Time-resolved SAXS Research Articles

Early Events in G-quadruplex Folding Captured by Time-Resolved Small-Angle X-Ray Scattering.

Time-resolved small-angle X-ray experiments (TR-SAXS) are reported here that capture and quantify a previously unknown rapid collapse of the unfolded oligonucleotide as an early step in G4 folding of hybrid 1 and hybrid 2 telomeric G-quadruplex structures. The rapid collapse, initiated by a pH jump, is characterized by an exponential decrease in the radius of gyration from 20.6 to 12.6 Å. The collapse is monophasic and is complete in less than 600 ms. Additional hand-mixing pH-jump kinetic studies show that slower kinetic steps follow the collapse. The folded and unfolded states at equilibrium were further characterized by SAXS studies and other biophysical tools, to show that G4 unfolding was complete at alkaline pH, but not in LiCl solution as is often claimed. The SAXS Ensemble Optimization Method (EOM) analysis reveals models of the unfolded state as a dynamic ensemble of flexible oligonucleotide chains with a variety of transient hairpin structures. These results suggest a G4 folding pathway in which a rapid collapse, analogous to molten globule formation seen in proteins, is followed by a confined conformational search within the collapsed particle to form the native contacts ultimately found in the stable folded form.

Branched Polymeric Prenucleation Assemblies Initiate Calcium Phosphate Precipitation.

The formation of crystalline calcium phosphate (CaP) has recently gained ample attention as it does not follow the classic nucleation-and-growth mechanism of solid formation. Instead, the precipitation mechanisms can involve numerous intermediates, including soluble prenucleation species. However, structural features, stability, and transformation of such solution-state precursors remain largely undisclosed. Herein, we report a detailed and comprehensive characterization of the sequential events involved in calcium phosphate crystallization starting from the very early prenucleation stage. We integrated an extensive set of time-resolved methods, including NMR, turbidimetry, SAXS, cryo-TEM, and calcium-potentiometry to show that CaP nucleation is initiated by the transformation of "branched" polymeric prenucleation assemblies into amorphous calcium phosphate spheres. Such a mineralization process starts with the spontaneous formation of so-called nanometric prenucleation clusters (PNCs) that later assemble into those branched polymeric assemblies without calcium ion uptake from the solution. Importantly, the branched macromolecular species are invisible to many techniques (NMR, turbidity, calcium-potentiometry) but can readily be evidenced by time-resolved SAXS. We find that these polymeric assemblies constitute the origin of amorphous calcium phosphate (ACP) precipitation through an unexpected process: spontaneous dissolution is followed by local densification of 100-200 nm wide domains leading to ACP spheres of similar size. Finally, we demonstrate that the timing of the successive events involved in the CaP mineralization pathway can be kinetically controlled by the Ca2+/Pi molar ratio, such that the lifetime of the soluble transient species can be increased up to hours when decreasing it.

Understanding the effects of ethanol on the liposome bilayer structure using microfluidic-based time-resolved small-angle X-ray scattering and molecular dynamics simulations.

Lipid nanoparticles (LNPs) are essential carrier particles in drug delivery systems, particularly in ribonucleic acid delivery. In preparing lipid-based nanoparticles, microfluidic-based ethanol injection may produce precisely size-controlled nanoparticles. Ethanol is critical in LNP formation and post-treatment processes and affects liposome size, structure, lamellarity, and drug-loading efficiency. However, the effects of time-dependent changes in the ethanol concentration on the structural dynamics of liposomes are not clearly understood. Herein, we investigated ethanol-induced lipid bilayer changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations. The time-resolved SAXS measurement system revealed that single unilamellar liposomes were converted to multilamellar liposomes within 0.8 s of contact with ethanol, and the d-spacing was decreased from 6.1 (w/o ethanol) to 4.4 nm (80% ethanol) with increasing ethanol concentration. We conducted 1 μs MD simulations to understand the molecular-level structural changes in the liposomes. The MD simulations revealed that the changes in the lamellar structure caused by ethanol at the molecular level could explain the structural changes in the liposomes observed via time-resolved SAXS. Therefore, the post-treatment process to remove residual ethanol is critical in liposome formation.

Determination of Reaction Kinetics by Time-Resolved Small-Angle X-ray Scattering during Polymerization-Induced Self-Assembly: Direct Evidence for Monomer-Swollen Nanoparticles.

The kinetics of heterogeneous polymerization is determined directly using small-angle X-ray scattering (SAXS). This important advancement is exemplified for the synthesis of sterically-stabilized diblock copolymer nanoparticles by reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization of benzyl methacrylate (BzMA) in mineral oil at 90 °C. The principle of mass balance is invoked to derive a series of equations for the analysis of the resulting time-resolved SAXS patterns. Importantly, there is a continuous change in the X-ray scattering length density for the various components within the reaction mixture. This enables the volume fraction of unreacted BzMA monomer to be calculated at any given time point, which enables the polymerization kinetics to be monitored in situ directly without relying on supplementary characterization techniques. Moreover, SAXS enables the local concentration of both monomer and solvent within the growing swollen nanoparticles to be determined during the polymerization. Data analysis reveals that the instantaneous rate of BzMA polymerization is proportional to the local monomer concentration within the nanoparticles. In principle, this powerful new time-resolved SAXS approach can be applicable to other heterogeneous polymerization formulations.

Solvent selectivity governed self-assembly of block copolymer in nanofabrication

Block copolymer-based nanoproducts including particles, fibers and films show promise in high-end applications including nanotemplating, nanolithography, and nanoporous membranes due to their advantageous self-assembled structure. However, few studies probe the nanofabrication process in detail and monitor the concomitant self-assembled structural transitions. Herein, time-resolved small-angle X-ray scattering (SAXS) is exploited to follow such nanostructural changes. Evaporation-induced casting, electrospraying or electrospinning, followed by a final solvent vapor annealing step, have been used to create a series of nanoproducts with reversibly tunable morphologies from a model block copolymer, polystyrene-b-poly(ethylene-co-butylene)-b-polystyrene (SEBS). During evaporation-induced casting, which is considered as an unconfined system, a selective (co)solvent system composed of tetrahydrofuran (THF) and dimethylformamide (DMF) can lead to the formation of various SEBS structures. For DMF-based unconfined systems, the final SEBS lamellar structures were found to be different from the structure formed from THF only, demonstrating the critical role of solvent selection in evaporation-induced casting. The comparison of SEBS electrospun fibers reveals the unique and complex self-assembled structural transition occurring in a confined system. Time-resolved SAXS studies of our model triblock copolymer provide guidelines for a more general approach to access various nanostructures from self-assembling block copolymers.

Equilibrium and time-resolved SAXS on BioCAT

Equilibrium and time-resolved SAXS on BioCAT

In Situ Monitoring of Polymerization-Induced Self-Assembly and Gelation During the Synthesis of Triblock Copolymers via Time-Resolved Small-Angle X-ray Scattering and Rheology

<i>In Situ</i> Monitoring of Polymerization-Induced Self-Assembly and Gelation During the Synthesis of Triblock Copolymers <i>via</i> Time-Resolved Small-Angle X-ray Scattering and Rheology

Conceptual Design of a Synchrotron Beamline Dedicated to Ultrafast Time-Resolved Small-Angle X-Ray Scattering

Conceptual Design of a Synchrotron Beamline Dedicated to Ultrafast Time-Resolved Small-Angle X-Ray Scattering

Beyond the standard model of solubilization: Non-ionic surfactants induce collapse of lipid vesicles into rippled bilamellar nanodiscs

HypothesisAlthough solubilization of lipid membranes has been studied extensively, questions remain regarding the structural pathways and metastable structures involved. This study investigated whether the non-ionic detergent Triton X-100 follows the classical solubilization pathway or if intermediate nanostructures are formed. ExperimentsSmall angle X-ray and neutron scattering (SAXS/SANS) was used in combination with transmission electron cryo-microscopy and cryo-tomography to deduce the structure of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles and Triton X-100. Time-resolved SAXS and dynamic light scattering were used to investigate the kinetics of the process. FindingsUpon addition of moderate detergent amounts at low temperatures, the lipid vesicles implode into ordered rippled bilamellar disc structures. The bilayers arrange in a ripple phase to accommodate packing constraints caused by inserted TX-100 molecules. The collapse is suggested to occur through a combination of water structure destabilization by detergents flipping across the membrane and osmotic pressure causing interbilayer attraction internally. The subsequently induced ripples then stabilize the aggregates and prevent solubilization, supported by the observation that negatively charged vesicles undergo a different pathway upon TX-100 addition, forming large bicelles. The findings demonstrate the richness in assembly pathways of simple lipids and detergents and stimulate considerations for the use of certain detergents in membrane solubilization.

Time-Resolved Small-Angle X-Ray Scattering of Protein Cage Assembly.

