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.     

Multiplexed fluorescence resonance energy transfer aptasensor between upconversion nanoparticles and graphene oxide for the simultaneous determination of mycotoxins

We presented a new aptasensor for mycotoxins, which was based on multiplexed fluorescence resonance energy transfer (FRET) between multicolor upconversion fluorescent nanoparticles (UCNPs) as donors and graphene oxide (GO) as the entire and effective acceptor. BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.7), Tm(0.02) upconversion nanoparticles were synthesized and functionalized, respectively, with immobilized ochratoxin A (OTA)-aptamers and fumonisin B(1) (FB(1))-aptamers. On the basis of the strong π-π stacking effect between the nucleobases of the aptamers and the sp(2) atoms of GO, the aptamer modified-UCNPs can be brought in close proximity to the GO surface. The strong upconversion fluorescence both of BaY(0.78)F(5):Yb(0.2), Er(0.02) and BaY(0.78)F(5):Yb(0.2), Tm(0.02) can be completely quenched by the GO, because of a good overlap between the fluorescence emission of multicolor UCNPs and the absorption spectrum of GO. In contrast, in the presence of OTA and FB(1), the aptamers preferred to bind to their corresponding mycotoxins, which led to changes in the formation of aptamers, and therefore, aptamer modified-UCNPs were far away from the GO surface. Our study results showed that the fluorescence intensity of BaYF(5):Yb Er and BaYF(5):Yb Tm were related to the concentration of OTA and FB(1). We therefore developed a sensitive and simple platform for the simultaneous detection of OTA and FB(1) with multicolor UCNPs and GO as the FRET pair. The aptasensor provided a linear range from 0.05 to 100 ng·mL(-1) for OTA and 0.1 to 500 ng·mL(-1) for FB(1); the detection limit of OTA was 0.02 ng·mL(-1) and FB(1) was 0.1 ng·mL(-1). As a practical application, the aptasensor was used to monitor OTA and FB(1) level in naturally contaminated maize samples with the results consistent with that of a classic ELISA method. More importantly, the novel multiplexed FRET was established for the first time based on multiplexed energy donors to the entire energy acceptor; this work was expected to open up a new field of FRET system applications for various targets.     

A homogeneous and noncompetitive immunoassay based on the enhanced fluorescence resonance energy transfer by leucine zipper interaction

Fluorescence resonance energy transfer (FRET) between two GFP variants is a powerful technique to describe protein-protein interaction in a biological system. However, it has a limitation that the two variants tethered to the respective proteins have to be in sufficient proximity upon binding, which is often difficult to attain by simple N- or C-terminal fusions. Here we describe a novel method to significantly enhance FRET between GFP variant-tagged proteins with the use of leucine zippers. For the homogeneous sandwich immunoassay of a high molecular weight antigen human serum albumin (HSA), two separate single-chain Fvs recognizing distant epitopes of HSA were respectively fused with fluorescence donor ECFP or acceptor EYFP, and FRET between the two was analyzed by fluorescence spectrometry. Because these two proteins did not give any detectable FRET uponantigen addition, we tethered each protein with a leucine zipper motif (c-Jun or FosB) at the C-terminus to help the neighborhood of the GFP variants. Upon antigen addition, the new pairs showed significant antigen-dependent FRET. By exchanging the binding domains, the method will find a range of applications for the assay of other proteins and their interactions in vitro or in vivo.     

Tomographic imaging of ratiometric fluorescence resonance energy transfer in scattering media

A method to visualize and quantify fluorescence resonance energy transfer (FRET) in scattering media is proposed. It combines the ratiometric FRET method with fluorescence molecular tomography (FMT) in continuous wave (CW) mode. To evaluate the performance of the proposed method, experiments on a tissue-mimicking phantom are carried out. The results demonstrate that the proposed approach is capable of visualizing and quantifying the FRET distribution in scattering media, which implies the further application of the ratiometric assay in in vivo studies.     

Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer

A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.     

