Advances in sample environments for neutron scattering for colloid and interface science.

This review describes recent advances in sample environments across the full complement of applicable neutron scattering techniques to colloid and interface science. Temperature, pressure, flow, tensile testing, ultrasound, chemical reactions, IR/visible/UV light, confinement, humidity and electric and magnetic field application, as well as tandem X-ray methods, are all addressed. Consideration for material choices in sample environments and data acquisition methods are also covered as well as discussion of current and potential future use of machine learning and artificial intelligence.

Vortex fluidic regulated phospholipid equilibria involving liposomes down to sub-micelle size assemblies.

Conventional channel-based microfluidic platforms have gained prominence in controlling the bottom-up formation of phospholipid based nanostructures including liposomes. However, there are challenges in the production of liposomes from rapidly scalable processes. These have been overcome using a vortex fluidic device (VFD), which is a thin film microfluidic platform rather than channel-based, affording ∼110 nm diameter liposomes. The high yielding and high throughput continuous flow process has a 45° tilted rapidly rotating glass tube with an inner hydrophobic surface. Processing is also possible in the confined mode of operation which is effective for labelling pre-VFD-prepared liposomes with fluorophore tags for subsequent mechanistic studies on the fate of liposomes under shear stress in the VFD. In situ small-angle neutron scattering (SANS) established the co-existence of liposomes ∼110 nm with small rafts, micelles, distorted micelles, or sub-micelle size assemblies of phospholipid, for increasing rotation speeds. The equilibria between these smaller entities and ∼110 nm liposomes for a specific rotational speed of the tube is consistent with the spatial arrangement and dimensionality of topological fluid flow regimes in the VFD. The prevalence for the formation of ∼110 nm diameter liposomes establishes that this is typically the most stable structure from the bottom-up self-assembly of the phospholipid and is in accord with dimensions of exosomes.

Self-assembly and hydrogelation of a potential bioactive peptide derived from quinoa proteins.

In this work the identification of peptides derived from quinoa proteins which could potentially self-assemble, and form hydrogels was carried out with TANGO, a statistical mechanical based algorithm that predicts ß-aggregate propensity of peptides. Peptides with the highest aggregate propensity were subjected to gelling screening experiments from which the most promising bioactive peptide with sequence KIVLDSDDPLFGGF was selected. The self-assembling and hydrogelation properties of the C-terminal amidated peptide (KIVLDSDDPLFGGF-NH2) were studied. The effect of concentration, pH, and temperature on the secondary structure of the peptide were probed by circular dichroism (CD), while its nanostructure was studied by transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). Results revealed the existence of random coil, α-helix, twisted ß-sheet, and well-defined ß-sheet secondary structures, with a range of nanostructures including elongated fibrils and bundles, whose proportion was dependant on the peptide concentration, pH, or temperature. The self-assembly of the peptide is demonstrated to follow established models of amyloid formation, which describe the unfolded peptide transiting from an α-helix-containing intermediate into ß-sheet-rich protofibrils. The self-assembly is promoted at high concentrations, elevated temperatures, and pH values close to the peptide isoelectric point, and presumably mediated by hydrogen bond, hydrophobic and electrostatic interactions, and π-π interactions (from the F residue). At 15 mg/mL and pH 3.5, the peptide self-assembled and formed a self-supporting hydrogel exhibiting viscoelastic behaviour with G' (1 Hz) ~2300 Pa as determined by oscillatory rheology measurements. The study describes a straightforward method to monitor the self-assembly of plant protein derived peptides; further studies are needed to demonstrate the potential application of the formed hydrogels in food and biomedicine.

Role of Ca2+ in the pepsin-induced coagulation and in the dynamic in vitro gastric digestion behavior of casein micelles.