Recent improvements in X-ray detectors and synchrotron light sources have made it possible to measure time-resolved small-angle X-ray scattering (TR-SAXS) at millisecond time resolution. As an example, in this chapter we describe the beamline setup, experimental scheme, and the points that should be noted in stopped-flow TR-SAXS experiments for investigating the ferritin assembly reaction.

Time-Resolved Small-Angle X-ray Scattering Studies during the Aqueous Emulsion Polymerization of Methyl Methacrylate.

Recently, we reported time-resolved synchrotron small-angle X-ray scattering (TR-SAXS) studies during aqueous emulsion polymerization using a bespoke stirrable reaction cell (J. Am. Chem. Soc. 2021, 143, 1474-1484). This proof-of-concept study utilized a semifluorinated specialty monomer (2,2,2-trifluoroethyl methacrylate) to ensure high X-ray contrast relative to water. Herein, we extend this approach to emulsion polymerization of methyl methacrylate (MMA) in the presence or absence of sodium dodecyl sulfate (SDS) at 70 °C. Solution conductivity measurements for this anionic surfactant indicated a critical micelle concentration (CMC) of 10.9 mM at this temperature. Thus, SDS was employed at either 1.0 or 20.0 mM, which corresponds to well below or well above its CMC. Postmortem analysis by 1H NMR spectroscopy indicated MMA conversions of 93-95% for these three formulations. We demonstrate that the X-ray contrast between water and PMMA is sufficiently large to produce high-quality scattering patterns during TR-SAXS experiments. Such patterns were fitted using a hard-sphere scattering model to monitor the evolution in particle diameter. This enabled (i) determination of the time point for the onset of nucleation and (ii) the evolution in particle size to be monitored during the MMA polymerization. The final particle diameters obtained from such TR-SAXS studies were consistent with postmortem DLS analyses, while TEM studies confirmed that near-monodisperse latex particles were formed. Micellar nucleation occurs within just 2 min when the SDS concentration is well above its CMC, resulting in a high particle number concentration and relatively small latex particles. In contrast, when SDS is either absent or present below its CMC, particle nuclei are formed by homogeneous nucleation over significantly longer time scales (14-15 min). In the latter case, adsorption of SDS onto nascent particles reduces their coagulation, giving rise to a larger number of smaller particles compared to the surfactant-free polymerization. However, the characteristic time required for the onset of nucleation is barely affected because this is mainly controlled by the kinetics of homogeneous polymerization of the relatively water-soluble MMA monomer within the aqueous phase. These results suggest that the aqueous emulsion polymerization of several other (meth)acrylic monomers, and perhaps also vinyl acetate, may be amenable to TR-SAXS studies.

Investigations of structural and dynamical mechanisms of ice formation regulated by graphene oxide nanosheets.

Recent research indicates that graphene oxide (GO) nanosheets can be used to regulate ice formation by controlling critical ice nucleus growth in water at supercooling temperatures. In addition, the study of ice formation mechanisms regulated by GO nanosheets, a good model system for antifreeze proteins (AFPs), will shed light on how AFPs regulate ice formation in nature. In this work, time-resolved small-angle x-ray scattering (TR-SAXS) and quasi-elastic neutron scattering (QENS) experiments were carried out to investigate the structural and dynamical mechanisms of ice formation regulated by GO nanosheets. Strikingly, a transient intermediate state was observed in TR-SAXS experiments that only exists in the aqueous dispersions with a larger GO size (11 nm). This serves as evidence that the size of GO is critical for regulating ice formation. Elastic neutron scattering results indicate that ice is formed in all samples and thermal hysteresis occurs in GO aqueous dispersions in both H2O and D2O. The structural and dynamics information about water molecules in GO, extracted from QENS, reveals different dynamical behaviors of water molecules in GO aqueous dispersions when approaching the ice formation temperature.

Physiochemical Changes to TTCF Ensilication Investigated Using Time-Resolved SAXS

Successful eradication or control of prevailing infectious diseases is linked to vaccine efficacy, stability, and distribution. The majority of protein-based vaccines are transported at fridge (2–8 °C) temperatures, cold chain, to retain potency. However, this has been shown to be problematic. Proteins are inherently susceptible to thermal fluctuations, occurring during transportation, causing them to denature. This leads to ineffective vaccines and an increase in vaccine-preventable diseases, especially in low-income countries. Our research utilises silica to preserve vaccines at room temperature, removing the need for cold chain logistics. The methodology is based upon sol–gel chemistry in which soluble silica is employed to encapsulate and ensilicate vaccine proteins. This yields a protein-loaded silica nanoparticle powder which is stored at room temperature and subsequently released using a fast chemical process. We have previously shown that tetanus toxin C fragment (TTCF) ensilication is a diffusion-limited cluster aggregation (DLCA)-based process using time-resolved small-angle x-ray scattering (SAXS). Here, we present our expanded investigation on the modularity of this system to further the understanding of ensilication via time-resolved SAXS. Our results show that variations in the ensilication process could prove useful in the transition from batch to in-flow manufacturing of ensilicated nanoparticles.