Microcapsule biosensors using competitive binding resonance energy transfer assays based on apoenzymes

This paper reports the first demonstration of a fluorescence resonance energy transfer based glucose sensor, wherein a competitive binding (CB) assay is encapsulated into polyelectrolyte microcapsules. The work supports the concept that microcapsules are superior to hydrogel systems or other matrixes for competitive-binding-based system, as they provide free movement of the sensing elements within the capsule interior while constant total sensing assay concentration is maintained. The transduction approach employed in these preliminary experiments is also a novel CB system based on a model apoenzyme, apo-glucose oxidase (AG), which is highly specific to beta-d-glucose, as the model target-binding protein. The glucose sensitivity of the fluorescein isothiocyanate (FITC)-dextran and tetramethylrhodamine isothiocyanate-AG encapsulated in microcapsules showed 5 times greater specificity for beta-D-glucose over other sugars, with sensitivity (change in intensity ratio) in the range of 2-6%/mM. It was observed that the sensitivity and range of the response can be tailored by controlling the assay concentration using different FITC-dextran molecular weight and total capsule concentration. The findings support the concepts of using microcapsules to encapsulate CB assays for reversible and stable sensors and the use of apoenzymes as specific molecular recognition elements in CB assays. Further, characterization results for microcapsule glucose sensors demonstrate their suitability for monitoring physiological glucose levels.     

Intramolecular fluorescence resonance energy transfer system with coumarin donor included in beta-cyclodextrin

In aqueous solutions, the fluorescence of the intramolecular fluorescence resonance energy-transfer (FRET) system 1 was strongly quenched, because of close contact between the donor and acceptor moieties. FRET occurred, and the acceptor fluorescence was increased, by adding beta-cyclodextrin (beta-CD) to aqueous solutions of 1. Spectral analysis supported the idea that the FRET enhancement was due to the formation of an inclusion complex of the coumarin moiety in beta-CD, resulting in separation of the fluorophores. On the basis of this result, we propose that covalent binding of coumarin to beta-CD will provide a FRET cassette molecule. So, compound 2 bearing beta-CD covalently was designed and synthesized. Fluorescence intensity of 2 was enhanced markedly compared to the intensity of 3. Applying this FRET system, various FRET probes that will be useful for ratio imaging and also the high-throughput screening will be provided.     

Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis-Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD-thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.     

Quantum dot-fluorescent protein pairs as novel fluorescence resonance energy transfer probes

Fluorescence resonance energy transfer (FRET) characteristics, including the efficiency, donor-acceptor distance, and binding strength of six fluorescent protein (FP)-quantum dot (QD) pairs were quantified, demonstrating that FPs are efficient acceptors for QD donors with up to 90% quenching of QD fluorescence and that polyhistidine coordination to QD core-shell surface is a straightforward and effective means of conjugating proteins to commercially available QDs. This provides a novel approach to developing QD-based FRET probes for biomedical applications.     

Controlled fluorescence resonance energy transfer between ZnO nanoparticles and fluorophores in layer-by-layer assemblies

We controlled the fluorescence resonance energy transfer (FRET) between ZnO nanoparticles and rhodamine B (RB) within multilayered thin films prepared by the layer-by-layer (LbL) assembling method. Positively charged ZnO nanoparticles and RB-labeled poly(allyamine hydrochloride) (RB-PAH) were accurately incorporated into LbL assemblies of polyelectrolytes. The distance between ZnO nanoparticles and RB-PAH was adjusted by varying the number of layers of pure polyelectrolytes, leading to the controlled FRET from ZnO nanoparticles to RB-PAH.     

