Supramolecular helical porphyrin arrays using DNA as a scaffold

A diphenyl porphyrin substituted nucleotide was incorporated site specifically into DNA, leading to helical stacked porphyrin arrays in the major groove of the duplexes. The porphyrins show an electronic interaction which is significantly enhanced compared to the analogous tetraphenyl porphyrin (TPP) as shown in the large exciton coupling of the porphyrin B-band absorbance. Analogous to the TPP-DNA, an induced helical secondary structure is observed in the single strand porphyrin-DNA. The modified DNA can be hybridised to an immobilised complementary strand leading to fluorescent beads.     

Monitoring the conformational fluctuations of DNA hairpins using single-pair fluorescence resonance energy transfer.

We present single-pair fluorescence resonance energy transfer (spFRET) observations of individual opening and closing events of surface-immobilized DNA hairpins. Two glass-surface immobilization strategies employing the biotin-streptavidin interaction and a third covalent immobilization strategy involving formation of a disulfide bond to a thiol-derivatized glass surface are described and evaluated. Results from image and time-trace data from surface-immobilized molecules are compared with those from freely diffusing molecules, which are unperturbed by surface interactions. Using a simple two-state model to analyze the open and closed time distributions for immobilized hairpins, we calculate the lifetimes of the two states. For hairpins with a loop size of 40 adenosines and a stem size of either seven or nine bases, the respective closed-state lifetimes are 45 +/- 2.4 and 103 +/- 6.0 ms, while the respective open-state lifetimes are 133 +/- 5.5 and 142 +/- 22 ms. These results show that the open state of the hairpin is favored over the closed state of the hairpin under these conditions, consistent with previous diffusion fluorescence correlation spectroscopy (FCS) experiments on poly(A)-loop hairpins. The measured open-state lifetime is about 30 times longer than the calculated 3 ms open-state lifetime for both hairpins based on a closing rate scaling factor derived from a previous FCS study for hairpins in diffusion with 12-30 thymidines in their loops. As predicted, the closed-state lifetime is dependent on the stem length and is independent of the loop characteristics. Our findings indicate that current models should consider sequence dependence in calculating ssDNA thermostability. The surface immobilization chemistries and other experimental techniques described here should prove useful for studies of single-molecule populations and dynamics.     

Detection of DNA based on fluorescence resonance energy transfer of polyelectrolyte-protected CdTe quantum dots as energy donors

The approach for DNA detection was established by using a fluorescence resonance energy transfer (FRET) system, in which the energy donor was poly-diallyldimethylammonium chloride-protected quantum dots and the energy receptor was ethidium bromide (EB) inserting into the double stranded DNA. The concentration of the probe DNA, EB and NaCl was optimized. Under the optimized conditions, the FRET system has a stable signal and good reproducibility. The linear range is 7.7-61.6 nM with the correlation coefficient of 0.998 and the limit of detection is 7.7 nM. This method is simple and sensitive, and makes the label-free DNA detection come true.     

Fast and sensitive analysis of DNA hybridization in a PDMS micro-fluidic channel using fluorescence resonance energy transfer

Fluorescence resonance energy transfer has been used to illustrate its applicability to the sensitive detection of DNA hybridization reactions in a PDMS microfluidic channel.     

Carbazole-based organogel as a scaffold to construct energy transfer arrays with controllable fluorescence emission

A diaryldiketopyrrolopyrrole derivative functionalized with phenothiazine moieties (DPPP) was synthesized and introduced into the ordered 4-(3,6-di-tert-butyl-9H-carbazol-9-yl)benzamide (TBCB) organogel system. It was found that TBCB-based gel became a scaffold to make DPPP molecules line up along the gel fibers, resulting in new self-assembled arrays, whose XRD patterns were quite different from those of the neat TBCB gel and DPPP crystal. In the composite gel, the occurrence of a partial energy transfer from the excited light-harvesting antenna of TBCB to the DPPP acceptor was confirmed on the basis of time-dependent and time-resolved fluorescence investigations. Remarkably, the composite gel could emit intense red light or purplish white light by tuning the excitation wavelength. Such ordered soft materials with color-tunable emission may possess potential applications in sensor and photonic devices.     

Ultrafast fluorescence resonance energy transfer in a micelle

Ultrafast fluorescence resonance energy transfer (FRET) from coumarin 153 (C153) to rhodamine 6G (R6G) is studied in a neutral PEO(20)-PPO(70)-PEO(20) triblock copolymer (P123) micelle and an anionic micelle (sodium dodecyl sulfate, SDS) using a femtosecond up-conversion setup. Time constants of FRET were determined from the rise time of the acceptor emission. It is shown that a micelle increases efficiency of FRET by holding the donor and the acceptor at a close distance (intramicellar FRET) and also by tuning the donor and acceptor energies. It is demonstrated that in the P123 micelle, intramicellar FRET (i.e., donor and acceptor in same micelle) occurs in 1.2 and 24 ps. In SDS micelle, there are two ultrafast components (0.7 and 13 ps) corresponding to intramicellar FRET. The role of diffusion is found to be minor in the ultrafast components of FRET. We also detected a much longer component (1000 ps) for intramicellar FRET in the larger P123 micelle.     

General and reliable quantitative measurement of fluorescence resonance energy transfer using three fluorescence channels

In this paper, we describe a comprehensive general system adapted for quantitative fluorescence resonance energy transfer (FRET) measurement using signals from three channels of a fluorescence instrument. The general FRET measurement system involves two established methods, as well as two novel approaches. Unlike the previous measurements, which can be taken correctly only when the quantity of the acceptor is greater than or equal to that of the donor, one of our novel methods can overcome this obstacle and take quantitative FRET measurements when the donor is in excess of the acceptor. Hence the general FRET measurement system allowed one to determine the exact distance when the donor and acceptor were present in different quantities, and integrated the methods for quantitative FRET measurements. The uniformity of measured values and utility of each method were validated using molecular standards based on DNA oligonucleotide rulers. We also discussed and validated the use of a novel method for estimating the relative quantities of the donor and acceptor fluorophores when they were not known before an appropriate method of this system can be selected.     

