Bulge‐like Asymmetric Heterodye Clustering in DNA Duplex Results in Efficient Quenching of Background Emission Based on the Maximized Excitonic Interaction

Asymmetric dye clusters with a single fluorophore (Cy3) and multiple quenchers (4′‐methylthioazobenzene‐4‐carboxylate, methyl red, and 4′‐dimethylamino‐2‐nitroazobenzene‐4‐carboxylate) were prepared. The dye and one‐to‐five quenchers were tethered through D ‐threoninol to opposite strands of a DNA duplex. NMR analysis revealed that the clusters with a single fluorophore and two quenchers formed a sandwich‐like structure (antiparallel H‐aggregates). The melting temperatures of all the heteroclusters were almost the same, although structural distortion should become larger, as the number of quenchers increased. An asymmetric heterocluster of a single fluorophore and two quenchers showed larger excitonic interaction (i.e., hypochromicity of Cy3), than did a single Cy3 and a single quencher. Due to the larger exciton coupling between the dyes, the 1:2 heterocluster suppressed the background emission more efficiently than the 1:1 cluster. However, more quenchers did not enhance quenching efficiency due to the saturation of exciton coupling with two quenchers. Finally, this asymmetric 1:2 heterocluster was introduced into the stem region of a molecular beacon (MB; also known as an in‐stem MB) targeting the fusion site in the L6 BCR‐ABL fusion gene. With this MB design, the signal/background ratio was as high as 68 due to efficient suppression of background emission resulting from the maximized excitonic interaction.     

Using circularly polarized luminescence to probe exciton coherence in disordered helical aggregates

Circularly polarized emission from helical MOPV4 aggregates is studied theoretically based on a Hamiltonian including excitonic coupling, exciton phonon coupling, and site disorder. The latter is modeled via a Gaussian distribution of site energies. The frequency dependence of the circularly polarized luminescence dissymmetry g(lum)(omega) contains structural information about the low-energy-neutral (excitonic) polaron from which emission originates. Near the 0-0 emission frequency, g(lum)(omega) provides a measure of the exciton coherence length, while at lower energies, in the vicinity of the sideband frequencies, g(lum)(omega) probes the polaron radius. The present work focuses on how the 0-0 dissymmetry, g(lum)(0-0), relates to the emitting exciton's coherence function, from which the coherence length is deduced. In the strong disorder limit where the exciton is localized on a single chromophore, g(lum)(0-0) is zero. As disorder is reduced and the coherence function expands, /g(lum)(0-0)/ increases more rapidly than the sideband dissymmetries, resulting in a pronounced surge in g(lum)(omega) near the 0-0 transition frequency. The resulting spectral shape of g(lum)(omega) is in excellent agreement with recent experiments on MOPV4 aggregates. In the limit of very weak disorder, corresponding to the motional narrowing regime, the coherence function extends over the entire helix. In this region, g(lum)(0-0) undergoes a surprising sign reversal but only for helices which are between n+12 and n+1 complete turns (n = 0,1,...). This unusual sign change is due to the dependence of the rotational line strength on long-range exciton coherences which are also responsible for a heightened sensitivity of g(lum)(omega) to long-range excitonic coupling.     

The magnetic polaron modulated luminescence bands of organic-inorganic hybrid ferroelectric anti-perovskite (C3H9N)3Cd2Cl7 doped with Mn2+

Recently, the excellent optical properties of organic-inorganic hybrid metal halides have attracted much attention in the optoelectronic field. However, their complicated preparation processes seriously influence their properties and applications. In this work, we developed a series of organic-inorganic hybrid metal halides (C₃H₉N)₃Cd₂Cl₇:x%Mn²⁺ with an antiperovskite structure with ferroelectrics in an early report, giving tunable emissions contemporarily with different manganese (Mn)²⁺ concentrations via a simple mechanochemical method. Meanwhile, their single crystals were also grown by a slow thermal evaporation method. The as-grown products with Mn dopants exhibited diluted magnetic semiconductor behavior and varied emission profiles by different excitation wavelengths, which could be modified by the heat treatment. All the emission bands come from the different magnetic polarons with enhanced electron-phonon coupling or self-trapped exciton formation. Ferromagnetic coupling Mn–Mn pairs or clusters in the doped lattice favor the magnetic polaron and red emission at room temperature and even give much stronger emission above room temperature. The excitonic magnetic polaron and local excitonic magnetic polaron were detected at about 309 nm and 398 nm, respectively, with Mn doping. Without Mn²⁺ dopant, the weak emission band at about 398 nm can also be detected from an intrinsically bound exciton or confined exciton from the amine incorporated metal chlorides. This Mn-doped anti-perovskite Cd halides may find applications in the solid display and lighting, as well as the magneto-optical devices.     