Quantum dot-based fluorescence resonance energy transfer with improved FRET efficiency in capillary flows

Fluorescence resonance energy transfer (FRET)-based nanosensors with quantum dots (QDs) as donors and organic dyes as acceptors have long been of interest for the detection of biomolecules such as nucleic acids, but their low FRET efficiency in bulk solution has prevented the sensitive detection of nucleic acids due to the large size of the QDs and the long length of nucleic acids. Here we describe a novel approach to improve the detection sensitivity of QD-based nanosensors using single-molecule detection in a capillary flow. In comparison with bulk measurement, single-molecule detection in a capillary flow possesses the unique advantages of improved FRET efficiency, high sensitivity, prevention of photobleaching, and low sample consumption. Greater FRET efficiency was obtained due to the deformation of DNA in the capillary stream. This technique can be easily extended to sensitive bimolecular analysis in microfluidic chips, and it may also offer a promising approach to study the deformation of small nucleic acids in fluid flow.     

Highly adaptable and sensitive protease assay based on fluorescence resonance energy transfer

Proteases are widely used in analytical sciences and play a central role in several widespread diseases. Thus, there is an immense need for highly adaptable and sensitive assays for the detection and monitoring of various proteolytic enzymes. We established a simple protease fluorescence resonance energy transfer (pro-FRET) assay for the determination of protease activities, which could in principle be adapted for the detection of all proteases. As proof of principle, we demonstrated the potential of our method using trypsin and enteropeptidase in complex biological mixtures. Briefly, the assay is based on the cleavage of a FRET peptide substrate, which results in a dramatic increase of the donor fluorescence. The assay was highly sensitive and fast for both proteases. The detection limits for trypsin and enteropeptidase in Escherichia coli lysate were 100 and 10 amol, respectively. The improved sensitivity for enteropeptidase was due to the application of an enzyme cascade, which leads to signal amplification. The pro-FRET assay is highly specific as even high concentrations of other proteases did not result in significant background signals. In conclusion, this sensitive and simple assay can be performed in complex biological mixtures and can be easily adapted to act as a versatile tool for the sensitive detection of proteases.     

Homogeneous noncompetitive immunoassay for 17beta-estradiol based on fluorescence resonance energy transfer

We previously presented a homogeneous noncompetitive assay principle based on quenching of the fluorescence of europium(III) chelate. This assay principle has now been applied to the measurement of 17beta-estradiol (E2) using europium(III) chelate labeled estradiol specific antibody Fab fragment (Eu(III)-Fab) as a donor and E2 conjugated with nonfluorescent QSY21 dye as an acceptor. Fluorescence could be measured only from those Eu(III)-Fab that were bound to E2 of the sample, while the fluorescence of the Eu(III)-Fab that were not occupied by E2 were quenched by E2-QSY21 conjugates. The performance of the assay was tested both in buffer and in the presence of serum. Because approximately half of the Fabs were incapable of binding to E2, the maximum quenching efficiency was only 55%. The lowest limits of detection were 18 and 64 pM in buffer and serum-based calibrators, respectively. The highest measurable concentrations were 0.4 nM in buffer and 1 nM in serum. The presented noncompetitive assay principle requires only one binder, and it can be applied to other haptens as well, providing that a suitable antibody is available.     

Single-molecule fluorescence resonance energy transfer in nanopipets: improving distance resolution and concentration range

In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of approximately 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations. The flow of the donor/acceptor (Cy3B/ATTO647N) labeled double-stranded DNA conjugates was established by electrokinetic forces. Because of the small inner diameter of the nanopipet, every molecule passing the tip is detected applying alternating laser excitation (ALEX). Thus, the technique offers the advantage to study interactions with smaller association constants (<10(9) M-1) using minute sample amounts (<5 microL). The improved photon statistics reduces shot-noise contributions and results in sharper FRET distributions. Experimental results are supported by Monte Carlo simulations which also explain the occurrence of two populations in burst size distributions measured in nanopipet experiments. Because of the confinement of the molecules in nanopipets, the widths of FRET histograms are reduced to a degree where shot-noise is not the only limiting factor but also conformational dynamics of the linkers used to attach the chromophores have to be considered. In addition, our experiments emphasize the influence of photoinduced dark states on both the mean energy transfer efficiency and the width of FRET histograms.     

