Elemental fingerprinting of mineral species in iron-fortified milk: anomalous small-angle X-ray scattering and resonant soft X-ray scattering studies.

Anomalous small-angle X-ray scattering (ASAXS) and resonant soft X-ray scattering (RSoXS) are two related techniques that can enable element-specific structural information to be obtained. The development of iron-fortified milk products can greatly benefit from such techniques, allowing the structure of iron and other minerals (such as native calcium) within the casein micelle to be determined. Each method has advantages and disadvantages: for ASAXS, the sample preparation is straightforward, but the signal is relatively low and information about the structure of Ca is difficult to access. RSoXS can be used to study both Ca and Fe, and the element-specific signals observed are proportionally much higher; however, the measurements are challenging due to the difficulty of precise control of the solution thickness using currently available vacuum-compatible liquid cells. Nevertheless, complementary results from both techniques indicate Fe is co-located with Ca, i.e. within the colloidal calcium phosphate nanoclusters that are present within native casein micelles in milk.

Absolute Intramolecular Distance Measurements with Angstrom-Resolution Using Anomalous Small-Angle X-ray Scattering.

Accurate determination of molecular distances is fundamental to understanding the structure, dynamics, and conformational ensembles of biological macromolecules. Here we present a method to determine the full distance distribution between small (∼7 Å radius) gold labels attached to macromolecules with very high-precision (≤1 Å) and on an absolute distance scale. Our method uses anomalous small-angle X-ray scattering close to a gold absorption edge to separate the gold-gold interference pattern from other scattering contributions. Results for 10-30 bp DNA constructs achieve excellent signal-to-noise and are in good agreement with previous results obtained by single-energy SAXS measurements without requiring the preparation and measurement of single labeled and unlabeled samples. The use of small gold labels in combination with ASAXS read out provides an attractive approach to determining molecular distance distributions that will be applicable to a broad range of macromolecular systems.

Quantitative Determination of Metal Ion Adsorption on Cellulose Nanocrystals Surfaces.

Nanocellulose is a bio-based material that holds significant potential in the field of water purification. Of particular interest is their potential use as a key sorbent material for the removal of metal ions from solution. However, the structure of metal ions adsorbed onto cellulose surfaces is not well understood. The focus of this work is to determine quantitatively the three-dimensional distribution of metal ions of different valencies surrounding negatively charged carboxylate functionalized cellulose nanocrystals (CNCs) using anomalous small-angle X-ray scattering (ASAXS). These distributions can affect the water and ionic permeability in these materials. The data show that increasing the carboxylate density on the surface of the CNCs from 740 to 1100 mmol/kg changed the nature of the structure of the adsorbed ions from a monolayer into a multilayer structure. The monolayer was modeled as a Stern layer around the CNC nanoparticles, whereas the multilayer structure was modeled as a diffuse layer on top of the Stern layer around the nanoparticles. Within the Stern layer, the maximum ion density increases from 1680 to 4350 mmol of Rb+/(kg of CNC) with the increase in the carboxylate density on the surface of the nanoparticles. Additionally, the data show that CNCs can leverage multiple mechanisms, such as electrostatic attraction and the chaotropic effect, to adsorb ions of different valencies. By understanding the spatial organization of the adsorbed metal ions, the design of cellulose-based sorbents can be further optimized to improve the uptake capacity and selectivity in separation applications.

Anomalous small-angle X-ray scattering analyses on hierarchical structures of rubber-filler systems.

The hierarchical structures of poly(styrene-ran-butadiene) (SBR) rubber/carbon black (CB) systems vulcanized with sulfur and ZnO have been clarified using anomalous small-angle X-ray scattering (ASAXS) near the Zn absorption edge. In the case of SBR/CB systems vulcanized with peroxide, it has been found previously that the hierarchical structures formed by CB consist of aggregates of primary particles and agglomerates of those aggregates with mass-fractal dimensions. However, to date the hierarchical structures in SBR/CB systems vulcanized with sulfur and ZnO have not been well investigated, despite being commonly used. This is because the strong scattering contrast of Zn prevents the quantitative analyses of the hierarchical structures of CB using X-ray scattering. In this study, the effects of Zn on the scattering intensity were eliminated and the structure factors of CB in SBR/CB systems were obtained using the ASAXS method. By extrapolating to the zero volume fraction of CB, the particle structure factor of the CB aggregates was estimated and it was found that the CB aggregates consist of closely packed CB primary particles. The presence of large particles of ZnO and particles of ZnS on the order of 10 nm in size is confirmed.

