Sergio Dominguez-Medina

In situ measurement of bovine serum albumin interaction with gold nanospheres

We present in situ observations of adsorption of bovine serum albumin (BSA) on citrate-stabilized gold nanospheres. We implemented scattering correlation spectroscopy as a tool to quantify changes in the nanoparticle brownian motion resulting from BSA adsorption onto the nanoparticle surface. Protein binding was observed as an increase in the nanoparticle hydrodynamic radius. Our results indicate the formation of a protein monolayer at similar albumin concentrations as those found in human blood. Additionally, by monitoring the frequency and intensity of individual scattering events caused by single gold nanoparticles passing the observation volume, we found that BSA did not induce colloidal aggregation, a relevant result from the toxicological viewpoint. Moreover, to elucidate the thermodynamics of the gold nanoparticle-BSA association, we measured an adsorption isotherm which was best described by an anticooperative binding model. The number of binding sites based on this model was consistent with a BSA monolayer in its native state. In contrast, experiments using poly(ethylene glycol)-capped gold nanoparticles revealed no evidence for adsorption of BSA.

Probing a Century Old Prediction One Plasmonic Particle at a Time

In 1908, Gustav Mie solved Maxwells equations to account for the absorption and scattering of spherical plasmonic particles. Since then much attention has been devoted to the size dependent optical properties of metallic nanoparticles. However, ensemble measurements of colloidal solutions generally only yield the total extinction cross sections of the nanoparticles. Here, we show how Mies prediction on the size dependence of the surface absorption and scattering can be probed separately for the same gold nanoparticle by using two single particle spectroscopy techniques, (1) dark-field scattering and (2) photothermal imaging, which selectively only measure scattering and absorption, respectively. Combining the optical measurements with correlated scanning electron microscopy furthermore allowed us to measure the size of the spherical gold nanoparticles, which ranged from 43 to 274 nm in diameter. We found that even though the trend predicted by Mie theory is followed well by the experimental data over a large range of nanoparticle diameters, for small size variations changes in scattering and absorption intensities are dominated by factors other than those considered by Mie theory. In particular, spectral shifts of the plasmon resonance due to deviations from a spherical particle shape alone cannot explain the observed variation in absorption and scattering intensities.

Plasmon Emission Quantum Yield of Single Gold Nanorods as a Function of Aspect Ratio

We report on the one-photon photoluminescence of gold nanorods with different aspect ratios. We measured photoluminescence and scattering spectra from 82 gold nanorods using single-particle spectroscopy. We found that the emission and scattering spectra closely resemble each other independent of the nanorod aspect ratio. We assign the photoluminescence to the radiative decay of the longitudinal surface plasmon generated after fast interconversion from excited electron-hole pairs that were initially created by 532 nm excitation. The emission intensity was converted to the quantum yield and was found to approximately exponentially decrease as the energy difference between the excitation and emission wavelength increased for gold nanorods with plasmon resonances between 600 and 800 nm. We compare this plasmon emission to its molecular analogue, fluorescence.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Radiative and nonradiative properties of single plasmonic nanoparticles and their assemblies.

A surface plasmon is the coherent oscillation of the conduction band electrons. When a metal nanoparticle is excited to produce surface plasmons, incident light is both scattered and absorbed, giving rise to brilliant colors. One available technique for measuring these processes, ensemble extinction spectroscopy, only measures the sum of scattering and absorption. Although the spectral responses of these processes are closely related, their relative efficiencies can differ significantly as a function of nanoparticle size and shape. For some applications, researchers may need techniques that can quantitatively measure absorption or scattering alone. Through advances in single particle spectroscopy, researchers can overcome this problem, separately determining the radiative (elastic and inelastic scattering) and nonradiative (absorption) properties of surface plasmons. Furthermore, because we can use the same sample preparation for both single particle spectroscopy measurements and electron microscopy, this technique provides detailed structural information and a direct correlation between optical properties and nanostructure morphology. In this Account, we present our quantitative investigations of both radiative (scattering and one-photon luminescence) and nonradiative (absorption) properties of the same individual plasmonic nanostructures employing different single particle spectroscopy techniques. In particular, we have used a combined setup to study the same structure with dark-field scattering spectroscopy, photothermal heterodyne imaging, confocal luminescence microscopy, and scanning electron microscopy. While Mie theory thoroughly describes the overall size dependence of scattering and absorption for nanospheres, our real samples deviate significantly from the predicted trend: their particle shape is not perfectly spherical, especially when supported on a substrate. Because of the high excitation rate in laser based single particle measurements, we can efficiently detect one-photon luminescence despite a low quantum yield. For gold nanoparticles, the luminescence spectrum follows the scattering response, and therefore we assigned it to the emission of a plasmon. Due to strong near-field interactions the plasmonic response of closely spaced nanoparticles deviates significantly from that of the constituent nanoparticles. This response arises from coupled surface plasmon modes that combine those of the individual nanoparticles. Our correlated structural and optical imaging strategy is especially powerful for understanding these collective modes and their dependence on the assembly geometry.

Photoluminescence of a Plasmonic Molecule

Photoluminescent Au nanoparticles are appealing for biosensing and bioimaging applications because of their non-photobleaching and non-photoblinking emission. The mechanism of one-photon photoluminescence from plasmonic nanostructures is still heavily debated though. Here, we report on the one-photon photoluminescence of strongly coupled 50 nm Au nanosphere dimers, the simplest plasmonic molecule. We observe emission from coupled plasmonic modes as revealed by single-particle photoluminescence spectra in comparison to correlated dark-field scattering spectroscopy. The photoluminescence quantum yield of the dimers is found to be surprisingly similar to the constituent monomers, suggesting that the increased local electric field of the dimer plays a minor role, in contradiction to several proposed mechanisms. Aided by electromagnetic simulations of scattering and absorption spectra, we conclude that our data are instead consistent with a multistep mechanism that involves the emission due to radiative decay of surface plasmons generated from excited electron-hole pairs following interband absorption.

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Measuring the Hydrodynamic Size of Nanoparticles Using Fluctuation Correlation Spectroscopy.

Fluctuation correlation spectroscopy (FCS) is a well-established analytical technique traditionally used to monitor molecular diffusion in dilute solutions, the dynamics of chemical reactions, and molecular processes inside living cells. In this review, we present the recent use of FCS for measuring the size of colloidal nanoparticles in solution. We review the theoretical basis and experimental implementation of this technique and its advantages and limitations. In particular, we show examples of the use of FCS to measure the size of gold nanoparticles, monitor the rotational dynamics of gold nanorods, and investigate the formation of protein coronas on nanoparticles.