ABSTRACT
An advanced design of the analytical ultracentrifuge with multiwavelength emission detection (MWE-AUC) is presented which offers outstanding performance concerning the spectral resolution and range flexibility as well as the quality of the data acquired. The excitation by a 520 nm laser is complemented with a 405 nm laser. An external spectrograph with three switchable tunable gratings permits optimisation of the spectral resolution in an order of magnitude range while keeping the spectral region broad. The new system design leads also to a significant reduction of systematic signal noise and allows the assessment and control of inner filter effects. Details regarding the very large signal dynamic range are presented, an important aspect when studying samples in a broad concentration range of up to five orders of magnitude. Our system is validated by complementary studies on two biological systems, fluorescent BSA and GFP, using the commercial Optima AUC with absorbance detection for comparison. Finally, we demonstrate the capabilities of our second generation MWE-AUC with respect to multiwavelength characterisation of gold nanoclusters, which exhibit specific fluorescence depending on their structure. Overall, this work depicts an important stepping stone for the concept of multiwavelength emission detection in AUC. The MWE-AUC developed, being to our knowledge the first and sole one of its kind, has reached the development level suitable for the future in-depth studies of size-, shape- and composition-dependent emission properties of colloids.
ABSTRACT
The Stokes-Einstein-Sutherland (SES) equation is at the foundation of statistical physics, relating a particle's diffusion coefficient and size with the fluid viscosity, temperature, and the boundary condition for the particle-solvent interface. It is assumed that it relies on the separation of scales between the particle and the solvent, hence it is expected to break down for diffusive transport on the molecular scale. This assumption is however challenged by a number of experimental studies showing a remarkably small, if any, violation, while simulations systematically report the opposite. To understand these discrepancies, analytical ultracentrifugation experiments are combined with molecular simulations, both performed at unprecedented accuracies, to study the transport of buckminsterfullerene C60 in toluene at infinite dilution. This system is demonstrated to clearly violate the conditions of slow momentum relaxation. Yet, through a linear response to a constant force, the SES equation can be recovered in the long time limit with no more than 4% uncertainty both in experiments and in simulations. This nonetheless requires partial slip on the particle interface, extracted consistently from all the data. These results, thus, resolve a long-standing discussion on the validity and limits of the SES equation at the molecular scale.
ABSTRACT
Multidimensional particle properties determine the product properties in numerous advanced applications. Accurate and statistically meaningful measurements of complex particles and their multidimensional distributions are highly challenging but strongly needed. 2D particle size distributions of plasmonic nanoparticles of complex regular shape can be obtained from analytical ultracentrifugation experiments via the optical back coupling method. A workflow for the calculation of frictional properties of arbitrarily shaped nanoparticles was developed based on bead shell models and applied to gold bipyramids with a pentagonal cross-section. The obtained 2D particle length-diameter distributions and the reduced cumulative 1D length and diameter distributions were compared to transmission electron microscopy measurements. While we find very good agreement for most measurements, the obtained length and diameter distributions were shifted by a few nanometers for some samples. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, electron tomography, and finite element modeling indicate that the shift originated from a slight mismatch between the assumed shape of the simulated perfect bipyramids and the real particle shape and composition due to the presence of silver in the particles. This study demonstrates the feasibility of the applied techniques for complex shape analysis of nanoparticle ensembles with unmatched particle count numbers.
ABSTRACT
The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils.In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.
