Single Dot Spectroscopy of Two-Color Quantum Dot/Quantum Shell Nanostructures

Single dot spectroscopy is performed on two-color CdSe/ZnS/CdSe core/barrier/shell nanostructures. Unlike quantum dots cores, these systems have two phases with which to emit and ultimately examine for blinking analysis. These particles are brighter than conventional quantum dots and also show the photoluminescence (PL) intensity and energy fluctuations characteristic of quantum dots. Single dot spectral diffusion analysis yields no measureable energy shift correlation between the core and the shell on the 200 ms time scale. In contrast, the single dot PL from the CdSe shell has narrower linewidths than the CdSe core, indicating differences in its spectral diffusion on shorter timescales

The Role of Charge in the Surfactant-Assisted Stabilization of the Natural Product Curcumin

Colloidal solutions of surfactants that form micelles or vesicles are useful for solubilizing and stabilizing hydrophobic molecules that are otherwise sparingly soluble in aqueous solutions. In this paper we investigate the use of micelles and vesicles prepared from ionic surfactants for solubilizing and stabilizing curcumin, a medicinal natural product that undergoes alkaline hydrolysis in water. We identify spectroscopic signatures to evaluate curcumin partitioning and deprotonation in surfactant mixtures containing micelles or vesicles. These spectroscopic signatures allow us to monitor the interaction of curcumin with charged surfactants over a wide range of pH values. Titration data are presented to show the pH dependence of curcumin interactions with negatively and positively charged micelles and vesicles. In solutions of cationic micelles or positively charged vesicles, strong interaction between the Cur−1 phenoxide ion and the positively charged surfactants results in a change in the acidity of the phenolic hydrogen and a lowering of the apparent lowest pKa value for curcumin. In the microenvironments formed by anionic micelles or negatively charged bilayers, our data indicates that curcumin partitions as the Cur0 species, which is stabilized by interactions with the respective surfactant aggregates, and this leads to an increase in the apparent pKa values. Our results may explain some of the discrepancies within the literature with respect to reported pKa values and the acidity of the enolic versus phenolic protons. Hydrolysis rates, quantum yields, and molar absorption coefficients are reported for curcumin in a variety of solutions

Wavelength-Resolved Studies of Förster Energy Transfer in Azobenzene-Modified Conjugated Polymers: The Competing Roles of Exciton Migration and Spectral Resonance

We report results from wavelength-resolved fluorescence lifetime measurements of azobenzene-modified poly(p-phenylenevinylene) (PPV). The introduction of an azobenzene side chain enables reversible phototriggered modulation of the PPV emission. Intensity modulation is possible because Förster-type energy transfer from the PPV backbone to the azobenzene side-chain acceptors is more efficient for the cis-azobenzene isomer than for the trans. Here we explore how side-chain quenching competes with intrinsic PPV exciton dynamics. By probing the red and blue edges of the PPV emission, we evaluate the photophysical effects of the side chain on different exciton populations. Prior to UV exposure, when the azobenzene side chains are trans, three exciton subpopulations are detected:  (i) rapidly diffusing and unquenched, (ii) nondiffusing and quenched, and (iii) low-energy, nondiffusing and unquenched. Exciton diffusion on the far blue edge is extremely efficient and mitigates the effects of side-chain quenching, giving rise to the first population. The second population is due to spectral resonance between side chains and nondiffusing excitons. The third population consists of excitons residing on the longest PPV chain segments. After UV-induced trans → cis photoisomerization, we observe a greater degree of quenching on the red edge as spectral overlap improves and the lowest-energy chromophores become efficiently quenched. Hence, cis-azobenzene quenching of (iii) contributes greatly to the efficient photomodulation of these PPV derivatives. In addition, rapid exciton migration on the blue edge may help to improve modulation efficiencies by effectively competing with energy transfer to trans-azobenzene. These findings provide improved understanding of the underlying mechanisms of energy transfer to side chains in this important class of polymers

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

Magnetic Iron Oxide Nanoparticles for Biorecognition: Evaluation of Surface Coverage and Activity

Modifying the surfaces of magnetic nanoparticles (MNPs) by the covalent attachment of biomolecules will enable their implementation as contrast agents for magnetic resonance imaging or as media for magnetically assisted bioseparations. In this paper we report both the surface coverage and the activity of IgG antibodies on MNPs. The antibodies were immobilized on γ-Fe2O3 nanoparticles by conventional methods using aminopropyltriethoxy silane and subsequent activation by glutaraldehyde. Novel fluorescence methods were used to provide a quantitative evaluation of this well-known approach. Our results show that surface coverage can be stoichiometrically adjusted with saturated surface coverage occurring at ∼36% of the theoretical limit. The saturated surface coverage corresponds to 34 antibody molecules bound to an average-sized MNP (32 nm diameter). We also show that the immobilized antibodies retain ∼50% of their binding capacity at surface-saturated levels