The effect of Ca2+ on pepsin-induced hydrolysis of κ-casein and subsequent coagulation of casein micelles was studied in a micellar casein (MC) solution at pH ≈ 6.0 at 37 °C without stirring. An NaCl-supplemented MC solution was used as a positive control to assess the effect of increased ionic strength after CaCl2 addition. Quantitative determination of the released para-κ-casein during the reaction using reverse-phase high-performance liquid chromatography showed that specific hydrolysis of κ-casein by pepsin was little affected by the addition of either CaCl2 or NaCl. However, rheological properties and microstructures of curds induced by pepsin hydrolysis depended markedly on the addition of salts. Addition of CaCl2 up to 17.5 mM facilitated coagulation, with decreases in coagulation time and critical hydrolysis degree, and increases in firming rate and maximum storage modulus (G'max); further addition of CaCl2 (22.5 mM) resulted in a lower G'max. Increased ionic strength to 52.5 mM by adding NaCl retarded the coagulation and resulted in a looser curd structure. In a human gastric simulator, MC, without the addition of CaCl2, did not coagulate until the pH decreased to ≈5.0 after ≈50 min of digestion. Addition of CaCl2 facilitated coagulation of casein micelles and resulted in more cohesive curds with dense structures during digestion, which slowed the emptying rate of caseins. At the same CaCl2 concentration, a sample with higher ionic strength coagulated more slowly. This study provides further understanding on the effect of divalent (Ca2+) ions and ionic strength on the coagulation of casein micelles and the digestion behavior of milk.

Pepsin-induced coagulation of casein micelles: Effect of whey proteins and heat treatment.

The effect of whey proteins and heat treatment (90 °C, 5 min) on pepsin-induced hydrolysis of κ-casein, and subsequent coagulation of casein micelles, was investigated at pH 6.3 and 6.0 using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. Whey proteins did not affect the hydrolysis of κ-casein but retarded the coagulation process. Heat treatment did not affect the hydrolysis kinetics in whey protein (WP)-free samples, but slightly impaired the hydrolysis rate in WP-containing samples. The coagulation process of WP-free samples was little affected by heat-treatment. However, compared with unheated WP-contained sample at the same pH, the coagulation process of the heated sample was retarded at pH 6.3 but enhanced at pH 6.0. The curd in heated samples with smaller pores had higher water holding capacity. This knowledge provides further understanding on the role of whey proteins and heat treatment on the coagulation mechanisms of milk under gastric conditions.

Building blocks of ß-sitosterol-γ-oryzanol gels revealed by small-angle neutron scattering and real space modelling.

Mixtures of ß-sitosterol and γ-oryzanol form gels in a range of organic solvents. Despite being widely studied, particularly as potential oleogels for food application, details of the intrinsic gel-forming building blocks remain unclear. Small-angle neutron scattering (SANS) combined with solvent contrast variation has been used to evaluate potential structural models. While evidence exists that the building blocks are hollow cylinders (tubules), the simultaneous fitting of twelve contrast-varied SANS data sets indicates that the previously proposed model of double walled tubules is incorrect. Predicted scattering based on real space models provides compelling evidence that the origin of the gelling behaviour is the limited assembly of adjacent tubules to form a space-filling network of fibrils.

Kinetics of pepsin-induced hydrolysis and the coagulation of milk proteins.

Hydrolysis-induced coagulation of casein micelles by pepsin occurs during the digestion of milk. In this study, the effect of pH (6.7-5.3) and pepsin concentration (0.110-2.75 U/mL) on the hydrolysis of κ-casein and the coagulation of the casein micelles in bovine skim milk was investigated at 37°C using reverse-phase HPLC, oscillatory rheology, and confocal laser scanning microscopy. The hydrolysis of κ-casein followed a combined kinetic model of first-order hydrolysis and putative pepsin denaturation. The hydrolysis rate increased with increasing pepsin concentration at a given pH, was pH dependent, and reached a maximum at pH ∼6.0. Both the increase in pepsin concentration and decrease in pH resulted in a shorter coagulation time. The extent of κ-casein hydrolysis required for coagulation was independent of the pepsin concentration at a given pH and, because of the lower electrostatic repulsion between para-casein micelles at lower pH, decreased markedly from ∼73% to ∼33% when pH decreased from 6.3 to 5.3. In addition, the rheological properties and the microstructures of the coagulum were markedly affected by the pH and the pepsin concentration. The knowledge obtained from this study provides further understanding on the mechanism of milk coagulation, occurring at the initial stage of transiting into gastric conditions with high pH and low pepsin concentration.

Advanced structural characterisation of agar-based hydrogels: Rheological and small angle scattering studies.

