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.
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.
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 dendrimer's 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.
Anionic meso-tetrakis(4-sulfonatophenyl)porphine (TSPP) interacts with poly(amido) amine (PAMAM) dendrimers of generations 2.0 and 4.0 in aqueous solution to form several dendrimer-associated species, which depend on the relative concentrations of dendrimer to porphyrin (D/P ≡ [PAMAM]/[TSPP]). At D/P ratios above the isoelectric point of the charge balance between the dendrimer and porphyrin, only two spectroscopic species were detected. An equilibrium model assuming a dendrimer-induced TSPP H-dimer coexisting with dendrimer-associated TSPP monomer afforded a good description of the experimental data, giving equilibrium constants (K d) for the dimer dissociation of 0.78 and 2.67 for generations 2.0 and 4.0 dendrimers, respectively. The decomposition of the Soret band afforded TSPP H-aggregates' spectra with bandwidth values in the range 910−1210 cm-1, clearly larger than the monomer bandwidth around 711−800 cm-1. The fluorescence decays are nearly exponential with a long decay time component, which varies from 10 to 12 ns depending on the D/P ratio. However, at pH 2, there are striking differences between generations 2.0 and 4.0, which should be related to a more hydrophobic environment provided by the latter one. Time-resolved fluorescence anisotropy gave rotational times for the porphyrin−dendrimer complex from which hydrodynamic radii of approximately 14 and 21 Å, similar to those of generations 2.0 and 4.0 PAMAM dendrimers, were retrieved.
We demonstrate correlation analysis of the photothermal signal from gold nanoparticles diffusing in solution. Our experimental setup is primarily optimized for large detection volumes and therefore has a relatively low numerical aperture (0.25). This requirement limits light collection and detection efficiency, but we can nevertheless detect particles as small as 20 nm in diameter in 100 μs. Photothermal correlation spectroscopy (PCS) in water and in water:glycerol mixtures gives access to characteristic diffusion times of some tens of milliseconds. Diffusion is enhanced for high pump intensities, due to the increased temperature around the particle. The photothermal signal at the detector arises from the interference of the scattered probe beam and a reference field. In the forward configuration, the reference is the transmitted probe beam itself. In the backward configuration, the reference wave is a reflection by a nearby interface. It has an optical phase difference depending on the distance of the particle to the reflecting interface. The backward signal also includes the directly modulated scattering signal. Potential applications of photothermal correlation in biological labeling are demonstrated on bacteriophage virus particles carrying 20 nm gold labels.
We have performed atomistic molecular dynamics simulations of PAMAM dendrimers of generations 0.5, 1.5, 2.5, 3.5, and 4.5. The simulated systems comprise the charged dendrimer and its counterions embedded in a dielectric continuum (i.e., without explicit solvent). Structural properties of these dendrimers, like the radius of gyration, the principal moments of inertia, and the segment density profiles, were evaluated from the simulations. The average radius of gyration obtained for the intermediate half-generations 2.5, 3.5, and 4.5 follows the same scaling law that was previously inferred from simulations of full-generation PAMAMs, Rg approximately M1/3, and is characteristic of space-filling objects. The low half-generations 0.5 and 1.5 deviate, however, to greater Rg values. The shape of the smaller dendrimers is approximately that of a prolate ellipsoid, which becomes more spherical for higher generations. The segment density profiles show features identical to those obtained in other simulations of flexible-chain dendrimers, like dendron-backfolding. Two slightly different configurations, in terms of size and shape, were identified for generation 2.5. The radial distributions of counterions extracted from the simulations compare well with the solutions of Poisson-Boltzmann cell model, and the dendrimer's effective charge was estimated using the Bjerrum criterion. The influence of electrostatic interactions in the dendrimer's conformation due to repulsion between the charged end-groups and its relation to counterion effects is discussed for the several generations simulated. The form factors calculated from the simulations are compared with the model of a homogeneous ellipsoid of revolution. The overall results are in agreement with the previously established morphological transition of PAMAM dendrimers toward a more spherical and compact conformation above generations 3 or 4.
Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod's surface. The model sensors were further characterized by measuring their dose-response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response.
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