Single-molecule spectroscopic study of dynamic nanoscale DNA bending behavior of HIV-1 nucleocapsid protein.

We have studied the conformational dynamics associated with the nanoscale DNA bending induced by human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein using single-molecule Förster resonance energy transfer (SM-FRET). To gain molecular-level insights into how the HIV-1 NC locally distorts the structures of duplexed DNA segments, the dynamics, reversibility, and sequence specificity of the DNA bending behavior of NC have been systematically studied. We have performed SM-FRET measurements on a series of duplexed DNA segments with varying sequences, lengths, and local structures in the presence of the wide-type HIV-1 NC and NC mutants lacking either the basic N-terminal domain or the zinc fingers. On the basis of the SM-FRET results, we have proposed a possible mechanism for the NC-induced DNA bending in which both NC's zinc fingers and N-terminal domain are found to play crucial roles. The SM-FRET results reported here add new mechanistic insights into the biological behaviors and functions of HIV-1 NC as a retroviral DNA-architectural protein which may play critical roles in the compaction, nuclear import, and integration of the proviral DNA during the retroviral life cycle.

Mimicking conjugated polymer thin-film photophysics with a well-defined triblock copolymer in solution.

Conjugated polymers (CPs) are promising materials for use in electronic applications, such as low-cost, easily processed organic photovoltaic (OPV) devices. Improving OPV efficiencies is hindered by a lack of a fundamental understanding of the photophysics in CP-based thin films that is complicated by their heterogeneous nanoscale morphologies. Here, we report on a poly(3-hexylthiophene)-block-poly(tert-butyl acrylate)-block-poly(3-hexylthiophene) rod-coil-rod triblock copolymer. In good solvents, this polymer resembles solutions of P3HT; however, upon the addition of a poor solvent, the two P3HT chains within the triblock copolymer collapse, affording a material with electronic spectra identical to those of a thin film of P3HT. Using this new system as a model for thin films of P3HT, we can attribute the low fluorescence quantum yield of films to the presence of a charge-transfer state, providing fundamental insights into the condensed phase photophysics that will help to guide the development of the next generation of materials for OPVs.

Conformation and energy transfer in single conjugated polymers.

In contrast to the detailed understanding of inorganic materials, researchers lack a comprehensive view of how the properties of bulk organic materials arise from their individual components. For conjugated polymers to eventually serve as low cost semiconductor layers in electronic devices, researchers need to better understand their functionality. For organics, traditional materials science measurements tend to destroy the species of interest, especially at low concentrations. However, fluorescence continues to be a remarkably flexible, relatively noninvasive tool for probing the properties of individual molecules and allows researchers to carry out a broad range of experiments based on a relatively simple concept. In addition, the sensitivity of single-molecule spectroscopy allows researchers to see the properties of an individual component that would be masked in the bulk phase. In this Account, we examine several photophysical properties of different conjugated polymers using single-molecule spectroscopy. In these experiments, we probed the relationship between the conformation of single conjugated polymer chains and the distance scale and efficiency of energy transfer within the polymer. Recent studies used polarization anisotropy measurements on single polymer chains to study chain folding following spin-casting from solution. This Account summarizes the effects of monomer regioregularity and backbone rigidity, by comparing a regiorandom phenylene vinylene (MEH-PPV) with both a regiorandom and regioregular thiophene (P3HT). Synthesis of novel polymers allowed us to explore the role of different conformation-directing inclusions in a PPV backbone. We showed that these inclusions control the conformation of individual chains and that molecular dynamics can predict these structural effects. In situ solvent vapor annealing studies explored the dynamics of polymer chains as well as the effect of solvent evaporation on the structural equilibrium of the polymer. We observed that a slower rate of solvent evaporation results in a narrow population of highly ordered polymer chains. These highly ordered single chains serve as a model system to probe the effect of conformation on energy transfer following excitation in single MEH-PPV polymer chains in two distinct experiments. In the first, we correlated the anisotropy of the fluorescence emission of individual chains with the anisotropy of their fluorescence excitation. Using this data, we derived a model for energy transfer in a conjugated polymer, simulating chromophores along a chain, coupled via Förster energy transfer. In the second experiment, super-resolution measurements demonstrated the ability of single-molecule spectroscopy to directly visualize energy transfer along a polymer chain embedded in a model device environment. A capacitive device allowed for controlled localization of hole polarons onto the polymer chain. These positive charges subsequently quenched local excitations, providing insight into the range of energy transfer in these single polymer molecules. As researchers continue to characterize conjugated polymer films and develop methods for creating multichain systems, single-molecule techniques will provide a greater understanding of how polymer morphology influences interchain interactions and will lead to a richer description of the electronic properties of bulk conjugated polymer films.

Conformational effect on energy transfer in single polythiophene chains.

Herein we describe the use of regioregular (rr-) and regiorandom (rra-) P3HT as models to study energy transfer in ordered and disordered single conjugated polymer chains. Single molecule fluorescence spectra and excitation/emission polarization measurements were compared with a Förster resonance energy transfer (FRET) model simulation. An increase in the mean single chain polarization anisotropy from excitation to emission was observed for both rr- and rra-P3HT. The peak emission wavelengths of rr-P3HT were at substantially lower energies than those of rra-P3HT. A simulation based on FRET in single polymer chain conformations successfully reproduced the experimental observations. These studies showed that ordered conformations facilitated efficient energy transfer to a small number of low-energy sites compared to disordered conformations. As a result, the histograms of spectral peak wavelengths for ordered conformations were centered at much lower energies than those obtained for disordered conformations. Collectively, these experimental and simulated results provide the basis for quantitatively describing energy transfer in an important class of conjugated polymers commonly used in a variety of organic electronics applications.

Unmasking bulk exciton traps and interchain electronic interactions with single conjugated polymer aggregates.

For conjugated polymer materials, there is currently a major gap in understanding between the fundamental properties observed in single molecule measurements and the bulk electronic properties extracted from measurements of highly heterogeneous thin films. New materials and methodologies are needed to follow the evolution from single chain to bulk film properties as multiple chains begin to interact. In this work, we used a controlled solvent vapor annealing process to assemble single chains of phenylene-vinylene conjugated polymers into aggregates that can be individually spectroscopically interrogated. This approach allowed us to probe the effects of interchain coupling in isolated conjugated polymer nanodomains of controlled size. By assembling these aggregates from building blocks of both pristine MEH-PPV and MEH-PPV derivatives containing structure-directing ortho- or para-terphenyl inclusions, we were able to control the ordering of these nanodomains as measured by single aggregate polarization anisotropy measurments. Depending on the individual chain constituents, these aggregates varied from highly anisotropic to nearly isotropic, respectively facilitating or inhibiting interchain coupling. From the single chain fluorescence lifetimes, we demonstrated that these structure directing inclusions effectively break the phenylene-vinylene conjugation, allowing us to differentiate interchain electronic effects from those due to hyper-extended conjugation. We observed well-defined bathochromic shifts in the fluorescence spectra of the aggregates containing extensive interchain interactions, indicating that low-energy exciton traps in MEH-PPV are the result of coupling interactions between neighboring chain segments. These results demonstrate the power of the synthetic inclusion approach to control properties at not just the single chain level, but as a comprehensive approach toward ground-up design of bulk electronic properties.

Self-assembly of highly ordered conjugated polymer aggregates with long-range energy transfer.

Applications of conjugated polymers (CP) in organic electronic devices such as light-emitting diodes and solar cells depend critically on the nature of electronic energy transport in these materials. Single-molecule spectroscopy has revealed their fundamental properties with molecular detail, and recent reports suggest that energy transport in single CP chains can extend over extraordinarily long distances of up to 75 nm. An important question arises as to whether these characteristics are sustained when CP chains agglomerate into a neat solid. Here, we demonstrate that the electronic energy transport in aggregates composed of tens of polymer chains takes place on a similar distance scale as that in single chains. A recently developed molecular-level understanding of solvent vapour annealing has allowed us to develop a technique to control the CP agglomeration process. Aggregates with volumes of at least 45,000 nm(3) (molecular weight ≈ 21 MDa) maintain a highly ordered morphology and show pronounced fluorescence blinking behaviour, indicative of substantially long-range energy transport. Our findings provide a new lens through which the ordering of single CP chains and the evolution of their morphological and optoelectronic properties can be observed, which will ultimately enable the rational design of improved CP-based devices.

Electronic energy transfer in highly aligned MEH-PPV single chains.

This paper describes the simultaneous measurement of excitation and emission anisotropy to visualize energy transfer in single chains of the prototypical conjugated polymer MEH-PPV, for samples with >70% of the single chains organized into extended, rod-like conformations. The uniformity and high degree of order of the single molecules in these experiments has allowed direct comparison of our experimental data to energy-transfer simulations in model polymer chains. Increases in average anisotropy from 0.62 to 0.74 from excitation to emission and average changes of <15° to the in-plane dipole principal orientation axis confirmed that energy was transferred to a relatively small number of sites in these highly ordered chains. This organization persisted even at large molecular weights (M(n) = 850 kDa). Electronic energy transfer in highly anisotropic model chains was simulated using an incoherent Förster-type mechanism to generate modulation depth histograms in good agreement with the observed data, as well as ensemble emission energies consistent with previously reported results. In these ordered model chains, excitons migrated an average of 6 nm before emission. This distance, far larger than the radius for single-step FRET, implies that energy transfer in MEH-PPV is a multistep funneling process.

