Pedro M. R. Paulo

Optical detection of single non-absorbing molecules using the surface plasmon resonance of a gold nanorod

Existing methods for the optical detection of single molecules require the molecules to absorb light to produce fluorescence or direct absorption signals. This limits the range of species that can be detected, because most molecules are purely refractive. Metal nanoparticles or dielectric resonators can be used to detect non-absorbing molecules because local changes in the refractive index produce a resonance shift. However, current approaches only detect single molecules when the resonance shift is amplified by a highly polarizable label or by a localized precipitation reaction on the surface of a nanoparticle. Without such amplification, single-molecule events can only be identified in a statistical way. Here, we report the plasmonic detection of single molecules in real time without the need for labelling or amplification. Our sensor consists of a single gold nanorod coated with biotin receptors, and the binding of single proteins is detected by monitoring the plasmon resonance of the nanorod with a sensitive photothermal assay. The sensitivity of our device is ∼700 times higher than state-of-the-art plasmon sensors and is intrinsically limited by spectral diffusion of the surface plasmon resonance.

Resonant plasmonic enhancement of single-molecule fluorescence by individual gold nanorods

Enhancing the fluorescence of a weak emitter is important to further extend the reach of single-molecule fluorescence imaging to many unexplored systems. Here we study fluorescence enhancement by isolated gold nanorods and explore the role of the surface plasmon resonance (SPR) on the observed enhancements. Gold nanorods can be cheaply synthesized in large volumes, yet we find similar fluorescence enhancements as literature reports on lithographically fabricated nanoparticle assemblies. The fluorescence of a weak emitter, crystal violet, can be enhanced more than 1000-fold by a single nanorod with its SPR at 629 nm excited at 633 nm. This strong enhancement results from both an excitation rate enhancement of ∼130 and an effective emission enhancement of ∼9. The fluorescence enhancement, however, decreases sharply when the SPR wavelength moves away from the excitation laser wavelength or when the SPR has only a partial overlap with the emission spectrum of the fluorophore. The reported measurements of fluorescence enhancement by 11 nanorods with varying SPR wavelengths are consistent with numerical simulations.

Chemical Interface Damping in Single Gold Nanorods and Its Near Elimination by Tip‐Specific Functionalization

Tip-functionalized nanorods: Single-particle spectroscopy shows that functionalization of small gold nanorods with thiol groups leads to a broadening of the plasmon resonance by chemical interface damping. By specifically functionalizing the tips of the nanorod (see picture) this broadening is nearly eliminated while the sensing performance is maintained relative to fully functionalized particles.

Non-covalent dendrimer–porphyrin interactions: the intermediacy of H-aggregates?

The interaction of anionic meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) with poly(amidoamine) (PAMAM) dendrimers of generations 2 and 4 in aqueous solution was investigated with steady-state absorption and fluorescence techniques. At low dendrimer concentrations, the formation of low absorptive/emissive species occurs in these systems. With an increase of dendrimer concentration, the porphyrins rearrange at the dendritic outer shell and the absorption and emission spectra suggest the presence of H-aggregates of TSPP. These aggregates dissociate to yield monomeric complexed species in the limit of high dendrimer concentration, which presents very similar spectra for both generations studied. On the other hand, the emission of the same systems at pH 2 shows significant differences between dendrimer generations. While for generation 2 the fluorescence spectra practically coincide with that of the diacid TSPP and its J-aggregate, for generation 4 the spectra obtained are similar to that at high pH. This was interpreted according to a morphologic transition in PAMAM dendrimers around generation 3, to give a more compact structure, which provides a hydrophobic environment for the associated TSPP. At low pH, an increase in J-aggregation is observed in the dendrimers presence. Aging effects were observed, in particular for the systems where different aggregated forms of TSPP coexist, showing that for intermediate dendrimer concentrations these are thermodynamically labile systems.

Reorganization of self-assembled dipeptide porphyrin J-aggregates in water-ethanol mixtures.

The self-assembly of a neutral meso-methoxyphenylporphyrin functionalized with a dipeptide glycilglycine substituent (MGG) in water and in water-ethanol mixtures was studied by absorption and fluorescence spectroscopy. In water, hydrophobic interactions and the noncovalent intermolecular hydrogen bonding between the terminal carboxylate group of one porphyrin and the hydrogen atoms of the pyrrolic nitrogens of another porphyrin originate nonspecific disorganized H- and J-aggregates. The addition of ethanol (0.1-25% v/v) to the water creates small clusters within which porphyrin J-aggregates reorganize as revealed by a narrow intense band detected by the Rayleigh light scattering (RLS) at 443 nm. Similar phenomenology is detected in SDS premicellar aggregates. Computational DFT calculations of a model dimer formation stabilized via intermolecular hydrogen bonding estimate an energy gain of -22 kJ mol(-1) and a center-to-center and interplane distances between porphyrin moieties of 16.8 and 3.7 Å, respectively. The kinetics of the J-aggregate formation could be fitted with a time-dependent model, and an activation energy of 96 kJ mol(-1) was estimated. The aggregates morphology of MGG was followed by transmission electron microscopy (TEM) which showed rod-type structures of 5-8 μm evolving to spherical particles with increased ethanol content. Similar images and sizes were obtained in analogous samples using fluorescence lifetime imaging microscopy (FLIM) and dynamic light scattering (DLS). The formation of excitonically coupled supramolecular MGG structures of brickwork or staircase types is proposed in these water-ethanol mixtures.

