Advanced manufacturing provides tailor-made solutions for crystallography with x-ray free-electron lasers.

Serial crystallography at large facilities, such as x-ray free-electron lasers and synchrotrons, evolved as a powerful method for the high-resolution structural investigation of proteins that are critical for human health, thus advancing drug discovery and novel therapies. However, a critical barrier to successful serial crystallography experiments lies in the efficient handling of the protein microcrystals and solutions at microscales. Microfluidics are the obvious approach for any high-throughput, nano-to-microliter sample handling, that also requires design flexibility and rapid prototyping to deal with the variable shapes, sizes, and density of crystals. Here, we discuss recent advances in polymer 3D printing for microfluidics-based serial crystallography research and present a demonstration of emerging, large-scale, nano-3D printing approaches leading into the future of 3D sample environment and delivery device fabrication from liquid jet gas-dynamic virtual nozzles devices to fixed-target sample environment technology.

Low-Loading Mixed Matrix Materials: Fractal-Like Structure and Peculiarly Enhanced Gas Permeability.

Mixed matrix materials (MMMs) containing metal-organic framework (MOF) nanoparticles are attractive for membrane carbon capture. Particularly, adding <5 mass % MOFs in polymers dramatically increased gas permeability, far surpassing the Maxwell model's prediction. However, no sound mechanisms have been offered to explain this unusual low-loading phenomenon. Herein, we design an ideal series of MMMs containing polyethers (one of the leading polymers for CO2/N2 separation) and discrete metal-organic polyhedra (MOPs) with cage sizes of 2-5 nm. Adding 3 mass % MOP-3 in a polyether increases the CO2 permeability by 100% from 510 to 1000 Barrer at 35 °C because of the increased gas diffusivity. No discernible changes in typical physical properties governing gas transport properties are detected, such as glass transition temperature, fractional free volume, d-spacing, etc. We hypothesize that this behavior is attributed to fractal-like networks formed by highly porous MOPs, and for the first time, we validate this hypothesis using small-angle X-ray scattering analysis.

Thinner than a knife's edge: 3D-printed liquid sheet jet technology for solution phase XFEL experiments.

In this commentary, we explore the pioneering implementation of 3D-printed thin liquid sheet devices for advanced X-ray scattering and spectroscopy experiments at high-repetition rate XFELs.

Pressure-controlled magnetism in 2D molecular layers.

Long-range magnetic ordering of two-dimensional crystals can be sensitive to interlayer coupling, enabling the effective control of interlayer magnetism towards voltage switching, spin filtering and transistor applications. With the discovery of two-dimensional atomically thin magnets, a good platform provides us to manipulate interlayer magnetism for the control of magnetic orders. However, a less-known family of two-dimensional magnets possesses a bottom-up assembled molecular lattice and metal-to-ligand intermolecular contacts, which lead to a combination of large magnetic anisotropy and spin-delocalization. Here, we report the pressure-controlled interlayer magnetic coupling of molecular layered compounds via chromium-pyrazine coordination. Room-temperature long-range magnetic ordering exhibits pressure tuning with a coercivity coefficient up to 4 kOe/GPa, while pressure-controlled interlayer magnetism also presents a strong dependence on alkali metal stoichiometry and composition. Two-dimensional molecular interlayers provide a pathway towards pressure-controlled peculiar magnetism through charge redistribution and structural transformation.

Palladium-Percolated Networks Enabled by Low Loadings of Branched Nanorods for Enhanced H2 Separations.

Nanoparticles (NPs) at high loadings are often used in mixed matrix membranes (MMMs) to improve gas separation properties, but they can lead to defects and poor processability that impede membrane fabrication. Herein, it is demonstrated that branched nanorods (NRs) with controlled aspect ratios can significantly reduce the required loading to achieve superior gas separation properties while maintaining excellent processability, as demonstrated by the dispersion of palladium (Pd) NRs in polybenzimidazole for H2 /CO2 separation. Increasing the aspect ratio from 1 for NPs to 40 for NRs decreases the percolation threshold volume fraction by a factor of 30, from 0.35 to 0.011. An MMM with percolated networks formed by Pd NRs at a volume fraction of 0.039 exhibits H2 permeability of 110 Barrer and H2 /CO2 selectivity of 31 when challenged with simulated syngas at 200 °C, surpassing Robeson's upper bound. This work highlights the advantage of NRs over NPs and nanowires and shows that right-sizing nanofillers in MMMs is critical to construct highly sieving pathways at minimal loadings. This work paves the way for this general feature to be applied across materials systems for a variety of chemical separations.