Agar-based extracts from Gelidium sesquipedale were generated by heat and combined heat-sonication, with and without the application of alkali pre-treatment. Pre-treatment yielded extracts with greater agar contents; however, it produced partial degradation of the agar, reducing its molecular weight. Sonication produced extracts with lower agar contents and decreased molecular weights. A gelation mechanism is proposed based on the rheological and small angle scattering characterization of the extracts. The formation of strong hydrogels upon cooling was caused by the association of agarose chains into double helices and bundles, the sizes of which depended on the agar purity and molecular weight. These different arrangements at the molecular scale consequently affected the mechanical performance of the obtained hydrogels. Heating of the hydrogels produced a gradual disruption of the bundles; weaker or smaller bundles were formed upon subsequent cooling, suggesting that the process was not completely reversible.

Interfacial Structures of Droplet-Stabilized Emulsions Formed with Whey Protein Microgel Particles as Revealed by Small- and Ultra-Small-Angle Neutron Scattering.

Droplet-stabilized emulsions (DSEs) were made from oil droplets coated with whey protein microgel (WPM) particles. The WPM particles with z-average hydrodynamic diameters of 270.9 ± 4.7 and 293.8 ± 6.7 nm were obtained by heating whey proteins with 10 mM phosphate buffer, pH 5.9 (-PB) and no buffer (-NPB), respectively. The primary emulsions coated by WPM-NPB and WPM-PB particles had mass fractal dimensions of ∼2.75, as determined by small- and ultra-small-angle neutron scattering (SANS and USANS). The size of the subsequently formed DSEs (D32 ≈ 7-23 µm), which were stabilized by the primary emulsion droplets, made with either WPM-NPB (termed DSE-NPB) or WPM-PB (termed DSE-PB) was dependent on the concentration of the primary emulsion (10-60 wt %) in the aqueous phase. At the DSE-NPB interface, the adsorbed primary emulsion droplets formed a fractal network with a surface fractal dimension of about 3, indicating a rough interfacial layer. Combined SANS and USANS allowed a comprehensive understanding of the multilength scale structures from WPM particles to DSEs.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

The Effect of a Two-Stage Heat-Treatment on the Microstructural and Mechanical Properties of a Maraging Steel.

Maraging steels gain many of their beneficial properties from heat treatments which induce the precipitation of intermetallic compounds. We consider here a two-stage heat-treatment, first involving austenitisation, followed by quenching to produce martensite and then an ageing treatment at a lower temperature to precipitation harden the martensite of a maraging steel. It is shown that with a suitable choice of the initial austenitisation temperature, the steel can be heat treated to produce enhanced toughness, strength and creep resistance. A combination of small angle neutron scattering, scanning electron microscopy, electron back-scattered diffraction, and atom probe tomography were used to relate the microstructural changes to mechanical properties. It is shown that such a combination of characterisation methods is necessary to quantify this complex alloy, and relate these microstructural changes to mechanical properties. It is concluded that a higher austenitisation temperature leads to a greater volume fraction of smaller Laves phase precipitates formed during ageing, which increase the strength and creep resistance but reduces toughness.

Magnetic scattering in the simultaneous measurement of small-angle neutron scattering and Bragg edge transmission from steel.

Pulsed neutron sources enable the simultaneous measurement of small-angle neutron scattering (SANS) and Bragg edge transmission. This simultaneous measurement is useful for microstructural characterization in steel. Since most steels are ferromagnetic, magnetic scattering contributions should be considered in both SANS and Bragg edge transmission analyses. An expression for the magnetic scattering contribution to Bragg edge transmission analysis has been derived. The analysis using this expression was applied to Cu steel. The ferrite crystallite size estimated from this Bragg edge transmission analysis with the magnetic scattering contribution was larger than that estimated using conventional expressions. This result indicates that magnetic scattering has to be taken into account for quantitative Bragg edge transmission analysis. In the SANS analysis, the ratio of magnetic to nuclear scattering contributions revealed that the precipitates consist of body-centered cubic Cu0.7Fe0.3 and pure Cu, which probably has 9R structure including elastic strain and vacancies. These results show that effective use of the magnetic scattering contribution allows detailed analyses of steel microstructure.