Molecular Dynamics Simulations of Porphyrin−Dendrimer Systems: Toward Modeling Electron Transfer in Solution

We have performed computational simulations of porphyrin-dendrimer systems--a cationic porphyrin electrostatically associated to a negatively charged dendrimer--using the method of classical molecular dynamics (MD) with an atomistic force field. Previous experimental studies have shown a strong quenching effect of the porphyrin fluorescence that was assigned to electron transfer (ET) from the dendrimers tertiary amines (Paulo, P. M. R.; Costa, S. M. B. J. Phys. Chem. B 2005, 109, 13928). In the present contribution, we evaluate computationally the role of the porphyrin-dendrimer conformation in the development of a statistical distribution of ET rates through its dependence on the donor-acceptor distance. We started from simulations without explicit solvent to obtain trajectories of the donor-acceptor distance and the respective time-averaged distributions for two dendrimer sizes and different initial configurations of the porphyrin-dendrimer pair. By introducing explicit solvent (water) in our simulations, we were able to estimate the reorganization energy of the medium for the systems with the dendrimer of smaller size. The values obtained are in the range 0.6-1.5 eV and show a linear dependence with the inverse of the donor-acceptor distance, which can be explained by a two-phase dielectric continuum model taking into account the medium heterogeneity provided by the dendrimer organic core. Dielectric relaxation accompanying ET was evaluated from the simulations with explicit solvent showing fast decay times of some tens of femtoseconds and slow decay times in the range of hundreds of femtoseconds to a few picoseconds. The variations of the slow relaxation times reflect the heterogeneity of the dendrimer donor sites which add to the complexity of ET kinetics as inferred from the experimental fluorescence decays.

Optical spectroscopy and photochemistry of porphyrins and phthalocyanines

Studies of excited singlet and triplet states of porphyrins and phthalocyanines in organized media of reverse micelles, vesicles, monolayers, and Langmuir-Blodgett films along with more complex supramolecular organizates with proteins and dendrimers, are reported. Self-assembly in these systems was followed by imaging and temporal fluorescence techniques.

Deswelling and Electrolyte Dissipation in Free Diffusion of Charged PAMAM Dendrimers.

The diffusion coefficient of charged PAMAM dendrimers was measured by fluorescence correlation spectroscopy in aqueous solution at submicromolar concentrations. The solution pH was varied for conditions ranging from a fully charged to neutral charge dendrimer to infer about electrostatic swelling in the dilute regime. The diffusion coefficient of generation G4 increases by as much as 20% between high and low charge conditions due to the combined effects of polyelectrolyte deswelling and loss of electrolyte dissipation. By taking into account the electrolyte dissipation in the friction factor, we have found that the observed deswelling corresponds to a change of hydrodynamic radius between 7-13% for generation G4 and about 12% for generation G7. Simulations of molecular dynamics of dendrimer G4 show that counterion uptake by the dendrimer structure upon full protonation induces a 16% increase of its radius of gyration. The change in dendrimer size is slightly larger than that previously reported from neutron scattering techniques, thereby suggesting that electrostatic swelling is more pronounced at dilute dendrimer concentration and low ionic strength. It is confirmed that even higher generations, which have more congested molecular structures, can experience some degree of conformational change in response to a change of the dendrimer charge density.

Encapsulation of photoactive porphyrinoids in polyelectrolyte hollow microcapsules viewed by fluorescence lifetime imaging microscopy (FLIM)

Fluorescence Lifetime Imaging Microscopy (FLIM) was used to investigate the encapsulation of porphyrinoids in multilayer hollow microcapsules assembled layer by layer with poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH). The lifetime colour contrast enables the discrimination of fluorophore interactions at the microcapsule interface or deeper localized in the microcapsule shell. The coupling of functionalized porphyrin derivatives with oppositely charged polyelectrolyte made the microcapsule pH responsive. FLIM images were also obtained from aluminium monosulfonate phthalocyanine (AlPcS1) included in a lipid vesicle deposited on the surface of a modified microcapsule with dispersed gold nanoparticles (AuNP). In this case, a heterogeneous distribution of both quenched and enhanced fluorescence intensities was mapped from various fluorescence spots. These effects, undetected in the absence of AuNP, were accompanied by a decrease of lifetimes attributed to the contribution of plasmonic effects induced by AuNP. Fluorescence lifetime contrast-based imaging provides new insights in the field of polyelectrolyte microcapsules.

Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein.