Liposome are increasingly better developed as efficient drug carriers. Structural characterization of the functional liposomes with and without drug-uptake and the consequent drug-transport and conditional drug-delivery, is of interest yet not well-stablished. Here, we report an established combined methods using high-performance liquid chromatography(HPLC), asymmetric flow field-flow fractionation (AF4), UV-Vis absorption, refractive index (RI), multi-angle laser light scattering (MALLS), dynamic light scattering (DLS), and small-angle X-ray scattering for structural characterization of liposome solutions. We will demonstrate an example of using the integrated system to successfully determine hydrodynamic radius and its distribution, molecular mass, lipid aggregation number, of a model liposome system. The radius of gyration and detailed bilayer structures of the liposome system are determined using simultaneous small- and wide-angle X-ray scattering, incorporated with HPLC/UV-vis/RI, at the high-flux 13A BioSAXS undulator beamline of the 3.0 GeV Taiwan Photon Source. We expect this fast structural characterization system would contribute greatly on drug screening of biomedical industrials.

Often found in the gene chromosome 9 open reading frame 72 (C9ORF72) in the patients of familial frontotemporal dementia (a progressive disorder of the brain) and amyotrophic lateral sclerosis (muscles decreasing in size, resulting in difficulty in speaking, swallowing, and eventually breathing) are segments of abnormal dipeptide repeats, which serve as a signature of the diseases mentioned. Such dipeptide repeating of 10 – 1000 times can be found in the brain or spinal cord of the patients, including toxic Glycine-Arginine (GR)_n. Using the biological small-angle X-ray scattering beamline 13A at the Taiwan Photon Source (TPS), covering a wide range of scattering vector q = 0.01 to 1.0 Å^-1, we observed gradually ordered solution structures of (GR)_n (n = 5, 10, 15, 20, 25, 30) when the n value increases over 20. The model structures of the dipeptide repeats reconstructed based on the SAXS data analysis combined with molecular simulation suggest a possible formation mechanism of the ordered structures. Effect of intervening Prolines into the GR dipeptide repeats is also observed. We note that up to date, there are no crystal structures available for the intrinsically disorder dipeptide repeats.

Incomplete recrystallization after the melt processing of thermoplastics leads to a kinetically frustrated state in plastic packaging. Release of this frustration by random chain scission during environmental exposure and the associated structural relaxation is a mechanism in the embrittlement and fragmentation for these materials.[1] This mechanism is therefore a major route of physical degradation for the majority of plastic waste. The loss of mechanical properties and barrier properties of the material may catalyse further oxidation of microplastic waste and ultimately limit the lifetime of waste in the environment. After establishing the important role of nanostructure in embrittlement and further degradation we discuss the characterization of this process by X-ray scattering, both in the wide (WAXS) and small angle (SAXS) domains using semi-crystalline polyethylene as a model. These two experimental techniques characterize the packing of polymer chains into crystallites and the arrangement of these crystallites into lamellae respectively. These non-destructive bulk characterization techniques with a minimum of sample preparation offers a rapid and convenient access to relevant nanostructural parameters in order to define the temporal relationship between environmental exposure and structural relaxation.[2,3] Thus the perspective of X-ray scattering provides important insight into the lifetime of thermoplastics in the environment and will allow the engineering of more sustainable materials with optimized and controlled degradation, and thus impact on the environment.

Keywords: commodity plastics; plastic packaging; wide angle X-ray scattering; small angle X-ray scattering

C.J.G. acknowledges the CNRS and Université Paris-Sud for financial support during his sabbaticals. We would like to thank the Cooperative Research Centre for Polymers for funding parts of this study under project 2.4, Polyolefin-Biopolymer Films for More Sustainable Agricultural Production.

[1] Garvey, C. J., Impéror-Clerc, M., Rouzière, S., Gouadec, G., Boyron, O., Rowenczyk, L., Mingotaud, A. F. & ter Halle, A. (2020). Environmental Science & Technology 54, 11173-11181.

[2] Hsu, Y. C., Truss, R. W., Laycock, B., Weir, M. P., Nicholson, T. M., Garvey, C. J. & Halley, P. J. (2017). Polymer 119, 66-75.

[3] Hsu, Y. C., Weir, M. P., Truss, R. W., Garvey, C. J., Nicholson, T. M. & Halley, P. J. (2012). Polymer 53, 2385-2393.

In this study, we present the microstructure of various materials, obtained by using small-angle neutron scattering (SANS) in combination with different complementary scattering techniques. SANS resolution enables the investigation of inhomogeneities or precipitates in mesoscopic range with excellent statistics through sample bulk, moreover, it can be applied in-situ. For an example of precipitation of ω and α phases in the β matrix of the metastable β titanium alloy (Ti with 15 wt.% Mo), we show how SANS data can describe temperature resolved evolution of these phases at various heating rates [1]. Small-angle scattering, in this example, allow to overcome detection limits of the neutron diffraction due to the small size of the nanoparticles, and, it helped to demonstrate the coexistence of all three phases at about 550 °C, and to explain the abnormal behaviour of resistivity during constant rate heating.