Magnetically-stimulated transformations in the nanostructure of PEGylated phytantriol-based nanoparticles for on-demand drug release

Lipid-based liquid crystalline (LLC) systems are formed by the self-assembly of lipid materials in aqueous environments. The internal nanostructures of LLC systems can be manipulated using remote stimuli and have the potential to serve as ‘on-demand’ drug delivery systems. In this study, a magnetically-responsive system that displayed a transition in nanostructure from liposomes to cubosomes/hexasomes under external alternating magnetic field (AMF) was established by the incorporation of iron oxide nanoparticles (IONPs) into a PEGylated phytantriol (PHYT)-based LLC system. Small angle X-ray scattering (SAXS) was utilized to assess the equilibrium phase behaviour of the systems with different compositions of the lipids to find the optimized formulation. Time-resolved SAXS was then used to determine the dynamic transformation of nanostructures of the IONP-containing systems with the activation of AMF. The formulation containing PHYT and DSPE-PEG2000 at a 95 to 5 molar percent ratio produced a transition from lamellar phase to bicontinuous cubic phase, showing a slow-to-fast drug release profile. Inclusion of either 5 nm or 15 nm IONPs imparted magnetic-responsiveness to the system. The magnetically-responsive system produced an ‘on-demand’ drug delivery system from which the drug release was able to be triggered externally by AMF-stimulation.

Molecular Weight Dependence of Block Copolymer Micelle Fragmentation Kinetics.

The effect of molecular weight (M) on the fragmentation kinetics of micelles formed by 1,2-polybutadiene-block-poly(ethylene oxide) (PB-PEO) copolymers was studied in the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. A series of six samples, with total M ranging from 104 to 105 g mol-1 and nearly constant composition (fPEO ≈ 0.4), were examined; all six formed spherical micelles with PEO coronas. Nonequilibrium PB-PEO micelles were prepared by direct dissolution, a process that systematically produces nanoparticles with mean aggregation numbers more than twice the equilibrium values. When subjected to high temperature annealing (170 °C), the average micelle radius was found to decrease substantially, as determined by temperature-jump dynamic light scattering (T-jump DLS) and time-resolved small-angle X-ray scattering (TR-SAXS). The characteristic fragmentation times (τ) were found to increase strongly with increasing degree of polymerization N, as τ ∼ N1.8. This result compares favorably with the prediction of a previously untested model.

"Host in Host" Supramolecular Core-Shell Type Systems Based on Giant Ring-Shaped Polyoxometalates.

Herein, we show how the chaotropic effect arising from reduced molybdate ions in acidified aqueous solution is able to amplify drastically weak supramolecular interactions. Time-resolved Small Angle X-ray Scattering (SAXS) analysis suggests that molybdenum-blue oligomeric species form huge aggregates in the presence of γ-cyclodextrin (γ-CD) which results in the fast formation of nanoscopic {Mo154 }-based host-guest species, while X-ray diffraction analysis reveals that the ending-point of the scenario results in an unprecedented three-component well-ordered core-shell-like motif. A similar arrangement was found by using preformed hexarhenium chalcogenide-type cluster [Re6 Te8 (CN)6 ]4- as exogenous guest. This seminal work brings better understanding of the self-assembly processes in general and gives new opportunities for practical applications in the design of complex multicomponent materials via the simplicity of the non-covalent chemistry.

Co-assembly and Structure of Sodium Dodecylsulfate and other n-Alkyl Sulfates in Glycerol: n-Alkyl Sulfate-Glycerol Crystal Phase

