Graphene-Sealed Flow Cells for In Situ Transmission Electron Microscopy of Liquid Samples.

We introduce a graphene-based nanofluidic cell that facilitates in situ imaging of liquid samples via transmission electron microscopy. The cell combines the benefits of graphene liquid cells-namely, high resolution, reduced charging effects, and excellent sample stability-with the ability to introduce reactants and control fluid concentrations as provided by conventional silicon-nitride-windowed flow cells. The graphene flow cell offers significantly less window bowing compared to existing commercial holders. We demonstrate the performance of the flow cell by imaging gold nanoparticle dynamics and uranyl acetate crystallization. Our results confirm the utility of graphene flow cells in obtaining the high spatial and temporal resolution required for probing the complex dynamics of nanoparticles and nucleation pathways in aqueous solutions.

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis.

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

Symmetry Controlled, Genetic Presentation of Bioactive Proteins on the P22 Virus-like Particle Using an External Decoration Protein.

Viruses use spatial control of constituent proteins as a means of manipulating and evading host immune systems. Similarly, precise spatial control of proteins encapsulated or presented on designed nanoparticles has the potential to biomimetically amplify or shield biological interactions. Previously, we have shown the ability to encapsulate a wide range of guest proteins within the virus-like particle (VLP) from Salmonella typhimurium bacteriophage P22, including antigenic proteins from human pathogens such as influenza. Expanding on this robust encapsulation strategy, we have used the trimeric decoration protein (Dec) from bacteriophage L as a means of controlled exterior presentation on the mature P22 VLP, to which it binds with high affinity. Through genetic fusion to the C-terminus of the Dec protein, either the 17 kDa soluble region of murine CD40L or a minimal peptide designed from the binding region of the "self-marker" CD47 was independently presented on the P22 VLP capsid exterior. Both candidates retained function when presented as a Dec-fusion. Binding of the Dec domain to the P22 capsid was minimally changed across designed constructs, as measured by surface plasmon resonance, demonstrating the broad utility of this presentation strategy. Dec-mediated presentation offers a robust, modular means of decorating the exposed exterior of the P22 capsid in order to further orchestrate responses to internally functionalized VLPs within biological systems.

Higher order assembly of virus-like particles (VLPs) mediated by multi-valent protein linkers.

Two- and three-dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom-up approaches. In this study, higher order assembly of virus-like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer-by-layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi-topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials.

Use of the interior cavity of the P22 capsid for site-specific initiation of atom-transfer radical polymerization with high-density cargo loading.

Virus-like particles (VLPs) have emerged as important and versatile architectures for chemical manipulation in the development of functional hybrid nanostructures. Here we demonstrate a successful site-selective initiation of atom-transfer radical polymerization reactions to form an addressable polymer constrained within the interior cavity of a VLP. Potentially, this protein-polymer hybrid of P22 and cross-linked poly(2-aminoethyl methacrylate) could be useful as a new high-density delivery vehicle for the encapsulation and delivery of small-molecule cargos. In particular, the encapsulated polymer can act as a scaffold for the attachment of small functional molecules, such as fluorescein dye or the magnetic resonance imaging (MRI) contrast agent Gd-diethylenetriaminepentacetate, through reactions with its pendant primary amine groups. Using this approach, a significant increase in the labelling density of the VLP, compared to that of previous modifications of VLPs, can be achieved. These results highlight the use of multimeric protein-polymer conjugates for their potential utility in the development of VLP-based MRI contrast agents with the possibility of loading other cargos.

Site-directed coordination chemistry with P22 virus-like particles.

Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.