor molecule (Rh B). The dye doped poly(methyl methacrylate)-based polymer optical fiber is pumped axially at one end of the fiber using a 532 nm pulsed laser beam from a Nd:YAG laser and the fluorescence emission is collected from the other end. At low pump energy levels, fluorescence emission is observed. When the energy is increased beyond a threshold value, laser emission occurs with a multimode structure. The optical feedback for the gain medium is provided by the cylindrical surface of the optical fiber, which acts as a cavity. This fact is confirmed by the mode spacing dependence on the diameter of the fiber.     

Tandem dye acceptor used to enhance upconversion fluorescence resonance energy transfer in homogeneous assays

In fluorescence resonance energy transfer (FRET)-based assays, spectral separation of acceptor emission from donor emission is a common problem affecting the assay sensitivity. The challenge derives from small Stokes shifts characteristic to conventional fluorescent dyes resulting in leakage of donor emission to the measurement window intended only to collect the acceptor emission. We have studied a FRET-based homogeneous bioaffinity assay utilizing a tandem dye acceptor with a large pseudo-Stokes shift (139 nm). The tandem dye was constructed using B-phycoerythrin as an absorber and multiple Alexa Fluor 680 dyes as emitters. As a donor, we employed upconverting phosphor particles, which uniquely emit at visible wavelengths under low-energy infrared excitation enabling the fluorescence measurements free from autofluorescence even without time-resolved detection. With the tandem dye, it was possible to achieve four times higher signal from a single binding event compared to the conventional Alexa Fluor 680 dye alone. Tandem dyes are widely used in cytometry and other multiplex purposes, but their applications can be expanded to fluorescence-based homogeneous assays. Both the optimal excitation and emission wavelengths of tandem dye can be tuned to a desired region by choosing appropriate fluorophores enabling specifically designed acceptor dyes with large Stokes shift.     

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.     

A molecular scale full adder based on controlled intramolecular electron and energy transfer

Electron and electronic energy transfer processes are ways by which different molecules can signal their state from one (the Donor, D) to the other (the Acceptor, A). This transfer is often studied as an intermolecular process but it can also occur intramolecularly, that is between two bridged parts of a bichromophoric molecule.We show that the donor part of a bichromophore molecule can communicate its logic output as input to the acceptor part of the bichromophoric molecule. Such transfer is achieved through electronic energy transfer between the chromophores. We discuss a specific pair, the rhodamine 6G-azulene, for which there is considerable data, but the scheme is general enough to allow a wide choice of D–A pairs. We present preliminary results pertaining to a newly synthesized bichromophoric molecule based on this pair, in which a full adder is implemented, utilizing intramolecular electronic energy transfer between the two moieties.     

Reversible ratiometric probe for quantitative DNA measurements

We have designed a reversible fluorescent DNA probe that can be used to determine the concentration of single-stranded DNA in solution by a ratiometric fluorescence measurement. The probe consists of a single-stranded dual fluorescently labeled DNA molecule that adopts a stem-loop conformation in its nonhybridized state. The stem length and the length of the loop region complementary to the target were chosen to allow for reversible binding. The excitation and emission wavelengths of the two labels Cy3 and Cy5 allow for fluorescence resonance energy transfer in the closed state. Upon hybridization, the probe opens up resulting in a fluorescence intensity increase of the donor and a fluorescence intensity decrease of the acceptor. The ratio of the acceptor-to-donor fluorescence intensities is independent of the amount of probe and provides a quantitative measure of the free target concentration.     

Energy transfer in Sm:Eu system in zinc sodium phosphate glasses

The mechanism of nonradiative energy transfer process in zinc sodium phosphate glass system co-doped with samarium and europium ion has been examined under cw laser excitation. Donor–acceptor distance and quantum efficiency of transfer have been evaluated using the relevant theoretical expressions. Transfer probabilities have been determined using the overlap integral and relative fluorescence methods. The Forster–Dexter theoretical predictions are found to be in excellent agreement with experimental results. The nonresonant energy transfer assisted by phonons is the dominant transfer mechanism in the concentration range taken. Excitation spectra and the decay profile of the samarium ion also support the energy transfer from samarium to europium.     