LacI-DNA-IPTG Loops: Equilibria among Conformations by Single-Molecule FRET

The <i>E. coli</i> Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the <i>lac</i> operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Our previous work on the conformations of LacI loops used a hyperstable model DNA construct, 9C14, that contains a sequence directed bend flanked by operators. Single-molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability, and the current work assesses loop population distributions for LacI-9C14 complexes containing the synthetic inducer IPTG. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. LacI titration experiments and FRET fluctuation analysis suggest that the addition of IPTG induces loop conformational dynamics and re-equilibration between loop population distributions that include a mixture of looped states that do not exhibit high-efficiency FRET. The results show that repression by looping even at saturating IPTG should be considered in models for regulation of the operon. We propose that persistent DNA loops near the operator function biologically to accelerate rerepression upon exhaustion of inducer

Toward Efficient Photomodulation of Conjugated Polymer Emission: Optimizing Differential Energy Transfer in Azobenzene-Substituted PPV Derivatives

We present fluorescence studies of quenching behavior in photoaddressable azobenzene-substituted derivatives of the fluorescent conjugated polymer poly(p-phenylenevinylene) (PPV). The azobenzene side chains partially quench the PPV fluorescence, and we have shown previously that the quenching efficiency is greater when the azobenzene side chains are cis than when they are trans. This effect provides a photoaddressable means of modulating the fluorescence intensity of PPV derivatives. To optimize the efficiency of photoinduced intensity modulation, it is important to understand the molecular nature of quenching by both trans- and cis-azobenzene side chains. Here we investigate the photophysical origins of quenching by the two isomers using steady-state and time-resolved fluorescence spectroscopy. We present results from the azobenzene-modified PPV derivative poly(2-methoxy-5-((10-(4-(phenylazo)phenoxy)decyl)oxy)-1,4-phenylenevinylene) (MPA-10-PPV) and two new related polymers, a copolymer lacking half of the azobenzene side chains and an analogue of MPA-10-PPV with a tert-butyl-substituted azobenzene. These studies reveal that steric interactions influence the extent of PPV emission quenching by trans-azobenzene but do not affect the efficient quenching by cis-azobenzene. The difference in dynamic quenching efficiencies between trans- and cis-azobenzene isomers is consistent with fluorescence resonance energy transfer. These results show that it is possible to control the efficiency of photoswitchable fluorescence modulation through specific structural variations designed to encourage or block quenching by trans-azobenzene. This is a promising approach to providing useful general guidelines for designing photomodulated PPV derivatives

Highly Efficient Capture and Long-Term Encapsulation of Dye by Catanionic Surfactant Vesicles

Vesicles formed from the cationic surfactant, cetyltrimethylammonium tosylate (CTAT) and the anionic surfactant, sodium dodecylbenzenesulfonate (SDBS), were used to sequester the anionic dye carboxyfluorescein. Carboxyfluorescein was efficiently sequestered in CTAT-rich vesicles via two mechanisms:  encapsulation in the inner water pool and electrostatic adsorption to the charged bilayer. The apparent encapsulation efficiency (22%) includes both encapsulated and adsorbed fractions. Entrapment of carboxyfluorescein by SDBS-rich vesicles was not observed. Results show the permeability of the catanionic membrane is an order of magnitude lower than that of phosphatidylcholine vesicles and the loading capacity is more than 10 times greater

Carbohydrate Modified Catanionic Vesicles: Probing Multivalent Binding at the Bilayer Interface

This article reports on the synthesis, characterization, and binding studies of surface-functionalized, negatively charged catanionic vesicles. These studies demonstrate that the distribution of glycoconjugates in the membrane leaflet can be controlled by small alterations of the chemical structure of the conjugate. The ability to control the glycoconjugate concentration in the membrane provides a method to explore the relationship between ligand separation distance and multivalent lectin binding at the bilayer interface. The binding results using the O-linked glucosyl conjugate were consistent with a simple model in which binding kinetics are governed by the density of noninteracting glucose ligands, whereas the N-linked glycoconjugate exhibited binding kinetics consistent with interacting or clustering conjugates. From the noninteracting ligand model, an effective binding site separation of the sugar sites for concanavalin A of 3.6−4.3 nm was determined and a critical ligand density above which binding kinetics are zeroth order with respect to the amount of glycoconjugate present at the bilayer was observed. We also report cryo-transmission electron microscopy (cryo-TEM) images of conjugated vesicles showing morphological changes (multilayering) upon aggregation of unilamellar vesicles with concanavalin A

A Reactive Peptidic Linker for Self-Assembling Hybrid Quantum Dot−DNA Bioconjugates

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe−ZnS core−shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD−DNA−dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle−biomolecule structures