Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase.

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Development of an atmospheric plasma jet device for versatile treatment of electron microscope sample grids.

Atmospheric-pressure plasmas have been widely applied for surface modification and biomedical treatment because of their ability to generate highly reactive radicals and charged particles. In negative-stain electron microscopy (Neg-EM) and cryogenic electron microscopy (cryo-EM), plasmas have been used to generate hydrophilic surfaces and eliminate surface contaminants to embed specimens onto grids. In addition, plasma treatment is a prerequisite for negative-stain and Quantifoil grids, whose surfaces are coated with hydrophobic amorphous carbon. Although the conventional glow discharge system has been used successfully in this purpose, there has been no further effort to take an advantage from the recent progress in the plasma field. Here, we developed a nonthermal atmospheric plasma jet system as an alternative tool for treatment of surfaces. The low-temperature plasma is a nonequilibrium system that has been widely used in biomedical area. Unlike conventional glow discharge systems, the plasma jet system successfully cleans and introduces hydrophilicity on the grid surface in the ambient environment without a vacuum. Therefore, we anticipate that the plasma jet system will have numerous benefits, such as convenience and versatility, as well as having potential applications in surface modification for both negative-stain and cryo-EM grid treatment.

A4 Paper Chemistry: Synthesis of a Versatile and Chemically Modifiable Cellulose Membrane.

Multifunctional cellulose membranes were developed from A4-sized printing paper via chemical modification. A4 paper is a widely used and easily accessible product with high cellulose content. Inspired by cellulose chemistry, we report a simple modification of the A4 paper, converting it from a common office supply to a user-modifiable functionalized cellulose membrane for practical applications. The hydroxyl groups of cellulose enable a facile tuning of its internal structure and polarity via chemical modification. In addition, the functionalized cellulose membrane has more stable mechanical strength compared to commercial cellulose-based filtration membranes. As a proof-of-concept, we demonstrate the separation of a water/oil mixture using the functionalized A4 membrane; we have extended this idea to origami-assisted membrane applications. Finally, this versatile A4 paper chemistry may offer a promising strategy for the development of functional membranes.

Layer-by-Layer Assembly: Recent Progress from Layered Assemblies to Layered Nanoarchitectonics.

As an emerging concept for the development of new materials with nanoscale features, nanoarchitectonics has received significant recent attention. Among the various approaches that have been developed in this area, the fixed-direction construction of functional materials, such as layered fabrication, offers a helpful starting point to demonstrate the huge potential of nanoarchitectonics. In particular, the combination of nanoarchitectonics with layer-by-layer (LbL) assembly and a large degree of freedom in component availability and technical applicability would offer significant benefits to the fabrication of functional materials. In this Minireview, recent progress in LbL assembly is briefly summarized. After introducing the basics of LbL assembly, recent advances in LbL research are discussed, categorized according to physical, chemical, and biological innovations, along with the fabrication of hierarchical structures. Examples of LbL assemblies with graphene oxide are also described to demonstrate the broad applicability of LbL assembly, even with a fixed material.

Reduction-Triggered Self-Cross-Linked Hyperbranched Polyglycerol Nanogels for Intracellular Delivery of Drugs and Proteins.

Owing to the unique advantages of combining the characteristics of hydrogels and nanoparticles, nanogels are actively investigated as a promising platform for advanced biomedical applications. In this work, a self-cross-linked hyperbranched polyglycerol nanogel is synthesized using the thiol-disulfide exchange reaction based on a novel disulfide-containing polymer. A series of structural analyses confirm the tunable size and cross-linking density depending on the type of polymer (homo- or copolymer) and the amount of reducing agent, dithiothreitol, used in the preparation of the nanogels. The nanogels retain not only small molecular therapeutics irrespective of hydrophilic and hydrophobic nature but also large enzymes such as ß-galactosidase by exploiting the self-cross-linking chemistry. Their superior biocompatibility together with the controllable release of active therapeutic agents suggests the applicability of nanogels in smart drug delivery systems.

Bioinspired Catecholic Primers for Rigid and Ductile Dental Resin Composites.

In the construction of dental restorative polymer composite materials, surface priming on mineral fillers is essential to improve the mechanical performance of the composites. Here we present bioinspired catechol-functionalized primers for a tougher dental resin composite containing glass fillers. The catecholic primers with different polymerizable end groups were designed and then coated on glass surfaces using a simple drop-casting or dip-coating process. The surface binding ability and possible cross-linking (coupling or chemical bridging between the glass substrate and the dental resin) of the catecholic bifunctional primers were evaluated using atomic force microscopy, contact angle measurements, and the knife shear bonding test and compared to a state-of-the-art silane-based coupling agent. Various mechanical tests including shrinkage and compression tests of the dental resin composites were also conducted. Compression tests of the composites containing the catecholic primed fillers exhibited enhanced mechanical properties, owing to the bidentate hydrogen bonding of catechol moieties to the oxide mineral surface. Furthermore, the superior biocompatibility of the primed surface was confirmed via cell attachment assay, thus providing applicability of catecholic primers for practical dental and biomedical applications.

Multidimensional Thin Film Hybrid Electrodes with MoS2 Multilayer for Electrocatalytic Hydrogen Evolution Reaction.

Hybrid electrodes are widely used in various energy storage and conversion devices. However, conventional fabrication methods like simple mixing allow only limited control over the internal electrode structure, and it is often difficult to elucidate the structure-property relationship among the electrode components. Taking advantage of the versatile layer-by-layer (LbL) assembly method, herein we report the preparation of electrocatalytic thin film electrodes for hydrogen evolution reaction (HER), highlighting the importance of nanoscale composition in multidimensional hybrid electrodes. The fabrication utilized the electrostatic interaction between the two components: catalytically active two-dimensional MoS2 nanosheets and conductive, one-dimensional multiwalled carbon nanotube (MWNT) support. The electrocatalytic activity was found to be highly tunable by adjusting the thickness of the electrode, suggesting structural dependence of electron transfer and mass transport between the electrolyte and electrode, which is otherwise difficult to investigate in electrodes fabricated by simple conventional methods. Furthermore, the detailed mechanism of HER on the hybrid electrode was also investigated, revealing the fine balance between the catalytic activity of MoS2 and conductivity of MWNT. We anticipate that this unique approach will offer new insights into the nanoscale control of electrode architecture and the development of novel electroactive catalysts.