Upconversion fluorescence resonance energy transfer in a homogeneous immunoassay for estradiol

We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17beta-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel principle of UC-FRET has unique advantages compared to present homogeneous luminescence-based methods and can enable an attractive assay system platform for clinical diagnostics and for high-throughput screening approaches.     

Emission Tuning with Size‐Controllable Polymer Dots from a Single Conjugated Polymer

Two conjugated polymers (CPs) with various compositions of phenylene and benzoselenadiazoben (BSD) are synthesized to have a special emitting property; different fluorescence colors in solution and in the solid states, allowing the resulting conjugated polymer dots (Pdots) to emit different fluorescence colors upon their size variation. The photophysical property of such different‐sized Pdots is investigated using fluorescence spectra and fluorescence lifetimes. A decrease in the fluorescence lifetime of Pdots is observed with an increase in the size of Pdots, caused by quantitative change in energy transfer from phenylene (energy donor) to the BSD unit (energy acceptor). The results provide that any CP can be used for the fabrication of Pdots with size‐tunable emission, as long as the CP shows different emissions according to its phases. Such emission of Pdots can even be observed when in the solid solution in polymer matrix, which emits different fluorescence colors depending on the size of embedded Pdots in the polymer matrix.     

A New Design Strategy for Efficient Thermally Activated Delayed Fluorescence Organic Emitters: From Twisted to Planar Structures

In the traditional molecular design of thermally activated delayed fluorescence (TADF) emitters composed of electron‐donor and electron‐acceptor moieties, achieving a small singlet–triplet energy gap (ΔE_ST) in strongly twisted structures usually translates into a small fluorescence oscillator strength, which can significantly decrease the emission quantum yield and limit efficiency in organic light‐emitting diode devices. Here, based on the results of quantum‐chemical calculations on TADF emitters composed of carbazole donor and 2,4,6‐triphenyl‐1,3,5‐triazine acceptor moieties, a new strategy is proposed for the molecular design of efficient TADF emitters that combine a small ΔE_ST with a large fluorescence oscillator strength. Since this strategy goes beyond the traditional framework of structurally twisted, charge‐transfer type emitters, importantly, it opens the way for coplanar molecules to be efficient TADF emitters. Here, a new emitter, composed of azatriangulene and diphenyltriazine moieties, is theoretically designed, which is coplanar due to intramolecular H‐bonding interactions. The synthesis of this hexamethylazatriangulene‐triazine (HMAT‐TRZ) emitter and its preliminary photophysical characterizations point to HMAT‐TRZ as a potential efficient TADF emitter.     

Enhanced light harvesting from Först-type resonance energy transfer in the quasi-solid state dye-sensitized solar cells

The demonstrated F?rst-type resonance energy transfer (FRET) is demonstrated in quasi-solid type dye-sensitized solar cells between organic fluorescence materials as an energy donor doped in polymeric gel electrolyte and a ruthenium complex as an energy acceptor on the surface of TiO2. Strong spectral overlap of emission/absorption of the energy donor and acceptor is required to obtain high FRET efficiency. The judicious choice of the energy donor allows the enhancement of the light harvesting characters of the energy acceptor (N3) in quasi-solid dye sensitized solar cells which increases the power conversion efficiency by 25% compare to that of a pristine cell. The optimized cell architecture fabricated with the quasi-solid type electrolyte containing fluorescence materials shows a maximum efficiency of 5.08% with a short-circuit current density (J(sc)) of 12.63 mA/cm2, and an open-circuit voltage (V(oc)) of 0.70 V under illumination of simulated solar light (AM 1.5, 100 mW/cm2).     

