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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Upconversion fluorescence resonance energy transfer based biosensor for ultrasensitive detection of matrix metalloproteinase-2 in blood

Matrix metalloproteinase-2 (MMP-2) is a very important biomarker in blood. Presently, sensitive and selective determination of MMP-2 directly in blood samples is still a challenging job because of the high complexity of the sample matrix. In this work, we reported a new homogeneous biosensor for MMP-2 based on fluorescence resonance energy transfer (FRET) from upconversion phosphors (UCPs) to carbon nanoparticles (CNPs). A polypeptide chain (NH(2)-GHHYYGPLGVRGC-COOH) comprising both the specific MMP-2 substrate domain (PLGVR) and a π-rich motif (HHYY) was designed and linked to the surface of UCPs at the C terminus. The FRET process was initiated by the π-π interaction between the peptide and CNPs, which thus quenched the fluorescence of the donor. Upon the cleavage of the substrate by the protease at the amide bond between Gly and Val, the donor was separated from the acceptor while the π-rich motif stayed on the acceptor. As a result, the fluorescence of the donor was restored. The fluorescence recovery was found to be proportional to the concentration of MMP-2 within the range from 10-500 pg/mL in an aqueous solution. The quantification limit of this sensor was at least 1 order of magnitude lower than that of other reported assays for MMP-2. The sensor was used to determine the MMP-2 level directly in human plasma and whole blood samples with satisfactory results obtained. Owing to the hypersensitivity of the method, clinical samples of only less than 1 μL were needed for accurate quantification, which can be meaningful in MMP-2-related clinical and bioanalytical applications.     

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry

We report a method to increase the resolution of single pair fluorescence resonance energy transfer (spFRET) measurements in aqueous solutions. Solution-based spFRET measurements of fluorescently labeled biological molecules (proteins, RNA, DNA) are often used to obtain histograms of molecular conformation without resorting to sample immobilization. However, for solution-phase spFRET studies, the number of photons detected from a single molecule as it diffuses through an open confocal volume element are quite limited. An "average" transit may yield on the order of 40 photons. Shot noise on the number of detected photons substantially limits the resolution of the measurement. The method reported here uses a hydrodynamically focused sample stream to ensure molecules traverse the full width of an excitation laser beam. This substantially increases the average number of photons detected per molecular transit (approximately 85 photons/molecule), which increases measurement precision. In addition, this method minimizes another source of heterogeneity present in diffusive measures of spFRET: the distribution of paths taken through the excitation laser beam. We demonstrate here using a FRET labeled protein sample (a FynSH3 domain) that superior resolution (a factor of approximately 2) can be obtained via molecular cytometry compared to spFRET measurements based upon diffusion through an open confocal volume element.     

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids.     

Design, synthesis, and application of particle-based fluorescence resonance energy transfer sensors for carbohydrates and glycoproteins

This paper describes the development of novel particle-based fluorescence resonance energy transfer (FRET) sensors to quantify the concentration and monitor the binding affinity of carbohydrates and glycoproteins to lectins, which are carbohydrate binding proteins. The sensing approach is based on FRET between fluorescein (donor)-labeled lectin molecules, adsorbed on the surface of micrometric polymeric beads, and polymeric dextran molecules labeled with Texas Red (acceptor). The FRET efficiency of the donor-acceptor pair decreases in the presence of carbohydrates or glycoproteins that compete with the Texas Red-labeled dextran molecules on the lectinic binding sites. The inhibitory effect is concentration and time dependent. The sensing technique enables the discrimination between carbohydrates and glycoproteins based on their binding affinity to the FRET sensing particles as well as quantitative analysis of carbohydrates and glycoproteins in aqueous samples. In the future, the newly developed sensors could enable screening glycoprotein-based drugs for their binding affinity toward selective receptors.     

Efficient single-molecule fluorescence resonance energy transfer analysis by site-specific dual-labeling of protein using an unnatural amino acid

Single-molecule fluorescence resonance energy transfer (smFRET) measurement provides a unique and powerful approach to understand complex biological processes including conformational and structural dynamics of individual biomolecules. For effective smFRET analysis of protein, site-specific dual-labeling with two fluorophores as an energy donor and an acceptor is crucial. Here we demonstrate that site-specific dual-labeling of protein via incorporation of unnatural amino acid provides a clearer picture for the folded and unfolded states of the protein in smFRET analysis than conventional labeling using double cysteines. As a model study, maltose-binding protein (MBP) was dually labeled via incorporation of ρ-azido-l-phenylalanine and cysteine at specific positions, immobilized on a surface, and subjected to smFRET analysis under native and denaturing conditions. The resulting histograms show that site-specific dual-labeling results in a more homogeneous distribution in protein populations, enabling a precise smFRET analysis of protein.