SANS is an effective tool for the investigation materials containing heavy elements such as W and Co. In vanadium (V)-doped tungsten carbide (WC)-Co composite material system, in-situ and ex-situ SANS and ultra-small-angle neutron scattering (USANS) experiments helped us to delineate how additions of V affect the nano- and microstructure during sintering and result in smaller WC grains [2, 3]. Whereas SANS quantified the nano-scale interfacial layers responsible of grain coarsening inhibition, USANS was applied to study microstructural refinement.

SANS was also applied for the investigation of the Sc-doped TiO₂ anatase as material for photocatalysis. Growth of Sc precipitations was observed with increasing aging temperature (Fig. 1) due to its expelling from anatase crystallites. It was proved by SANS, neutron diffraction, and electron microscopy measurements that whole scandium content at 800 °C was driven out of grains and formed particles at TiO₂ grain boundaries.

[1] Zháňal, P., Ryukhtin, V., Farkas, G., Kadletz, P., Keiderling, U., Wallacher, D., Harcuba, P, (2020) MATEC Web of Conf. 321, 12027. [2] Yildiz A. B., Weidow, J., Ryukhtin, V., Norgrend, S., Wahnström, G., Hedström, P. (2019). Scripta Mat. 173, 106-109.

[3] Yildiz A. B. et al (2021) Materials & Design, 109825, In Press

Metal oxide-based porous nanomaterials are widely synthesized and used for several technological developments based on energy storage, catalytic chemistry, and medical applications [1-3]. In the present study, the newly designed MeOx (Me: Ce, Mn, Si, Ti) nanoparticles were prepared and structurally investigated in molecular, nanoscopic, and microscopic scales by using several complementary experimental (SAXS, SEM) methods. The form factors for elliptical, core-shell oblate, and fractal models were used in SAXS analyses (it can be shown in Figure 1) to characterize the morphologies. Thermal processes were activated at T= 410, 450, and 500 °C to investigate nanostructural properties. The focused targets with the present R&D studies were increasing the surface area of the nanoparticles and reaching the stabilized monodispersed morphologies and uniform distributions. As a result of the study, it was obtained that, the size, shape, and distribution controlled synthesizing processes are possible with thermal treatments. Especially, a critical temperature value of about 400°C is effective on the nanomorphologies of MnO₂ particles. Ellipsoidal fractal units come together to form larger and more compact core-shell oblate shape nanoparticles. Electrochemical measurements were also performed by using a conventional three-electrode system to determine the physicochemical properties. So, it was obtained that, the larger electrochemical capacitance than the commercial electrolytic metal dioxides may be prepared with these nanoparticles. On the other hand, it was also determined that the larger surface area and high porosity of the synthesized TiO₂ nanoparticles besides their well-determined monodispersed and uniformly distributed nanomorphologies may cause various immunomodulatory effects when they are exposed to cells with the purpose of several biochemical and biophysical applications. The in-vitro and in-vivo examinations were also started on the determined nanoparticles made by choice according to their properties to investigate their potential usages in medical applications.

Polyurethane (PU) films are potential candidate substrate for next-generation stretchable electron devices that attract much attention. Both tensile modulus and self-healing of PU films are anticipated yet seemingly mutual excluded properties. Here, metal-ligand coordination is proposed to modify the crystalline and nanostructural features of PU films for concomitantly improved tensile modulus and self-healing. PU films of bpyPTD are prepared from reaction of bpy with PTD of different polarity of solvents such as THF or DMF/THF. Metal precursors solutions of Zn, Ni, were selectively mixed into the bpyPTD solution for cast of the final product films of M-bpyPTD, with M = Ni, Zn. X-ray absorption is used to elucidate how the critical metal-ligand coordination between Zinc (II)/Nickel (II) and bipyridine (bpy) could effectively crosslink the segmented PTD into network structures of Zn-bpyPTD and Ni-bpyPTD.

The network structures of the M-bpyPTD are tested under film stretching at low temperatures and high humidity, using concomitant small- and wide-angle X-ray scattering (SAXS and WAXS). Density (or mean spacing) of the metal-ligand coordination cites could be enriched with the higher polarity solvent for improved tensile modulus. By contrast. improvement of the self-healing capability is reached with enhanced metal-ligand coordination strength of bpy with Ni. Accordingly, Ni-bpyPTD could be fabricated into pyramidal pressure sensors, showing characteristic pressure response with good cyclability for promising applications as electronic skins.