Triphenylene‐Derived Electron Acceptors and Donors on Ag(111): Formation of Intermolecular Charge‐Transfer Complexes with Common Unoccupied Molecular States

Over the past years, ultrathin films consisting of electron donating and accepting molecules have attracted increasing attention due to their potential usage in optoelectronic devices. Key parameters for understanding and tuning their performance are intermolecular and molecule–substrate interactions. Here, the formation of a monolayer thick blend of triphenylene‐based organic donor and acceptor molecules from 2,3,6,7,10,11‐hexamethoxytriphenylene (HAT) and 1,4,5,8,9,12‐hexaazatriphenylenehexacarbonitrile (HATCN), respectively, on a silver (111) surface is reported. Scanning tunneling microscopy and spectroscopy, valence and core level photoelectron spectroscopy, as well as low‐energy electron diffraction measurements are used, complemented by density functional theory calculations, to investigate both the electronic and structural properties of the homomolecular as well as the intermixed layers. The donor molecules are weakly interacting with the Ag(111) surface, while the acceptor molecules show a strong interaction with the substrate leading to charge transfer and substantial buckling of the top silver layer and of the adsorbates. Upon mixing acceptor and donor molecules, strong hybridization occurs between the two different molecules leading to the emergence of a common unoccupied molecular orbital located at both the donor and acceptor molecules. The donor acceptor blend studied here is, therefore, a compelling candidate for organic electronics based on self‐assembled charge‐transfer complexes.     

Fluorescence enhancement from Eu3+ ions in CdSe nanocrystal containing silica matrix hosts

Semiconductor cadmium selenide particles together with europium ions were incorporated into the silica matrix using sol–gel method. Here, the effect of nanocrystals on the absorption and fluorescence features of europium ions is discussed. The fluorescence spectra reveal that the intensity of characteristic emission of europium increases considerably in the presence of CdSe particles. This phenomenon can be explained as due to the energy transfer resulting from electron–hole recombination in the CdSe to the rare earth ion. These zero-dimensional materials, along with the effect of matrix incorporating europium ions, are found to have increased the optical gain.     

Quantum Dot-Based Biosensor for Detection of Human Cardiac Troponin I Using a Liquid-Core Waveguide

Human cardiac Troponin I is one of three subunits of the cardiac Troponin complex that are released into the bloodstream upon injury to cardiac muscle, particularly myocardial infarction, where it is absent under normal conditions. Rapid, sensitive detection of blood borne Troponin I is extremely important for early detection of myocardial infarction. An optical biosensor has been proposed as a versatile, adaptable, and effective method for detection of Troponin I. The biosensor architecture utilizes fluorescence resonance energy transfer (FRET), a distance-dependent chemical signal transduction method that occurs between two fluorescent molecules, termed the donor and acceptor. In order to launch FRET, a donor-labeled protein A molecule is bound to an acceptor-labeled capture antibody. When exposed to the Troponin I antigen, the antibody-antigen binding event initiates a conformational change within the structure of the antibody. As this morphological change in the antibody takes place, the distance between the donor and acceptor changes, resulting in a measurable shift in energy transfer. In this study, quantum dots were utilized as the FRET donors to further increase the efficiency of the biosensor system and organic dyes were utilized as the acceptors. This sensing mechanism was then interfaced in a liquid-core waveguide (LCW) platform that was able to capture the resulting fluorescence to achieve highly sensitive and accurate measurements. The biosensor demonstrated an ample sensitivity to the analyte, achieving a lower limit of detection of approximately 32 nM in phosphate buffered saline and 55 nM in human plasma. A high degree of specificity was also observed when the response to cardiac Troponin I is compared with that of a nonspecific protein. Response time of the biosensor was determined to be less than 1 min; an expeditious time compared with other Troponin diagnostic assays.     

Electrical control of Förster energy transfer

Bringing together compounds of intrinsically different functionality, such as inorganic nanostructures and organic molecules, constitutes a particularly powerful route to creating novel functional devices with synergetic properties found in neither of the constituents. We introduce nanophotonic functional elements combining two classes of materials, semiconductor nanocrystals and dyes, whose physical nature arises as a superposition of the properties of the individual components. The strongly absorbing rod-like nanocrystals focus the incident radiation by photopumping the weakly absorbing dye via energy transfer. The CdSe/CdS nanorods exhibit a large quantum-confined Stark effect on the single-particle level, which enables direct control of the spectral resonance between donor and acceptor required for nanoscopic F?rster-type energy transfer in single nanorod-dye couples. With this far-field manipulation of a near-field phenomenon, the emission from single dye molecules can be controlled electrically. We propose that this effect could lead to the design of single-molecule optoelectronic switches providing building blocks for more complex nanophotonic circuitry.     