Small-Angle Neutron Scattering Studies on the Multilamellae Formed by Mixing Lamella-Forming Cationic Diblock Copolymers with Lipids and Their Interaction with DNA.

We demonstrate that the lamella-forming polystyrene-block-poly(N-methyl-4-vinylpyridinium iodine) (PS-b-P4VPQ), with similar sizes of the PS and P4VPQ blocks, can be dispersed in the aqueous solutions by forming lipid/PS-b-P4VPQ multilamellae. Using small-angle neutron scattering (SANS) and 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (d62-DPPC) in D2O, a broad correlation peak is found in the scattering profile that signifies the formation of the loosely ordered d62-DPPC/PS-b-P4VPQ multilamellae. The thicknesses of the hydrophobic and hydrophilic layers of the d62-DPPC/PS-b-P4VPQ multilamellae are close to the PS layer and the condensed brush layer thicknesses as determined from previous neutron reflectometry studies on the PS-b-P4VPQ monolayer at the air-water interface. Such well-dispersed d62-DPPC/PS-b-P4VPQ multilamellae are capable of forming multilamellae with DNA in aqueous solution. It is found that the encapsulation of DNA in the hydrophilic layer of the d62-DPPC/PS-b-P4VPQ multilamellae slightly increases the thickness of the hydrophilic layer. Adding CaCl2 can enhance the DNA adsorption in the hydrophilic brush layer, and it is similar to that observed in the neutron reflectometry study of the DNA adsorption by the PS-b-P4VPQ monolayer.

Correlation of thermostability and conformational changes of catechol 2, 3-dioxygenases from two disparate micro-organisms.

We have investigated the structure of recombinant catechol 2, 3-dioxygenase (C23O) purified from two species in which the enzyme has evolved to function at different temperature. The two species are mesophilic bacterium Pseudomonas putida strain mt-2 and thermophilic archaea Sulfolobus acidocaldariusDSM639. Using the primary sequence analysis, we show that both C23Os have only 30% identity and 48% similarity but contain conserved amino acid residues forming an active site area around the iron ion. The corresponding differences in homology, but structural similarity in active area residues, appear to provide completely different responses to heating the two enzymes. We confirm this by small angle X-ray scattering and demonstrate that the overall structure of C23O from P. putida is slightly different from its crystalline form whereas the solution scattering of C23O from S. acidocaldarius at temperatures between 4 and 85°C ideally fits the calculated scattering from the single crystal structure. The thermostability of C23O from S. acidocaldarius correlates well with conformation in solution during thermal treatment. The similarity of the two enzymes in primary and tertiary structure may be taken as a confirmation that two enzymes have evolved from a common ancestor.

Effect of enzymatic hydrolysis on native starch granule structure.

Enzymatic digestion of six starches of different botanical origin was studied in real time by in situ time-resolved small-angle neutron scattering (SANS) and complemented by the analysis of native and digested material by X-ray diffraction, differential scanning calorimetry, small-angle X-ray scattering, and scanning electron microscopy with the aim of following changes in starch granule nanostructure during enzymatic digestion. This range of techniques enables coverage over five orders of length-scale, as is necessary for this hierarchically structured material. Starches studied varied in their digestibility and displayed structural differences in the course of enzymatic digestion. The use of time-resolved SANS showed that solvent-drying of digested residues does not induce any structural artifacts on the length scale followed by small-angle scattering. In the course of digestion, the lamellar peak intensity gradually decreased and low-q scattering increased. These trends were more substantial for A-type than for B-type starches. These observations were explained by preferential digestion of the amorphous growth rings. Hydrolysis of the semicrystalline growth rings was explained on the basis of a liquid-crystalline model for starch considering differences between A-type and B-type starches in the length and rigidity of amylopectin spacers and branches. As evidenced by differing morphologies of enzymatic attack among varieties, the existence of granular pores and channels and physical penetrability of the amorphous growth ring affect the accessibility of the enzyme to the substrate. The combined effects of the granule microstructure and the nanostructure of the growth rings influence the opportunity of the enzyme to access its substrate; as a consequence, these structures determine the enzymatic digestibility of granular starches more than the absolute physical densities of the amorphous growth rings and amorphous and crystalline regions of the semicrystalline growth rings.