Carbon nanotube-quenched fluorescent oligonucleotides: probes that fluoresce upon hybridization

We report an effective, novel self-assembled single-wall carbon nanotube (SWNT) complex with an oligonucleotide and demonstrate its feasibility in recognizing and detecting specific DNA sequences in a single step in a homogeneous solution. The key component of this complex is the hairpin-structured fluorescent oligonucleotide that allows the SWNT to function as both a "nanoscaffold" for the oligonucleotide and a "nanoquencher" of the fluorophore. Given this functionality, this carbon nanotube complex represents a new class of universal fluorescence quenchers that are substantially different from organic quenchers and should therefore have many applications in molecular engineering and biosensor development. Competitive binding of a DNA target and SWNTs with the oligonucleotide results in fluorescence signal increments relative to the fluorescence without a target as well as in marked fluorescence quenching. In contrast to the common loop-and-stem configuration of molecular beacons (MBs), this novel fluorescent oligonucleotide needs only one labeled fluorophore, yet the emission can be measured with little or no background interference. This property greatly improves the signal-to-background ratio compared with those for conventional MBs, while the DNA-binding specificity is still maintained by the MB. To test the interaction mechanisms of the fluorescent oligonucleotide with SWNTs and target DNA, thermodynamic analysis and fluorescence anisotropy measurements, respectively, were applied. Our results show that MB/SWNT probes can be an excellent platform for nucleic acid studies and molecular sensing.     

A Complex Comprising a Cyanine Dye Rotaxane and a Porphyrin Nanoring as a Model Light-Harvesting System

A nanoring-rotaxane supramolecular assembly with a Cy7 cyanine dye (hexamethylindotricarbocyanine) threaded along the axis of the nanoring was synthesized as a model for the energy transfer between the light-harvesting complex LH1 and the reaction center in purple bacteria photosynthesis. The complex displays efficient energy transfer from the central cyanine dye to the surrounding zinc porphyrin nanoring. We present a theoretical model that reproduces the absorption spectrum of the nanoring and quantifies the excitonic coupling between the nanoring and the central dye, thereby explaining the efficient energy transfer and demonstrating similarity with structurally related natural light-harvesting systems.     

A Complex Comprising a Cyanine Dye Rotaxane and a Porphyrin Nanoring as a Model Light‐Harvesting System

A nanoring‐rotaxane supramolecular assembly with a Cy7 cyanine dye (hexamethylindotricarbocyanine) threaded along the axis of the nanoring was synthesized as a model for the energy transfer between the light‐harvesting complex LH1 and the reaction center in purple bacteria photosynthesis. The complex displays efficient energy transfer from the central cyanine dye to the surrounding zinc porphyrin nanoring. We present a theoretical model that reproduces the absorption spectrum of the nanoring and quantifies the excitonic coupling between the nanoring and the central dye, thereby explaining the efficient energy transfer and demonstrating similarity with structurally related natural light‐harvesting systems.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

Simultaneous photoconjugation of methylene blue and cis-Rh(phen)2Cl2+ to DNA via a synergistic effect

Irradiation of the red-light absorbing dye, methylene blue (MB), in the presence of the metal complex, cis-Rh(phen)2Cl2+ (BISPHEN), leads to irreversible photobinding of both reagents to DNA. Evidence from absorption and emission spectroscopy indicates that the dye is strongly complexed to the DNA at the concentrations used in the experiments and that this complex is unaffected by the presence of BISPHEN. The level of covalent binding is proportional to the absorbed light dose, with the quantum efficiency for covalent binding of BISPHEN to the DNA with 633 nm light equal to 3.5 x 10(-4). Electrospray ionization mass spectrum of a mixture of DNA fragments created by enzymatic degradation of DNA isolated following irradiation indicates that purine adducts are formed with both BISPHEN and the dye. In addition, UV-Vis and high-performance liquid chromatography analyses of the irradiated MB/BISPHEN/DNA mixture and isolated adducts show extensive conversion of the dye and metal complex to the corresponding N-demethylated and aquated derivatives, respectively. Triplet quenchers for MB, for example oxygen and benzoquinone, inhibit both the photoconjugation and the photochemistry of BISPHEN. A mechanism for the synergistic interaction is proposed that involves photoconjugation of both partners to the DNA following oxidation and reduction via electron transfer between 1MB*/DNA and 3MB*/BISPHEN.     

