Surface-templated nanobubbles were studied for their ability to provide protective coatings for protein stabilization. Nanobubles are stable gas pockets measuring tens of nm in height and up to thousands of nm in diameter that can form at solid-liquid interfaces such as glass surfaces that are used to store protein therapeutics; the denaturation of which can lead to protein deactivation or aggregation.<br/>To study the effect of surface chemistry on nanobubble stability, glass surfaces were functionalized with copolymer brushes containing mixtures of hydrophobic and hydrophilic segments, exhibiting static water contact angles (CA) ranging from 10 to 75°. On methylated glass, dissolution and redeposition of nanobubbles resulted in reformation in mostly the same locations, consistent with the contact line pinning hypothesis. However, for copolymer-stabilized surfaces the presence of stable nanobubbles was correlated with global surface wettability, rather than local topography. Nanobubbles appeared to be stable when the surface exceeded a critical water CA of 50-60° for polymers containing carboxyl or sulfobetaine groups, respectively, as hydrophilic side chains. The critical CAs were insensitive to the identity of the hydrophobic segments.<br/>The behavior of proteins on the nanobubbles was examined using single-molecule total internal reflection fluorescence (SM-TIRF) microscopy combined with intramolecular Förster Resonance Energy Transfer (FRET). To study protein conformational dynamics, nitroreductase (NfsB) labeled with FRET dyes was prepared by engineering NfsB with noncanonical amino acid substitution and subsequent dye conjugation. Next, nanobubbles formed by a solvent exchange method were imaged using reflection brightfield microscopy, permitting the observation of spatial co-localization of nanobubbles and NfsB adsorption. Virtually all (96%) of NfsB molecules that interacted with nanobubbles remained folded, whereas less than 50% of NfsB molecules remained folded in the absence of nanobubbles, either on unmodified silica or methylated glass surfaces. Ensemble-average fluorometer TIRF experiments were consistent with the SM observations. We hypothesize that nanobubbles reduced protein damage by specifically passivating strongly denaturing topographical surface defects. As such, the nucleation of nanobubbles on surfaces has potential important implications for designing anti-fouling surfaces as well as for stabilizing therapeutic proteins during storage.

Reversible nanobubble surface modifications form protective surface layers at solid/liquid interfaces

2019 Fall National Meeting

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