The facilitated surface diffusion of transiently adsorbing molecules in a planar confined microenvironment (i.e., slit-like confinement) is highly relevant to biological phenomena, such as extracellular signaling, as well as numerous biotechnology systems. Here, we studied the surface diffusion of individual proteins confined between two symmetric lipid bilayer membranes, under a continuum of confinement heights, using single-molecule tracking and convex lens-induced confinement as well as hybrid, kinetic Monte Carlo simulations of a generalized continuous time random walk process. Surface diffusion was observed to vary non-monotonically with confinement height, exhibiting a maximum at a height of ∼750 nm, where diffusion was nearly 40% greater than that for a semi-infinite system. This demonstrated that planar confinement can, in fact, increase surface diffusion, qualitatively validating previous theoretical predictions. Simulations reproduced the experimental results and suggested that confinement enhancement of surface diffusion for symmetric systems is limited to cases where the adsorbate exhibits weak surface sticking.
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