We report on the development of a reference standard for q calibration of small angle scattering measurements. The standard consists of a 100 nm pitch line grating on a silicon nitride membrane. The grating is 100 nm tall and 40 nm wide tungsten lines. Tungsten was selected to give strong scattering intensity while not having absorption features around the carbon k-edge. The silicon nitride membrane allows measurements over a large range of beam energies. The test structure has a 1 µm two-dimensional grating super-imposed on the 100 nm line grating. The superlattice provides additional scattering peaks that can only be resolved in high-resolution configurations. The test structure allows evaluation of q-resolution in addition to calibration of q.

The prototype structure was tested between 250 eV and 24.5 keV and provided strong scattering at all energies. Figure 1 shows an example scattering pattern collected in a 60 s exposure on a laboratory SAXS system using Ga Kalpha. The scattering pattern allows calibration between 0.0006 Å^-1 and 0.1 Å^-1. We will also discuss measurements made at SAXS beamlines and at soft X-ray scattering beamlines.

Advanced composite materials will play a big role to meet growing challenges for future energy solutions. In the transportation sector polymer electrolyte membrane fuel cells (PEMFCs) are promising alternatives to combustion engines to reduce CO₂ emissions. PEMFC generate electricity by electrochemical reactions that take place in a complex environment within a so-called catalyst layer. In recent years, fundamental and applied PEMFC research has continuously been trying to improve its understanding of structure-performance relation of these layers. PEMFC catalyst layers are porous materials built from three different components; chemically active sites, electron conducting support and proton conducting binder. This study focusses on investigating Pt nanoparticles on carbon black support (PtC, HiSPEC3000) spray-coated on a polymer membrane (Nafion 211). We investigate three different samples with varying amounts of binder (PTFE) but an equal loading of catalyst (m_Pt = 0.2 mg/m²).

Scanning SAXS was performed at the cSAXS beamline of the Swiss Light Source (SLS) to study the meso and nanoscale of the samples based on simultaneous measurements of X-ray scattering and fluorescence (XRF) spectroscopy from small regions of 10x10 µm² within the catalyst layers. Figure 1A shows an exemplarily SAXS curve revealing statistical information of the nanostructure by e.g. evaluating the power law scaling at low q. The corresponding XRF spectroscopy data from XRF is particular interesting due to its ability to measure the distribution of Pt in the catalyst layer based on the Pt M_α emission line at 2.05 keV. A major advantage of scanning SAXS is also its capability to probe the data illustrated in Figure 1A/B on a macroscopic length scales, to possibly use these two features, power law scaling exponent at low q (Figure 1C) and the intensity of the Pt M_α (Figure 1D), to generate 2D scattering maps for a 1x1 mm² area. Based on the results obtained from these maps, specific areas were chosen to mill out cylindrical µm-sized pillars using FIB/SEM (see Figure 1F). These pillars were then investigated with the OMNI setup [1] at cSAXS to reveal the 3D nanostructure with ptychography X-ray nanotomography (PXCT) down to a resolution of 26 nm. Figure 1E shows the rendered pore network from one of the catalyst layer color-coded with the pore size distribution (threshold segmented). Currently, we explore different approaches to correlate the imaging data (PXCT) with the statistical data (SAXS and XRF). Our vision is to obtain 3D representative models for the catalyst layers based on the real structure (3D nanostructure PXCT, resolution 26nm), complemented with statistical data from SAXS and XRF down to single nanometer length scale.

[1] M. Holler, M. Guizar-Sicairos, et. Al. (2017). Rev. Sci. Instrum. 88(11),113701.

Keywords: IUCr2020; abstracts; PXCT; SAXS; PEMFC

This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 701647 (PSI-FELLOW-III-3i) and funding from the Chalmers initiative for advancement of neutron and x-ray techniques. The authors acknowledge the funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 681719). We also acknowledge the Paul Scherrer Institut, Villigen, Switzerland for provision of synchrotron radiation beamtime at beamline cSAXS of the SLS.

Small-angle neutron scattering (SANS) has been effectively utilized for structural analysis of biomacromolecular complex in solution because each component can be distinguished by using contrast matching method with selectively deuterated molecules. In particular, inverse contrast matching (iCM) method is quite useful because it can suppress incoherent scattering from hydrogen of solvent water by measuring nearly 75% deuterated biomolecules in 100% heavy water. Meanwhile, SEC-SAS, a combination of inline size-exclusion chromatography (SEC) and small-angle scattering measurements (SAS), has recently been developed to address the problem that undesirable contamination of aggregates and dissociated fragments prevent the precise analysis of target molecules. Currently SEC-SAS has become a popular option for small-angle X-ray scattering (SAXS), but not widely available yet for SANS. In this study, we applied the SEC-SAS technique to the iCM-SANS measurements (SEC-iCM-SANS) of antibody interaction systems: Immunoglobulin G (IgG) or its Fc fragment and 75% deuterated Fc-binding proteins. As a result, we could confirm that bound species were successfully fractionated by SEC excluding aggregates and unbound molecules and immediately subsequent iCM-SANS measurements provided the scattering profiles of the target complexes alone, in which hydrogenated components in the complexes were selectively observable.