DNA scaffold supports long-lived vibronic coherence in an indodicarbocyanine (Cy5) dimer

Vibronic coupling between pigment molecules is believed to prolong coherences in photosynthetic pigment–protein complexes. Reproducing long-lived coherences using vibronically coupled chromophores in synthetic DNA constructs presents a biomimetic route to efficient artificial light harvesting. Here, we present two-dimensional (2D) electronic spectra of one monomeric Cy5 construct and two dimeric Cy5 constructs (0 bp and 1 bp between dyes) on a DNA scaffold and perform beating frequency analysis to interpret observed coherences. Power spectra of quantum beating signals of the dimers reveal high frequency oscillations that correspond to coherences between vibronic exciton states. Beating frequency maps confirm that these oscillations, 1270 cm⁻¹ and 1545 cm⁻¹ for the 0-bp dimer and 1100 cm⁻¹ for the 1-bp dimer, are coherences between vibronic exciton states and that these coherences persist for ∼300 fs. Our observations are well described by a vibronic exciton model, which predicts the excitonic coupling strength in the dimers and the resulting molecular exciton states. The energy spacing between those states closely corresponds to the observed beat frequencies. MD simulations indicate that the dyes in our constructs lie largely internal to the DNA base stacking region, similar to the native design of biological light harvesting complexes. Observed coherences persist on the timescale of photosynthetic energy transfer yielding further parallels to observed biological coherences, establishing DNA as an attractive scaffold for synthetic light harvesting applications.

Dyes coupled to DNA display distance-dependent vibronic couplings that prolongs quantum coherences detected with 2D spectroscopy.     

Optical modulation and selective recovery of Cy5 fluorescence

Fluorescence modulation offers the opportunity to detect low-concentration fluorophore signals within high background. Applicable from the single-molecule to bulk levels, we demonstrate long-wavelength optical depopulation of dark states that otherwise limit Cy5 fluorescence intensity. By modulated excitation of a long-wavelength Cy5 transient absorption, we dynamically modulate Cy5 emission. The frequency dependence enables specification of the dark-state timescales enabling optical-demodulation-based signal recovery from high background. These dual-laser illumination schemes for high-sensitivity fluorescence-signal recovery easily improve signal-to-noise ratios by well over an order of magnitude, largely by discrimination against background. Previously limited to very specialized dyes, our utilization of long-lived dark states in Cy5 enables selective detection of this very common single-molecule and bulk fluorophore. Although, in principle, the "dark state" can arise from any photoinduced process, we demonstrate that cis-trans photoisomerization, with its unique transient absorption and lifetime enables this sensitivity boosting, long-wavelength modulation to occur in Cy5. Such studies underscore the need for transient absorption studies on common fluorophores to extend the impact of fluorescence modulation for high-sensitivity fluorescence imaging in a much wider array of applications.     

Linear absorbance of the pheophorbide-a butanediamine dendrimer P(4) in solution: computational studies using a mixed quantum classical methodology

The linear absorbance of a particular chromophore complex P(4) dissolved in ethanol is computed. P(4) is formed by a butanediamine dendrimer to which four pheophorbide-a molecules are covalently linked. The computations utilize a mixed quantum classical methodology and different approximations are compared. The electronic states of the P(4) chromophores which form Frenkel excitons in the excited states are treated quantum mechanically, whereas the intramolecular, intermolecular, as well as solvent coordinates are described classically. The computations use an improved exciton model, where the charge and transition densities of the chromophores are described by atomic partial charges, derived from a fit of the respective ab initio electrostatic potentials. Room temperature molecular dynamics simulations of all nuclear coordinates result in a time-dependent exciton model. It includes modulations of chromophore excitation energies due to charge density coupling between all chromophores as well as between the chromophores and solvent molecules, and, finally, modulations of the interchromophore excitonic couplings. The different approximations to the absorbance agree rather well. In particular, they confirm the reliability of adiabatic excitonic states which energies and oscillator strengths are altered by the overall temporal evolution of P(4) conformations. The fluctuations of solute-solvent interactions have a significantly larger effect on the absorbance broadening than the excitonic couplings but cannot completely explain the measured spectrum. The additional account for intrachromophore vibrations overcomes this discrepancy.     