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

Microfluidic polyimide gas dynamic virtual nozzles for serial crystallography.

Free liquid jets are a common sample delivery method in serial femtosecond x-ray (SFX) crystallography. Gas dynamic virtual nozzles (GDVNs) use an outer gas stream to focus a liquid jet down to a few micrometers in diameter. Such nozzles can be fabricated through various methods (capillary grinding, soft lithography, digital light processing, and two-photon polymerization) and materials, such as glass, polydimethylsiloxane, and photosensitive polyacrylates. Here, we present a broadly accessible, rapid prototyping laser ablation approach to micromachine solvent-resistant and inert Kapton polyimide foils with highly reproducible geometric features that result in 3D flow-focused GDVNs suitable for crystallography experiments at synchrotrons and free-electron laser facilities.

Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.

Sample deposition onto cryo-EM grids: from sprays to jets and back.

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

3D-MiXD: 3D-printed X-ray-compatible microfluidic devices for rapid, low-consumption serial synchrotron crystallography data collection in flow.

Serial crystallography has enabled the study of complex biological questions through the determination of biomolecular structures at room temperature using low X-ray doses. Furthermore, it has enabled the study of protein dynamics by the capture of atomically resolved and time-resolved molecular movies. However, the study of many biologically relevant targets is still severely hindered by high sample consumption and lengthy data-collection times. By combining serial synchrotron crystallography (SSX) with 3D printing, a new experimental platform has been created that tackles these challenges. An affordable 3D-printed, X-ray-compatible microfluidic device (3D-MiXD) is reported that allows data to be collected from protein microcrystals in a 3D flow with very high hit and indexing rates, while keeping the sample consumption low. The miniaturized 3D-MiXD can be rapidly installed into virtually any synchrotron beamline with only minimal adjustments. This efficient collection scheme in combination with its mixing geometry paves the way for recording molecular movies at synchrotrons by mixing-triggered millisecond time-resolved SSX.

Evaluation of serial crystallographic structure determination within megahertz pulse trains.

The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength range suitable for atomic resolution structure determination. An outstanding but crucial question is whether the use of a pulse repetition rate nearly four orders of magnitude higher than previously possible results in unwanted structural changes due to either radiation damage or systematic effects on data quality. Here, separate structures from the first and subsequent pulses in the European XFEL pulse train were determined, showing that there is essentially no difference between structures determined from different pulses under currently available operating conditions at the European XFEL.

A cryo-EM grid preparation device for time-resolved structural studies.

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Šreconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

Solution blow spinning of polymer/nanocomposite micro-/nanofibers with tunable diameters and morphologies using a gas dynamic virtual nozzle.

Uniform endless fibers are ubiquitous and their applications range from functional textiles over biomedical engineering to high-performance filtering and drug delivery systems. Here, we report a new method for the direct, reproducible fabrication of uniform polymer and composite micro-/nanofibers using a microfluidic gas flow focusing nozzle (Gas Dynamic Virtual Nozzle (GDVN)) relinquishing the need for external fiber pulling mechanisms. Compared to other methods, this technique is inexpensive, user-friendly and permits precise fiber diameter control (~250 nm to ~15 µm), high production rate (m/s-range) and direct fiber deposition without clogging due to stable, gas-focused jetting. Control over shape (flat or round) and surface patterning are achieved by simply tuning the air pressure and polymer concentration. The main thinning process happens after the polymer exits the device and is, therefore, mostly independent of the nozzle's internal geometry. Nevertheless, the lithography-based device design is versatile, allowing for precise flow-field control for operation stability as well as particle alignment control. As an example, we demonstrate the successful production of endless hematite nanocomposite fibers which highlights this technology's exciting possibilities that can lead to the fabrication of multifunctional/stimuli-responsive fibers with thermal and electrical conductivity, magnetic properties and enhanced mechanical stability.

3D Micromachined Polyimide Mixing Devices for in Situ X-ray Imaging of Solution-Based Block Copolymer Phase Transitions.