ASAXS measurements on ferritin and apoferritin at the bioSAXS beamline P12 (PETRA III, DESY).

Small-angle X-ray scattering is widely utilized to study biological macromol-ecules in solution. For samples containing specific (e.g. metal) atoms, additional information can be obtained using anomalous scattering. Here, measuring samples at different energies close to the absorption edges of relevant elements provides specific structural details. However, anomalous small-angle X-ray scattering (ASAXS) applications to dilute macromolecular solutions are challenging owing to the overall low anomalous scattering effect. Here, pilot ASAXS experiments from dilute solutions of ferritin and cobalt-loaded apoferritin are reported. These samples were investigated near the resonance X-ray K edges of Fe and Co, respectively, at the EMBL P12 bioSAXS beamline at PETRA III, DESY. Thanks to the high brilliance of the P12 beamline, ASAXS experiments are feasible on dilute protein solutions, allowing one to extract the Fe- or Co-specific anomalous dispersion terms from the ASAXS data. The data were subsequently used to determine the spatial distribution of either iron or cobalt atoms incorporated into the ferritin/apoferritin protein cages.

Experimental approaches for investigating ion atmospheres around nucleic acids and proteins.

Ionic interactions are crucial to biological functions of DNA, RNA, and proteins. Experimental research on how ions behave around biological macromolecules has lagged behind corresponding theoretical and computational research. In the 21st century, quantitative experimental approaches for investigating ionic interactions of biomolecules have become available and greatly facilitated examinations of theoretical electrostatic models. These approaches utilize anomalous small-angle X-ray scattering, atomic emission spectroscopy, mass spectrometry, or nuclear magnetic resonance (NMR) spectroscopy. We provide an overview on the experimental methodologies that can quantify and characterize ions within the ion atmospheres around nucleic acids, proteins, and their complexes.

Towards Real-Time Ion-Specific Structural Sensitivity in Nanoporous Carbon Electrodes Using In Situ Anomalous Small-Angle X-ray Scattering.

Current in situ techniques to study ion charge storage and electrical double-layer formation in nanoporous electrodes are either chemically sensitive to element-specific concentration changes or structurally sensitive to rearrangements of ions and solvent molecules; but rarely can they cover both. Here we introduce in situ anomalous small-angle X-ray scattering (ASAXS) as a unique method to extract both real-time structural and ion-specific chemical information from one single experiment. Using a 1 M RbBr aqueous electrolyte and a hierarchical micro- and mesoporous carbon electrode, we identify different charging mechanisms for positive and negative applied potentials. We are able not only to track the global concentration change of each ion species individually, but also to observe their individual local rearrangement within the pore space.

Elucidation of Spatial Distribution of Hydrophobic Aromatic Compounds Encapsulated in Polymer Micelles by Anomalous Small-Angle X-ray Scattering.

Spatial distribution of bromobenzene (BrBz) and 4-bromophenol (BrPh) as hydrophobic aromatic compounds incorporated in polymer micelles with vesicular structure consisting of poly(ethylene glycol)-b-poly(tert-butyl methacrylate) (PEG-b-PtBMA) in aqueous solution is investigated by anomalous small-angle X-ray scattering (ASAXS) analyses near Br K edge. Small-angle X-ray scattering (SAXS) intensities from PEG-b-PtBMA micelles containing BrBz and BrPh were decreased as the energy of incident X-ray approached to Br K edge corresponding to the energy dependence of anomalous scattering factor of Br. The analysis for the energy dependence of SAXS profiles from the PEG-b-PtBMA micelles containing BrBz revealed that BrBz molecules were located in hydrophobic layer of PEG-b-PtBMA micelles. On the contrary, it was found by ASAXS that BrPh existed not only in the hydrophobic layer but also in the shell layer. Since ASAXS analysis successfully accomplished to visualize the spatial distribution of hydrophobic molecules in polymer micelles, it should be expected to be a powerful tool for characterization of drug delivery vehicles.