DNA Encapsulation of Ten Silver Atoms Produces a Bright, Modulatable, Near Infrared-Emitting Cluster

Photostability, inherent fluorescence brightness, and optical modulation of fluorescence are key attributes distinguishing silver nanoclusters as fluorophores. DNA plays a central role both by protecting the clusters in aqueous environments and by directing their formation. Herein, we characterize a new near infrared-emitting cluster with excitation and emission maxima at 750 and 810 nm, respectively that is stabilized within C(3)AC(3)AC(3)TC(3)A. Following chromatographic resolution of the near infrared species, a stoichiometry of 10 Ag/oligonucleotide was determined. Combined with excellent photostability, the cluster's 30% fluorescence quantum yield and 180,000 M(-1)cm(-1) extinction coefficient give it a fluorescence brightness that significantly improves on that of the organic dye Cy7. Fluorescence correlation analysis shows an optically accessible dark state that can be directly depopulated with longer wavelength co-illumination. The coupled increase in total fluorescence demonstrates that enhanced sensitivity can be realized through Synchronously Amplified Fluorescence Image Recovery (SAFIRe), which further differentiates this new fluorophore.     

Synthesis of fluorescent probes based on stilbenes and diphenylacetylenes targeting β-amyloid plaques

Three fluorescent probes were synthesized aiming for optical imaging to detect amyloid plaques present in the patients with Alzheimer’s disease (AD). These compounds were prepared via Sonogashira coupling of a well-defined fluorophore (4-bora-3a,4a-diaza-s-indacene, BODIPY) with the pharmacophore possessing either a stilbene or a diphenylacetylene moiety. Different polyethylene glycol chain lengths were used as linkers between the fluorophore and the pharmacophore to adjust the lipophilicity of these probes. These compounds exhibit strong fluorescence emission between 665 and 680 nm and have very high extinction coefficients comparable to the parent fluorophore, BODIPY dye.     

Thiazole orange and Cy3: improvement of fluorescent DNA probes with use of short range electron transfer

Thiazole orange was synthetically incorporated into oligonucleotides by using the corresponding phosphoramidite as the building block for automated DNA synthesis. Due to the covalent fixation of the TO dye as a DNA base surrogate, the TO-modified oligonucleotides do not exhibit a significant increase of fluorescence upon hybridization with the counterstrand. However, if 5-nitroindole (NI) is present as a second artificial DNA base (two base pairs away from the TO dye) a fluorescence increase upon DNA hybridization can be observed. That suggests that a short-range photoinduced electron transfer causes the fluorescence quenching in the single strand. The latter result represents a concept that can be transferred to the commercially available Cy3 label. It enables the Cy3 fluorophore to display the DNA hybridization by a fluorescence increase that is normally not observed with this dye.     

Coherent Exciton-Phonon Coupling in CdSe/ZnS Nanocrystals Studied by Two-Dimensional Electronic Spectroscopy

Coherent exciton-phonon coupling in CdSe/ZnS nanocrystals have been investigated by temperature-dependent two-dimensional electronic spectroscopy (2DES) measurements. Benefiting from the ability of 2DES to dissect assembles in nanocrystal films, we have clearly identified experimental evidences of coherent coupling between exciton and phonon in CdSe/ZnS nanocrystals. In time domain, 2DES signals of excitonic transitions beat at a frequency resonant to a longitudinal optical phonon mode; in energy domain, phonon side bands are distinct at both Stokes and anti-Stokes sides. When temperature increases, phonon-induced exciton dephasing is observed with dramatic broadening of homogeneous linewidth. The results suggest exciton-phonon coupling is essential in elucidating the quantum dynamics of excitonic transitions in semiconductor nanocrystals.     