Hypothesis.Following the observation of a microfibrillar phase in sodium dodecylsulfate (SDS)-glycerol mixtures, it is hypothesized that this phase is a crystalline structure containing SDS and glycerol, where the interaction between sulfate and glycerol layers mediates the co-assembly, which also could be universal for similar systems formed by n-alkyl sulfate homologues.Experiment.n-alkyl sulfate glycerol solutions were studied using a combination of optical microscopy, small- and wide-angle X-ray scattering (SAXS/WAXS). Time-resolved SAXS was employed to determine the phase formation in SDS-glycerol-water mixtures.Findings.The microfibrillar crystalline phase was reproduced in even-chained n-alkyl sulfates with a chain length between 12 and 18 carbon atoms, where the phase lamellar period increased uniformly with the alkyl chain length. Reconstruction of electron density profiles from the diffraction patterns allowed the lamellar structural motif of the phase, the glycerol location and stoichiometry to be determined. When SDS-glycerol-water mixtures with water concentration below 6 wt% are isothermally solidified at 20 °C, SDS-glycerol crystals and/or anhydrous SDS form, where the former is inhibited by the latter at higher water concentrations. The learnings from the SDS-glycerol phase formation allows new gels to be created, utilising the glycerol-sulfate motif generating microfibrils. This expands the knowledge of the applicable formulation space for SDS-water containing mixtures.

Time-Resolved Small-Angle X-ray Scattering Studies during Aqueous Emulsion Polymerization.

The persulfate-initiated aqueous emulsion polymerization of 2,2,2-trifluoroethyl methacrylate (TFEMA) is studied by time-resolved small-angle X-ray scattering (SAXS) at 60 °C using a stirrable reaction cell. TFEMA was preferred to styrene because it offers much greater X-ray scattering contrast relative to water, which is essential for sufficient temporal resolution. The evolution in particle size is monitored by both in situ SAXS and ex situ DLS in the absence or presence of an anionic surfactant (sodium dodecyl sulfate, SDS). Post-mortem SAXS studies confirmed the formation of well-defined spherical latexes, with volume-average diameters of 353 ± 9 nm and 68 ± 4 nm being obtained for the surfactant-free and SDS formulations, respectively. 1H NMR spectroscopy studies of the equivalent laboratory-scale formulations indicated TFEMA conversions of 99% within 80 min and 93% within 60 min for the surfactant-free and SDS formulations, respectively. Comparable polymerization kinetics are observed for the in situ SAXS experiments and the laboratory-scale syntheses, with nucleation occurring after approximately 6 min in each case. After nucleation, scattering patterns are fitted using a hard sphere scattering model to determine the evolution in particle growth for both formulations. Moreover, in situ SAXS enables identification of the three main intervals (I, II, and III) that are observed during aqueous emulsion polymerization in the presence of surfactant. These intervals are consistent with those indicated by solution conductivity and optical microscopy studies. Significant differences between the surfactant-free and SDS formulations are observed, providing useful insights into the mechanism of emulsion polymerization.

Diatom-inspired self-assembly for silica thin sheets of perpendicular nanochannels

HypothesisMultistage silicate self-organization into light-weight, high-strength, hierarchically patterned diatom frustules carries hints for innovative silica-based nanomaterials. With sodium silicate in a biomimetic sol-gel system templated by a tri-surfactant system of hexadecyltrimethylammonium bromide, sodium dodecylsulfate, and poly(oxyethylene-b-oxypropylene-b-oxyethylene) (P123), mesoporous silica nanochannel plates with perpendicular channel orientation are synthesized. The formation process, analogous to that of diatom frustules, is postulated to be directed by an oriented self-assembly of the block copolymer micelles shelled with charged catanionic surfactants upon silication. ExperimentsThe postulated formation process for the oriented silica nanochannel plates was investigated using time-resolved small-angle X-ray and neutron scattering (SAXS/SANS) and freeze fracture replication transmission electron microscopy (FFR-TEM). FindingsWith fine-tuned molar ratios of the anionic, cationic, and nonionic surfactants, the catanionic combination and the nonionic copolymer form charged, prolate ternary micelles in aqueous solutions, which further develop into prototype monolayered micellar plates. The prolate shape and maximized surfactant adsorption of the complex micelles, revealed from combined SAXS/SANS analysis, are of critical importance in the subsequent micellar self-assembly upon silicate deposition. Time-resolved SAXS and FFR-TEM indicate that the silicate complex micelles coalesce laterally into the prototype micellar nanoplates, which further fuse with one another into large sheets of monolayered silicate micelles of in-plane lamellar packing. Upon silica polymerization, the in-plane lamellar packing of the micelles further transforms to 2D hexagonal packing of vertically oriented silicate channels. The unveiled structural features and their evolution not only elucidate the previously unresolved self-assembly process of through-thickness silica nanochannels but also open a new line of research mimicking free-standing frustules of diatoms.

Sub-100 µs time-resolved SAXS at BioCAT

Sub-100 µs time-resolved SAXS at BioCAT