Electrogenerated chemiluminescence of conjugated polymer films from patterned electrodes.

A new phenomenon is presented in which electrogenerated chemiluminescence (ECL) is generated and propagates laterally as self-reinforcing waves as a result of the oxidation of a poly(9,9-dioctylfluorene-co-benzothiadiazole) thin film. In an ordered array of Au electrode posts that act as effective ECL nucleation sites, soliton-like waves were observed to expand from each site and annihilate upon collision with each other. Simulations of the ECL response supported the experimental observations that the ECL waves propagate at a constant speed. A correlated diffusion mechanism involving the correlated motion of ions, injected holes, and solvent molecules is proposed to interpret the experimental data qualitatively. A rapid increase in the diffusion coefficient of these species in the polymer results in a sharp interface between non-oxidized and oxidized polymer phases wherein the electrochemical (EC) oxidation and mass transport of all pertinent species take place. EC oxidation of conjugated polymers of this type has important implications for the understanding of these materials and their modes of operation in EC conjugated polymer devices.

Hole localization in molecular crystals from hybrid density functional theory.

We use first-principles computational methods to examine hole trapping in organic molecular crystals. We present a computational scheme based on the tuning of the fraction of exact exchange in hybrid density functional theory to eliminate the many-electron self-interaction error. With small organic molecules, we show that this scheme gives accurate descriptions of ionization and dimer dissociation. We demonstrate that the excess hole in perfect molecular crystals forms self-trapped molecular polarons. The predicted absolute ionization potentials of both localized and delocalized holes are consistent with experimental values.

Controlling chain conformation in conjugated polymers using defect inclusion strategies.

The Horner method was used to synthesize random copolymers of poly(2-methoxy-5-(2'-ethylhexyloxy)-p-phenylene vinylene) (MEH-PPV) that incorporated different backbone-directing monomers. Single-molecule polarization absorption studies of these new polymers demonstrate that defects that preserve the linear backbone of PPV-type polymers assume the highly anisotropic configurations found in defect-free MEH-PPV. Rigid defects that are bent lower the anisotropy of the single chain, and saturated defects that provide rotational freedom for the chain backbone allow for a wide variety of possible configurations. Molecular dynamics simulations of model defect PPV oligomers in solution demonstrate that defect-free and linearly defected oligomers remain extended while the bent and saturated defects tend toward more folded, compact structures.

Electrogenerated chemiluminescence of pure polymer films and polymer blends.

We recently reported the discovery of soliton-like electrogenerated chemiluminescence (ECL) waves from pure conjugated polymer films and gold-nanoparticles-doped polymer films. In addition to a more detailed study of these polymer systems by changing the film thickness and the distribution of local leaks, we also apply the ECL wave phenomenon to polymer blends of conjugated and nonconjugated polymers. Poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT) is used as the active material that is oxidized and produces ECL with the presence of a co-reactant, tri-n-propylamine (TPA). Several factors such as film thickness, artificial leaks, and solubility of doped polymers are examined for their effects on the ECL behavior. When polystyrene (PS), less soluble in the electrolyte, is blended with F8BT, dotted ECL signals are observed and transported as waves. When poly(methyl methacrylate) (PMMA), more soluble in the electrolyte, is blended with F8BT, PMMA serves as local scratches and ECL waves are triggered simultaneously from the whole film.

Ultralong-range polaron-induced quenching of excitons in isolated conjugated polymers.

In conjugated polymers, radiative recombination of excitons (electron-hole pairs) competes with nonradiative thermal relaxation pathways. We visualized exciton quenching induced by hole polarons in single-polymer chains in a device geometry. The distance-scale for quenching was measured by means of a new subdiffraction, single-molecule technique--bias-modulated intensity centroid spectroscopy--which allowed the extraction of a mean centroid shift of 14 nanometers for highly ordered, single-polymer nanodomains. This shift requires energy transfer over distances an order of magnitude greater than previously reported for bulk conjugated polymers and far greater than predicted by the standard mechanism for exciton quenching, the unbiased diffusion of free excitons to quenching sites. Instead, multistep "energy funneling" to trapped, localized polarons is the probable mechanism for polaron-induced exciton quenching.

Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1.

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.

HIV-1 nucleocapsid protein bends double-stranded nucleic acids.

The human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein is believed to be unique among the nucleic acid (NA) binding proteins encoded by this retrovirus in being highly multifunctional and relatively nonsequence-specific. Underlying many of NC's putative functions, including for example its chaperon-like activity for various steps of HIV-1 reverse transcription, is NC's ability to partially melt short double-stranded regions of structured NAs, which is essentially a consequence of NC's general binding preference for single-stranded bases. Herein we report a different, previously undiscovered, mode of NC/NA interaction, i.e., NC-induced sharp bending of short segments of fully duplexed DNA/DNA and DNA/RNA. We use single-molecule fluorescence resonance energy transfer (SM-FRET) in vitro to probe NC-induced NA bending and associated heterogeneous conformational dynamics for model NC/NA complexes. NC-induced NA bending may have important biological roles in the previously reported NC-mediated condensation of duplex proviral DNA in the HIV-1 life cycle.