Resonant coupling of bound excitons with LO phonons in ZnO: excitonic polaron states and Fano interference

We report on a photoluminescence observation of robust excitonic polarons due to resonant coupling of exciton and longitudinal optical (LO) phonon as well as Fano-type interference in high quality ZnO crystal. At low enough temperatures, resonant coupling of excitons and LO phonons leads to not only traditional Stokes lines (SLs) but also up to second-order anti-Stokes lines (ASLs) besides the zero-phonon line (ZPL). The SLs and ASLs are found to be not mirror symmetric with respect to the ZPL, strongly suggesting that they are from different coupling states of exciton and phonons. Besides these spectral features showing the quasiparticle properties of exciton-phonon coupling system, the first-order SL is found to exhibit characteristic Fano lineshape, caused by quantum interference between the LO components of excitonic polarons and the continuous phonon bath. These findings lead to a new insight into fundamental effects of exciton-phonon interactions.     

A computational investigation on singlet and triplet exciton couplings in acene molecular crystals

Quantum chemical calculations (DFT, TDDFT and ZINDO/S) of singlet and triplet exciton couplings are presented and discussed for some acene derivatives (such as anthracene, tetracene, 9,10-di(phenyl)anthracene and 9,10-bis(phenylethynyl)anthracene). An accurate excited state single molecule characterization has been carried out followed by an analysis of the inter-molecular excitonic interactions, taking place in the crystalline phase. These have been correlated to exciton coupling terms obtaining guidelines for the choice of molecular materials with large exciton couplings. Such organic systems are likely to show multiexciton processes such as singlet fission (SF) and triplet-triplet annihilation (TTA) which are useful in energy conversion phenomena to be exploited in photonic and optoelectronic devices.     

Surface modified microarray chip and laser induced fluorescence microscopy to detect DNA cleavage

This work describes a quantitative method to detect DNA damage in the presence of Pb and Cd ions using a surface modified microarray chip and a laser induced fluorescence microscopy (LIFM). The detection was carried out by the immobilization of a single-stranded DNA oligomer, tagged with a Cy5 fluorophore on a polydimethylsiloxane (PDMS) microarray chip followed by LIFM. Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC) was attached as a cross-linker via the formation of covalent amide bonds. Then, the single-stranded DNA oligomer containing Cy5 as a fluorophore and thiol functional groups at both terminals, was bonded to the linker by reaction with sulfhydryl group. As the DNA oligomers were reacted with metal ions of Pb and Cd, the un-cleaved DNA oligomers were quantitatively identified by monitoring Cy5 fluorescence. Cadmium showed a quenching constant of 0.84 in the Stern–Volmer plot, whereas lead gave 0.22, indicating that cadmium ions suppress fluorescence more than lead ions. When optimized, fluorescence reductions of 23% (± 2.1) for Pb and 25% (± 1.4) for Cd were observed in air and decreased to almost < 5.0% in a radical scavenger of 5 mM. The cleaved DNA was also confirmed by MALDI-TOF-MS. In result, this experimental method using a microarray chip with surface modification provided quantitative determination of DNA oligomer damage with reproducible results, significantly reduced sample volumes and analysis times.     

Spectroscopic and photophysical properties of dUTP and internally DNA bound fluorophores for optimized signal detection in biological formats

Efficient signal generation in DNA-based assays requires understanding of the influence of fluorophore's interactions on the spectroscopic properties. The resulting changes in fluorescence intensity, quantum yield, emission anisotropy, and fluorescence lifetime provide straightforward tools for the study of molecular dynamics and interaction between labels and nucleic acids. Searching for bright fluorescent reporters for rolling circle amplification (RCA) as efficient signal enhancement strategy for biological formats, we investigated the spectroscopic properties of seven dyes: cyanines, rhodamines, and BODIPYs. They spectrally resemble Cy3, the most frequently used fluorophore in biodetection formats, and are measured in six samples (free dye, dye-dUTP, internally labeled ssDNA and dsDNA-single- and triple-labeled) using steady-state and time-resolved fluorometry. Special emphasis was dedicated to characterizing the nature of the interaction of these fluorophores differing in dye class, charge, and rigidity. Our results suggest dye charge and structure as main factors governing the dye's interactions, with DY-555 and Cy3B presenting the best candidates for our envisaged signal amplification strategy. This label comparison underlines the importance of a proper understanding of structure-property relations and dye-biomolecule interactions for reporter choice and presents a road map towards the design and interpretation of experiments using these labels on DNA of known sequence.     

Metal-enhanced fluorescence of nano-core–shell structure used for sensitive detection of prion protein with a dual-aptamer strategy

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore–metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core–shell Ag@SiO₂, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05–0.30 nM, which is successfully applied to the detection of PrP in mice brain homogenates.