Advances in modern interface- and material sciences often rely on the understanding of a system's structure-function relationship. Designing reproducible experiments that yield in situ time-resolved structural information at fast time scales is therefore of great interest, e.g., for better understanding the early stages of self-assembly or other phase transitions. However, it can be challenging to accurately control experimental conditions, especially when samples are only available in small amounts, prone to agglomeration, or if X-ray compatibility is required. We address these challenges by presenting a microfluidic chip for triggering dynamics via rapid diffusive mixing for in situ time-resolved X-ray investigations. This polyimide/Kapton-only-based device can be used to study the structural dynamics and phase transitions of a wide range of colloidal and soft matter samples down to millisecond time scales. The novel multiangle laser ablation three-dimensional (3D) microstructuring approach combines, for the first time, the highly desirable characteristics of Kapton (high X-ray stability with low background, organic solvent compatibility) with a 3D flow-focusing geometry that minimizes mixing dispersion and wall agglomeration. As a model system, to demonstrate the performance of these 3D Kapton microfluidic devices, we selected the non-solvent-induced self-assembly of biocompatible and amphiphilic diblock copolymers. We then followed their structural evolution in situ at millisecond time scales using on-the-chip time-resolved small-angle X-ray scattering under continuous-flow conditions. Combined with complementary results from 3D finite-element method computational fluid dynamics simulations, we find that the nonsolvent mixing is mostly complete within a few tens of milliseconds, which triggers initial spherical micelle formation, while structural transitions into micelle lattices and their deswelling only occur on the hundreds of milliseconds to second time scale. These results could have an important implication for the design and formulation of amphiphilic polymer nanoparticles for industrial applications and their use as drug-delivery systems in medicine.

Time-Resolved Analysis of the Structural Dynamics of Assembling Gold Nanoparticles.

The hydrophobic collapse is a structural transition of grafted polymer chains in a poor solvent. Although such a transition seems an intrinsic event during clustering of polymer-stabilized nanoparticles in the liquid phase, it has not been resolved in real time. In this work, we implemented a microfluidic 3D-flow-focusing mixing reactor equipped with real-time analytics, small-angle X-ray scattering (SAXS), and UV-vis-NIR spectroscopy to study the early stage of cluster formation for polystyrene-stabilized gold nanoparticles. The polymer shell dynamics obtained by in situ SAXS analysis and numerical simulation of the solvent composition allowed us to map the interaction energy between the particles at early state of solvent mixing, 30 ms behind the crossing point. We found that the rate of hydrophobic collapse depends on water concentration, ranging between 100 and 500 nm/s. Importantly, we confirmed that the polymer shell collapses prior to the commencement of clustering.

A microfluidic flow-focusing device for low sample consumption serial synchrotron crystallography experiments in liquid flow.

Serial synchrotron crystallography allows low X-ray dose, room-temperature crystal structures of proteins to be determined from a population of microcrystals. Protein production and crystallization is a non-trivial procedure and it is essential to have X-ray-compatible sample environments that keep sample consumption low and the crystals in their native environment. This article presents a fast and optimized manufacturing route to metal-polyimide microfluidic flow-focusing devices which allow for the collection of X-ray diffraction data in flow. The flow-focusing conditions allow for sample consumption to be significantly decreased, while also opening up the possibility of more complex experiments such as rapid mixing for time-resolved serial crystallography. This high-repetition-rate experiment allows for full datasets to be obtained quickly (∼1 h) from crystal slurries in liquid flow. The X-ray compatible microfluidic chips are easily manufacturable, reliable and durable and require sample-flow rates on the order of only 30 µl h-1.

Splitting and separation of colloidal streams in sinusoidal microchannels.

The control of the distribution of colloidal particles in microfluidic flows plays an important role in biomedical and industrial applications. A particular challenge is to induce cross-streamline migration in laminar flows, enabling the separation of colloidal particles according to their size, shape or elasticity. Here we show that viscoelastic fluids can mediate cross-streamline migration of deformable spherical and cylindrical colloidal particles in sinusoidal microchannels at low Reynolds numbers. For colloidal streams focused into the center of the channel entrance this leads to a symmetric stream-splitting and separation into four substreams. The degree of stream splitting and separation can be controlled via the flow rates, viscoelasticity of the focusing fluid, and the spatial microchannel modulation with an upper limit when reaching the microchannel walls. We demonstrate that this effect can be used to separate flexible particles of different size and shape. This methodology of cross-stream migration has thus great potential for the passive separation of colloids and cells in microfluidic channels.

Microfluidic nozzle device for ultrafine fiber solution blow spinning with precise diameter control.