An efficient and biocompatible fluorescence resonance energy transfer system based on lanthanide-doped nanoparticles

This work demonstrates an efficient and bio-friendly fluorescence resonance energy transfer (FRET) system based on lanthanide-doped inorganic nanoparticles. A facile aqueous route was used to synthesize the CePO(4):Tb nanorods with homogeneous colloidal dispersion, which emits a bright green light with a high quantum yield (～0.36) and a long fluorescence lifetime (～3.50 ms) upon UV excitation. Upon treatment of CePO(4):Tb with aqueous Rhodamine B (RhB), an efficient FRET occurs from the Tb(3+) to the RhB molecules, giving rise to well resolved and ratiometric emissions of donors and acceptors, respectively, with an energy transfer efficiency of up to 0.85. When incubated with HeLa cells at 37?°C, the CePO(4):Tb treated with RhB shows bright intracellular luminescence, indicating that it can be successfully internalized inside the cells and the FRET remains in the living cells. Moreover, the cytotoxic measurements demonstrate good biocompatibility and low cytotoxicity of our present FRET system. The advantages presented above including high quantum yield of donors, high energy transfer efficiency, ratiometric fluorescent emission and good biocompatibility, indicate the high potential of the CePO(4):Tb/RhB FRET system for monitoring biological events.     

Analysis of microRNA-induced silencing complex-involved microRNA-target recognition by single-molecule fluorescence resonance energy transfer

MicroRNAs (miRNAs) are important regulators of gene expression that control almost every physiological and pathological process. Although the complementarity between the seed region of a miRNA and its target mRNA is usually deemed as the key determinant in the miRNA-target recognition in animals, the mechanism of their recognition still remains enigmatic as more and more exceptions challenge the seed rule. Herein, we employ single-molecule fluorescence resonance energy transfer (smFRET) to investigate human miRNA-induced silencing complex (miRISC)-involved miRNA-target recognition with either perfect base pairing or poor seed match in real time. Our results demonstrate that the recognition between mammalian miRNA and its target with perfect base pairing proceeds in a two-state model as prokaryotic guide DNA-mediated recognition, suggesting a conserved pattern of guide RNA/DNA strand recognition. In addition to the general rule of miRNA-target recognition, our results reveal that annealing between miRNA and its target with poor seed match proceeds in a stepwise way, which is in accordance with the increase in the number of conformational states of miRNA-target duplex accommodated by the miRISC, suggesting the structural plasticity of human miRISC to conciliate the mismatches in seed region. This new dynamic information revealed by smFRET has an important implication for comprehensive understanding of the role of miRISC in the target recognition in mammals.     

Preparation of a transparent alumina film doped with fluorescence dye and its energy transfer laser emission

A transparent alumina film was prepared from an aqueous sol by the sol-gel process. Changes in the fluorescence of 7-azaindole were monitored during the processing from the aqueous sol to the dried gel by dehydration. Coumarin 1, which is insoluble in water, and an anionic dye, uranin, were doped into the film by utilization of surface-active reagents, Triton X-100 and cetyltrimethylammonium bromide respectively. This doped film exhibited energy-transfer-type laser emission by nitrogen laser pumping.     

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.     

Enhanced resonance energy transfer from PVK to MEH-PPV in nanoparticles

Organic semiconductor nanoparticles were prepared from poly(N-vinylcarbazole) (PVK), poly(2-methoxy-5-(2(')-ethyl-hexyloxy)-p-phenylene vinylene) (MEH-PPV) and their blend solution via a reprecipitation method, respectively. A wide photoluminescence band centred at 430?nm has been found in the PVK nanoparticles, which is obviously red-shifted by comparison with the PVK film. The red-shifted emission from the PVK nanoparticles has a good spectral superposition with the absorption of the MEH-PPV nanoparticles. However, the spectral superposition is very poor between the two polymers in the composite films. The markedly enhanced emission from MEH-PPV is experimentally observed in the composite polymer nanoparticles and attributed to F?rster energy transfer from PVK to MEH-PPV for excitation at the absorption maximum of PVK.