Buffering agents modify the hydration landscape at charged interfaces.

Buffering agents are widely used to stabilise the pH of solutions in soft matter and biological sciences. They are typically composed of weak acids and bases mixed in an aqueous solution, and can interact electrostatically with charged surfaces such as biomembranes. Buffers can induce protein aggregation and structural modification of soft interfaces, but a molecular-level picture is still lacking. Here we use high-resolution atomic force microscopy to investigate the effect of five commonly used buffers, namely 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), monosodium phosphate, saline sodium citrate (SSC) and tris(hydroxymethyl)aminomethane (Tris) on the hydration landscape of Muscovite mica in solution. Mica is an ideal model substrate due to its negative surface charge and identical lattice parameter when compared with gel-phase lipid bilayers. We show that buffer molecules can produce cohesive aggregates spanning over tens of nanometres of the interface. SSC, Tris and monosodium phosphate tend to create an amorphous mesh layer several molecules thick and with no preferential ordering. In contrast, MES and HEPES adopt epitaxial arrangements commensurate with the underlying mica lattice, suggesting that they offer the most suitable solution for high-resolution studies. To confirm that this effect persisted in biologically-relevant interfaces, the experiments were repeated on a silica-supported lipid bilayer. Similar trends were observed for this system using atomic force microscopy as well as ellipsometry. The effect of the buffering agents can be mitigated by the inclusion of salt which helps displace them from the interface