Distance-dependent energy transfer between indole and anthracene moieties in Langmuir–Blodgett films

1,2-Diphenyl indole (DPI) and 9,10-diphenyl anthracene (DPA) are non-amphiphilic molecules but form excellent LB films when mixed with stearic acid (SA). Spectroscopic investigations of these films indicate formation of aggregates of DPI and DPA in the mixed LB films. DPA has been used as the quencher of the fluorescence of the DPI donor. Distance-dependent energy transfer between donor and acceptor monolayers in the LB film, where they can be precisely separated by inert spacers of stearic acid layers of varied thickness, is shown to satisfy Khun's quadratic equation. This suggests that the donor excitations are delocalized. The large critical transfer distance estimated from the experimental results has been attributed to the formation of aggregates of the molecules in a LB monolayer.     

Investigation of an optical dual receptor method to detect HIV

A novel sensing method for detecting the human immunodeficiency virus (HIV) is reported. The method utilized a two receptor-binding event and required the integration of a chemical transducer system with two unique protein receptors, CD4 and Mab, a monoclonal antibody, which bind to gp120, a single-surface protein receptor on HIV. The chemical transduction system was based on the distance-dependent principle of fluorescence resonance energy transfer (FRET). During the binding event, the two fluorophore-labeled receptors docked at the surface of the gp120. The resulting close proximity of the two fluorophores upon binding initiated an energy transfer resulting in a detectable change of fluorescence. Donor fluorophore to acceptor fluorophore ratios were examined to ascertain any effects on energy transfer. The experiments demonstrated that higher ratios of acceptor fluorophores to donor fluorophores resulted in enhanced energy transfer. In-solution testing proved the feasibility of the dual receptor technique in detecting the presence of gp120 with a limit of detection of 5 ng/ml.     

Thermal-Lens Study on the Distance-Dependent Energy Transfer from Rhodamine 6G to Gold Nanoparticles

A study on energy transfer from the Rhodamine 6G (donor) to gold nanoparticles (acceptor) is investigated using a laser-based dual-beam thermal-lens technique. The nanoparticles are observed to quench the intrinsic fluorescence of the dye molecule via a nonradiative energy transfer mechanism. The influence of nanoparticle concentration $(0.09\,\hbox {nM}\,\hbox {to}\,0.24\,\hbox {nM})$ on the energy transfer mechanism with Rhodamine 6G $(1\;\upmu \hbox {M})$ is investigated. Analysis of the results indicates that the energy transfer efficiency is high (more than 50 %) in the presence of nanoparticles and the efficiency is enhanced with an increase in the nanoparticle concentration. The distance between the nanoparticle and dye molecule is evaluated on the basis of the nanomaterial surface energy transfer model. The thermal-lens studies probe the nonradiative path of de-excitation of the excited molecule, and the comparison between this technique and the conventional fluorescence method in measuring the distance as well as the energy-transfer efficiency clearly indicates that the thermal-lens technique is a complementary approach to study the energy-transfer mechanism between a donor and an acceptor.     

Organic host–guest systems for blue emission

The photoluminescence (PL) quantum yields (QYs) and lifetimes of the 1,6-diphenyl-1,3,5-hexatriene (DPH) blue emitter and of the acceptor–donor system, DPH and p-terphenyl (P3), are investigated embedded in the nanochannels of a perhydrotriphenylene (PHTP) matrix. In the co-inclusion system, light emission takes place from DPH, due to the efficient resonant energy transfer (RET) from the P3 donor molecules. The host matrix stabilizes the guest molecules against photooxidation degradation. The largest quantum yield, up to 100%, has been found in the co-inclusion compound (IC), where excitations are localized at the emissive acceptor sites.     

High-throughput DNA droplet assays using picoliter reactor volumes

The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.