Self-assembly of three-dimensional solid-state nanostructures containing approximately 33% by weight globular protein is demonstrated using a globular protein-polymer diblock copolymer, providing a route to direct nanopatterning of proteins for use in bioelectronic and biocatalytic materials. A mutant red fluorescent protein, mCherryS131C, was prepared by incorporation of a unique cysteine residue and site-specifically conjugated to end-functionalized poly(N-isopropylacrylamide) through thiol-maleimide coupling to form a well-defined model protein-polymer block copolymer. The block copolymer was self-assembled into bulk nanostructures by solvent evaporation from concentrated solutions. Small-angle X-ray scattering and transmission electron microscopy illustrated the formation of highly disordered lamellae or hexagonally perforated lamellae depending upon the selectivity of the solvent during evaporation. Solvent annealing of bulk samples resulted in a transition towards lamellar nanostructures with mCherry packed in a bilayer configuration and a large improvement in long range ordering. Wide-angle X-ray scattering indicated that mCherry did not crystallize within the block copolymer nanodomains and that the β-sheet spacing was not affected by self-assembly. Circular dichroism showed no change in protein secondary structure after self-assembly, while UV-vis spectroscopy indicated approximately 35% of the chromophore remained optically active.

Non‐regular, device‐oriented structures can be directed to assemble on chemically nanopatterned surfaces such that the density of features in the assembled pattern is multiplied by a factor of two or more compared to the chemical pattern. By blending the block copolymers with homopolymers and designing the chemical pattern rationally, complicated structures such as bends, jogs, junctions, terminations, and combined structures are fabricated. Previously, directed assembly of block copolymers has been shown to enhance the resolution of lithographic processes for hexagonal arrays of spots and parallel lines, corresponding to the bulk morphologies of block copolymer systems, but this is the first demonstration of enhanced resolution for more complicated, device‐oriented features. This fundamental knowledge broadens the range of technologies that can be served by the directed assembly of block copolymers.

Aqueous processing of globular protein-polymer diblock copolymers into solid-state materials and subsequent solvent annealing enables kinetic and thermodynamic control of nanostructure formation to produce block copolymer morphologies that maintain a high degree of protein fold and function. Using model diblock copolymers composed of mCherry-b-poly(N-isopropylacrylamide), orthogonal control over solubility of the protein block through changes in pH and the polymer block through changes in temperature is demonstrated during casting and solvent annealing. Hexagonal cylinders, perforated lamellae, lamellae, or hexagonal and disordered micellar phases are observed depending upon the coil fraction of the block copolymer and the kinetic pathway used for self-assembly. Good solvents for the polymer block produce ordered structures reminiscent of coil-coil diblock copolymers, while an unfavorable solvent results in kinetically trapped micellar structures. Decreasing solvent quality for the protein improves long-range ordering, suggesting that the strength of protein interactions influences nanostructure formation. Subsequent solvent annealing results in evolution of the nanostructures, with the best ordering and the highest protein function observed when annealing in a good solvent for both blocks. While protein secondary structure was found to be almost entirely preserved for all processing pathways, UV-vis spectroscopy of solid-state films indicates that using a good solvent for the protein block enables up to 70% of the protein to be retained in its functional form.

Relationships between leaf wetness and plant diseases have been studied for centuries. The progress and risk of many bacterial, fungal, and oomycete diseases on a variety of crops have been linked to the presence of free water on foliage and fruit under temperatures favorable to infection. Whereas the rate parameters for infection or epidemic models have frequently been linked with temperature during the wet periods, leaf wetness periods of specific time duration are necessary for the propagule germination of most phytopathogenic fungi and for their penetration of plant tissues. Using these types of relationships, disease-warning systems were developed and are now being used by grower communities for a variety of crops. As a component of Integrated Pest Management, disease-warning systems provide growers with information regarding the optimum timing for chemical or biological management practices based on weather variables most suitable for pathogen dispersal or host infection. Although these systems are robust enough to permit some errors in the estimates or measurements of leaf wetness duration, the need for highly accurate leaf wetness duration data remains a priority to achieve the most efficient disease management.