Electrogenerated chemiluminescence of soliton waves in conjugated polymers.

We report the discovery of soliton-like electrogenerated chemiluminescence (ECL) waves in the electrochemical oxidation of thin films (15-250 nm) of the semiconducting conjugated polymer poly(9,9-dioctylfluorene-co-benzothiadiazole) (F8BT). The ECL "wave fronts" were visualized and imaged in space and time by optical emission microscopy and observed to freely propagate parallel to the plane of the electrode. The data reveal that the EC solitons are triggered by the flux of electrolytes into the polymer film at nanoscale "leaks" (or defects) and propagate by a highly nonlinear, positive-feedback kinetic mechanism involving EC oxidation, mass transport of electrolyte anions, and oxidation-induced swelling, all taking place within the conjugated polymer film. Several model systems with spatially distributed reaction "triggers" were studied to elucidate the mechanistic details of this complex oxidation process in polymer films.

Detailed single-molecule spectroelectrochemical studies of the oxidation of conjugated polymers.

Single-particle fluorescence spectroelectrochemistry was used to investigate the electrochemical oxidation of isolated, immobilized particles of the conjugated polymers BEH-PPV and MEH-PPV at an indium tin oxide (ITO) electrode immersed in an electrolyte solution. Two types of particles were investigated: (i) polymer single molecules (SM) and (ii) nanoparticle (NP) aggregates of multiple polymer single molecules. For the BEH-PPV polymer, the observation of nearly identical lowest oxidation potentials for different SM in the ensemble is evidence for effective electrostatic screening by the surrounding electrolyte solution. A combination of Monte Carlo simulations and application of Poisson-Boltzmann solvers were used to model the charging of polymer single molecules and nanoparticles in the electrochemical environment. The results indicate that the penetration of electrolyte anions into the polymer nanoparticles is necessary to produce the observed narrow fluorescence quenching vs oxidation potential curves. Finally, fluorescence-lifetime single-molecule spectroelectrochemical (SMS-EC) data revealed that at low potential an excited state reduction process (i.e., electron transfer from ITO to the polymer) is probably the dominant fluorescence quenching process.

Factors controlling hole injection in single conjugated polymer molecules.

New insights on the molecular level details of the recently reported light-assisted injection of positive charge into single conjugated polymer chains are reported. Extensive new fluorescence-voltage single molecule spectroscopy (FV-SMS) measurements were performed on single chains of the archetypical conjugated polymer MEH-PPV embedded in a capacitor device to complement previous studies of the influence of the bias scan rate and optical excitation intensity. The use of a vacuum microscope allowed for the precise control of the device atmosphere, demonstrating the influence of triplet states in the MEH-PPV on the FV-SMS modulation. For identical device conditions, little variation was observed in the rate and yield of charging from molecule to molecule. Through the use of thicker supporting matrices and insulating polymer "blocking layers", it was determined that good electrical contact between the hole transport layers and the single molecules was necessary for charge injection. The results demonstrate the complexity of charge transfer processes at the interface of organic semiconductors and highlight the ability of single molecule methods to advance the understanding of such processes at the nanoscale.

Light-assisted deep-trapping of holes in conjugated polymers.

The injection of positive charge carriers (holes) into a single conjugated polymer chain was observed to be light-assisted. This effect may underlie critical, poorly understood organic electronic device phenomena such as the build-up of functional deeply trapped charge layers in polymer light emitting diodes. The charging/discharging dynamics were investigated indirectly by a variety of single molecule electro-optical spectroscopic techniques, including an "image-capture" approach.

Synthesis of CulnS2, CulnSe2, and Cu(InxGa(1-x))Se2 (CIGS) nanocrystal "inks" for printable photovoltaics.

Chalcopyrite copper indium sulfide (CuInS2) and copper indium gallium selenide (Cu(InxGa(1-x))-Se2; CIGS) nanocrystals ranging from approximately 5 to approximately 25 nm in diameter were synthesized by arrested precipitation in solution. The In/Ga ratio in the CIGS nanocrystals could be controlled by varying the In/Ga reactant ratio in the reaction, and the optical properties of the CulnS2 and CIGS nanocrystals correspond to those of the respective bulk materials. Using methods developed to produce uniform, crack-free micrometer-thick films, CulnSe2 nanocrystals were tested in prototype photovoltaic devices. As a proof-of-concept, the nanocrystal-based devices exhibited a reproducible photovoltaic response.