We present a microfluidic nozzle device for the controlled continuous solution blow spinning of ultrafine fibers. The device is fabricated by soft lithography techniques and is based on the principle of a gas dynamic virtual nozzle for precise three-dimensional gas focusing of the spinning solution. Uniform fibers with virtually endless length can be produced in a continuous process while having accurate control over the fiber diameter. The nozzle device is used to produce ultrafine fibers of perfluorinated copolymers and of polycaprolactone, which are collected and drawn on a rotating cylinder. Hydrodynamics and mass balance quantitatively predict the fiber diameter, which is only a function of flow rate and air pressure, with a small correction accounting for viscous dissipation during jet formation, which slightly reduces the jet velocity. Because of the simplicity of the setup, the precise control of the fiber diameter, the positional stability of the exiting ultrafine fiber and the potential to implement arrays of parallel channels for high throughput, this methodology offers significant benefits compared to existing solution-based fiber production methods.

Novel therapeutic intervention for osteoporosis prepared with strontium hydroxyapatite and zoledronic acid: In vitro and pharmacodynamic evaluation.

Osteoporosis therapeutics has been monopolized mainly by bisphosphonates, which are potent anti-osteoporotic drugs, while they do not promote bone formation or replenish the already resorbed bone. Although strontium substituted hydroxyapatite (SrHA) has been proclaimed to improve bone properties in an osteoporotic animal model, there is no published data on direct delivery of SrHA nanoparticles by bisphosphonate-like zoledronic acid (ZOL) to the bone. Therefore, this study was designed to investigate the potential of using SrHA/ZOL nanoparticle-based drug formulation in an ovariectomized rat model of postmenopausal osteoporosis. SrHA and SrHA/ZOL nanoparticles were prepared and characterized by field-emission scanning electron microscopy (FESEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). Twelve weeks after ovariectomy, rats were treated with either single intravenous dose of SrHA/ZOL (100, 50 or 25µg/kg); ZOL (100µg/kg); or SrHA (100µg/kg). Saline-treated OVX and SHAM-OVX groups served as controls. The energy-dispersive X-ray (EDX) microanalysis of bone specimen obtained from SrHA/ZOL groups yielded range between 64.3±6.7 to 66.9±6.8 of calcium weight (wt) % and 1.64±0.6 to 1.74±0.8 of calcium/phosphorus (Ca/P) ratio which was significantly higher when compared with 39.7±9.3 calcium and 1.30±0.2 Ca/P ratio for OVX group. Moreover, the strontium wt% in SrHA/ZOL group (between 3.1±0.5 and 6.8±0.4) was significantly higher than SrHA group (1.8±0.9). These results confirmed targeted delivery of SrHA nanoparticles by ZOL to the bone. Therapy with SrHA/ZOL showed significant improvements in trabecular bone microarchitecture and mechanical strength as compared to ZOL or SrHA (p<0.05). Moreover, treatment with SrHA/ZOL significantly precluded an increase in serum bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase than either ZOL or SrHA (p<0.05). These results strongly implicate that SrHA/ZOL nanoparticle-based drug formulation showed better efficacy at a much lower dose of ZOL. SrHA/ZOL drug formulation has a therapeutic advantage over ZOL or SrHA monotherapy for experimental osteoporosis.

A customizable software for fast reduction and analysis of large X-ray scattering data sets: applications of the new DPDAK package to small-angle X-ray scattering and grazing-incidence small-angle X-ray scattering.

X-ray scattering experiments at synchrotron sources are characterized by large and constantly increasing amounts of data. The great number of files generated during a synchrotron experiment is often a limiting factor in the analysis of the data, since appropriate software is rarely available to perform fast and tailored data processing. Furthermore, it is often necessary to perform online data reduction and analysis during the experiment in order to interactively optimize experimental design. This article presents an open-source software package developed to process large amounts of data from synchrotron scattering experiments. These data reduction processes involve calibration and correction of raw data, one- or two-dimensional integration, as well as fitting and further analysis of the data, including the extraction of certain parameters. The software, DPDAK (directly programmable data analysis kit), is based on a plug-in structure and allows individual extension in accordance with the requirements of the user. The article demonstrates the use of DPDAK for on- and offline analysis of scanning small-angle X-ray scattering (SAXS) data on biological samples and microfluidic systems, as well as for a comprehensive analysis of grazing-incidence SAXS data. In addition to a comparison with existing software packages, the structure of DPDAK and the possibilities and limitations are discussed.