Three small-angle X-ray scattering (SAXS) beamlines, BL-6A, BL-10C, and BL-15A2, are in operation at the Photon Factory, a synchrotron radiation facility in Japan, and are commonly utilized for versatile application to carry out the structural analysis and the structure-property correlation studies for soft and hard materials including biological macromolecules. The light source of BL-6A is a bending magnet, and the available X-ray energy is fixed at 8.3 keV (1.5 Å). The maximum camera length is 2.5 m, and SAXS/WAXS (wide-angle X-ray scattering) measurements are performed using PILATUS3 1M (Dectris) for SAXS and PILATUS 100K for WAXS as detectors, respectively. BL-10C is also the bending magnet beamline, and the available X-ray energy range is generally 7.0-14.0 keV (0.89-1.77 Å). The Max. camera length is 3.0 m, and SAXS/WAXS measurements can be performed with PILATUS3 2M for SAXS and PILATUS3 200K for WAXS. BL-15A is the short-period undulator beamline, and 15A1 and 15A2 are dedicated to XAFS and SAXS, respectively. BL-15A2 has two dedicated diffractometers, one for hard X-rays (5.7-15 keV, Max. camera length: 3.5 m) and the other for tender X-rays (2.1-5.4 keV, Max. camera length: 0.8 m), and these are tandemly installed against the beam in BL-15A2. PILATUS3 2M for SAXS and PILATUS3 300K-W for WAXS are installed as detectors. Because this PILATUS3 2M is vacuum compatible, it can be directly connected to the dedicated vacuum diffractometer for tender X-rays use under the vacuum condition. We have also installed a special setting to connect the hard X-ray system to the tender X-ray system. The max. camera length is 6.5 m at that time, and the SAXS resolution reaches 1500 nm using 2.1 keV. The devices for BioSAXS are installed in BL-10C and BL-15A2, which can be used not only for SEC-SAXS but also for titration-SAXS and time-resolved SAXS using microfluidic cells. We will introduce the latest measurement and analysis environment at these beamlines in this presentation.

Structural and functional biophysical studies often require temporal resolution to explore the kinetics of processes in macromolecular systems. The processes like amyloid fibrils formation and protein aggregation involve rapid consequent chemical reactions happening at native conditions [1]. Conformational changes caused by changing conditions have impact on the functionality of biological macromolecules and their complexes. Small-angle X-ray scattering (SAXS) is a structural method allowing one to capture conformational changes and measure kinetics of the macromolecules and complexes in near native solutions [2].

For functional biological complexes, it is important not only to observe structural changes, but also to recognise their biological implications with the help of additional information about the system. The sources if information can be e.g. an atomic model of a given state from cryo-electron microscopy or X-ray crystallography and/or simulated behaviour of the complex (molecular dynamics) under conditions of the time-resolved SAXS experiment [2, 3].

As the analysis of one dimensional SAXS data in terms of three-dimensional (3D) models is an ill-posed problem, and the analysis of kinetics needs the detection of time-dependent changes, characteristic times of the structural changes to need to be defined to analyse large amounts of time-resolved data. To do this, linear methods of reducing the dimensionality of data are being applied for obtaining time dependencies; statistical methods are utilised for the assessment of the importance of the contributing components and machine learning is used for data classification. The ATSAS software [4] is a powerful tool for small-angle scattering data analysis capable to extract rich structural information from the experimental data and also to fit the data with the available 3D models provided by other methods. This allows one to combine the structural information into a biophysical and biochemical evidence.

Although all the available ATSAS tools are straightforward to use, the data analysis still requires significant level of expertise to interactively utilize the tools when dealing with time-resolved studies. In order to optimise and simplify the data analysis procedures for the analysis of processes occurring in biomacromolecular systems, a new pipeline has been developed. The pipeline allows one to perform a comprehensive analysis and incorporates relevant components of ATSAS for the analysis of time-resolved. Its capacity is illustrated by the application to the time-resolved data on amyloid fibrils formation in solution.

[1] Michaels, T. C., et al. (2020) Nature chemistry, 12(5), 445-451.

[2] Svergun, D.I., et al. (2013), Small angle X-ray and neutron scattering from solutions of biological macromolecules. Vol. 19. Oxford University Press.

[3] Vestergaard, B. (2016), Archives of biochemistry and biophysics 602: 69-79.

[4] Manalastas-Cantos, K., et al. (2021) J. Appl. Cryst. 54, 343-355