Quantum dot-based multiplexed fluorescence resonance energy transfer

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

Shape-specific detection based on fluorescence resonance energy transfer using a flexible water-soluble conjugated polymer

We present the detection of the shape-specific conformation of DNA based on the fluorescence resonance energy transfer (FRET) by using a novel flexible water-soluble cationic conjugated polymer (CCP). The flexible backbone of CCP has more conformational freedom with the potential to be responsive to analyte shape by electrostatic interaction between flexible CCP and negatively charged DNA. The analyte shape dependent recognition is accomplished by structural changes that compressed or extended the flexible CCP. The morphology-dependent spectral properties of the novel flexible polymer related to the analyte shapes are investigated in detail, where two types of chromophores, referred to as "isolated" segment and "packed" segment aggregates, within the flexible polymer are identified by means of ensemble and single molecule measurements upon binding with different geometric DNA. The change in fluorescence intensity upon binding with shape-specific DNA without obvious color shifts makes this novel flexible polymer a suitable CCP donor for FRET measurements. The results provide insights for understanding the spectral properties of flexible water-soluble CCP and CCP/DNA interaction related to the geometry of target analyte.     

Two-dimensional fluorescence resonance energy transfer as a probe for protein folding: a theoretical study

We describe a two-dimensional (2D), four-color fluorescence resonance energy transfer (FRET) scheme, in which the conformational dynamics of a protein is followed by simultaneously observing the FRET signal from two different donor-acceptor pairs. For a general class of models that assume Markovian conformational dynamics, we relate the properties of the emission correlation functions to the rates of elementary kinetic steps in the model. We further use a toy folding model that treats proteins as chains with breakable cross-links to examine the relationship between the cooperativity of folding and FRET data and to establish what additional information about the folding dynamics can be gleaned from 2D, as opposed to one-dimensional FRET experiments. We finally discuss the potential advantages of the four-color FRET over the three-color FRET technique.     

Single lanthanide-doped oxide nanoparticles as donors in fluorescence resonance energy transfer experiments

We used lanthanide-ion doped oxide nanoparticles, Y(0.6)Eu(0.4)VO(4), as donors in fluorescent resonance energy transfer (FRET) experiments. The choice of these nanoparticles allows us to combine the advantages of the lanthanide-ion emission, in particular the long lifetime and the large Stokes shift between absorption and emission, with the detectability of the nanoparticles at the single-particle level. Using cyanine 5 (Cy5) organic molecules as acceptors, we demonstrated FRET down to the single-nanoparticle level. We showed that, due to the long donor lifetime, unambiguous and precise FRET measurements can be performed in solution even in the presence of large free acceptor concentrations. Highly efficient energy transfer was obtained for a large number of acceptor molecules per donor nanoparticle. We determined FRET efficiencies as a function of Cy5 concentration which are in good agreement with a multiple acceptor-multiple donor calculation. On the basis of the donor emission recovery due to acceptor photobleaching, we demonstrated energy transfer from single-nanoparticle donors in fluorescence microscopy experiments.     

Gold nanoparticle-quantum dot-polystyrene microspheres as fluorescence resonance energy transfer probes for bioassays

The paper describes the development of highly sensitive particle-based fluorescence resonance energy transfer (FRET) probes that do not use molecular fluorophores as donors and acceptors. In these probes, CdSe/ZnS luminescent quantum dots (QDs) were capped with multiple histidine-containing peptides to increase their aqueous solubility while maintaining their high emission quantum yield and spectral properties. The peptide-modified QDs (QD-His) were covalently attached to carboxyl-modified polystyrene (PS) microspheres to form highly emitting PS microspheres (QD-PS). Gold nanoparticles (AuNPs) were then covalently attached to the QD-PS surface to form AuNP-QD-PS composite microspheres that were used as FRET probes. Attachment of AuNPs to QD-PS completely quenched the QD emission through FRET interactions. The emission of QD-PS was restored when the AuNPs were removed from the surface by thiol ligand displacement. The new AuNP-QD-PS FRET platform is simple to prepare and highly stable, and it opens many new possibilities for carrying out FRET assays on microparticle-based platforms and in microarrays. The versatility of these assays could be greatly increased by replacing the linkers between the QDs and AuNPs with ones that selectively respond to specific cleaving agents or enzymes.     

Solution-phase single quantum dot fluorescence resonance energy transfer

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.     

Modulating fluorescence resonance energy transfer in conjugated liposomes

We report here a novel system where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism.     

Reversible modulation of quantum dot photoluminescence using a protein- bound photochromic fluorescence resonance energy transfer acceptor

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.     

Homogeneous time resolved fluorescence resonance energy transfer using rare earth cryptates as a tool for probing molecular interactions in biology

A homogeneous assay technology using time resolved fluorescence and fluorescence resonance energy transfer is described. A new class of fluorescent complexes, the cryptates, have been used as fluorescent donor with cross-linked allophycocyanin as acceptor. This new donor/acceptor shows an exceptionally high F?rster distance R0 of 9 nm. This allows to build up a set of strategies to probe the interactions of biomolecules in biology, particularly for high throughput screening applications. In this article, we describe the basics of the technology and review applications developed for studying different key molecular interactions involved in cellular processes.     

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors

The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD₆₀₅) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.