Subject(s)
Lactoglobulins/chemistry , Protein Aggregates , Ultracentrifugation , Molecular Dynamics Simulation , Protein Conformation , Stress, MechanicalABSTRACT
Brownian dynamics (BD) has been applied as a comprehensive tool to model sedimentation and diffusion of nanoparticles in analytical ultracentrifugation (AUC) experiments. In this article, we extend the BD algorithm by considering space-dependent diffusion and solvent compressibility. With this, the changes in the sedimentation and diffusion coefficient from altered solvent properties at increased pressures are accurately taken into account. Moreover, it is demonstrated how the concept of space-dependent diffusion is employed to describe concentration-dependent sedimentation and diffusion coefficients, in particular, through the Gralen coefficient and the second virial coefficient. The influence of thermodynamic nonideality on diffusional properties can be accurately simulated and agree with well-known evaluation tools. BD simulations for sedimentation equilibrium and sedimentation velocity (SV) AUC experiments including effects of hydrodynamic and thermodynamic nonideality are validated by global evaluation in SEDANAL. The interplay of solvent compressibility and retrieved nonideality parameters can be studied utilizing BD. Finally, the second virial coefficient is determined for lysozyme from SV AUC experiments and BD simulations and compared to membrane osmometry. These results are in line with DLVO theory. In summary, BD simulations are established for the validation of nonideal sedimentation in AUC providing a sound basis for the evaluation of complex interactions even in polydisperse systems.
ABSTRACT
In this study, a new detector for multiwavelength emission analytical ultracentrifugation (MWE-AUC) is presented, which allows measuring size- or composition-dependent fluorescence properties of nanoparticle ensembles. Validation of the new setup is carried out via comparison to a benchtop photoluminescence spectrometer and the established extinction-based multiwavelength analytical ultracentrifuge (MWL-AUC). The results on fluorescent proteins and silica particles demonstrate that the new device not only correctly reproduces sedimentation and diffusion coefficients of the particles but provides also meaningful fluorescence spectra. As an application example for a sample exhibiting a broad particle size distribution, spectra and size of graphene oxide nanoplatelets are extracted simultaneously. Narrowly distributed CdSe/ZnS quantum dots showing size- and structure-dependent shifts of their fluorescence spectra are analyzed as well. The combination of MWE- and MWL-AUC provides a comprehensive framework for the optical characterization for nanoparticles and macromolecules in terms of their extinction and emission properties.
ABSTRACT
Properties of nanoparticles are influenced by various parameters like size, shape or composition. Comprehensive high throughput characterization techniques are urgently needed to improve synthesis, scale up to production and make way for new applications of multidimensional particulate systems. In this study, we present a method for measuring two-dimensional size distributions of plasmonic nanorods in a single experiment. Analytical ultracentrifuge equipped with a multiwavelength extinction detector is used to record the optical and sedimentation properties of gold nanorods simultaneously. A combination of sedimentation and extinction properties, both depending on diameter and length of the dispersed nanorods, is used to measure two-dimensional distributions of gold nanorod samples. The length, diameter, aspect ratio, volume, surface and cross-sectional distributions can be readily obtained from these results. As the technique can be extended to other non-spherical plasmonic particles and can be used for determining relative amounts of particles of different shapes it provides complete and quantitative insights into particulate systems.
ABSTRACT
By combining analytical ultracentrifugation (AUC) in liquid phase and scanning mobility particle sizer (SMPS) in the gas phase, additional information on the particle size and morphology has been obtained for rigid particles. In this paper, we transfer this concept to soft particles, allowing us to analyze the size and molar mass of the short side chain perfluorosulfonic acid ionomer Aquivion® in a dilute aqueous suspension. The determination of the primary size and exact molar mass of this class of polymers is challenging since they are optically transparent and due to the formation of different aggregate structures depending on the concentration and solvent properties. First, validation of AUC and SMPS measurements was carried out using the well-defined biopolymers bovine serum albumin (BSA) and lysozyme (LYZ) to confirm the reliability of the results of the two unique and independent classifying methods. Then, the ionomer Aquivion® was studied using both techniques. From the mean molar mass of 185 ± 14 kDa obtained by AUC, a mean hydrodynamic diameter of 7.6 ± 0.5 nm was calculated. The particle size obtained from SMPS (7.1 nm) agrees very well with the results from AUC showing that the molecule was transferred into the gas phase without significantly changing its structure. In conclusion, the Aquivion® is molecularly dispersed in the used aqueous buffer solution without any aggregate formation in the investigated concentration range (< 2 g l-1).
