Single-Molecule Mechanochemical pH Sensing Revealing the Proximity Effect of Hydroniums Generated by an Alkaline Phosphatase.

Due to the fast diffusion, small molecules such as hydronium ions (H3 O+ ) are expected to be homogeneously distributed, even close to the site-of-origin. Given the importance of H3 O+ in numerous processes, it is surprising that H3 O+ concentration ([H3 O+ ]) has yet to be profiled near its generation site with nanometer resolution. Here, we innovated a single-molecule method to probe [H3 O+ ] in nanometer proximity of individual alkaline phosphatases. We designed a mechanophore with cytosine (C)-C mismatch pairs in a DNA hairpin. Binding of H3 O+ to these C-C pairs changes mechanical properties, such as stability and transition distance, of the mechanophore. These changes are recorded in optical tweezers and analyzed in a multivariate fashion to reduce the stochastic noise of individual mechanophores. With this method, we found [H3 O+ ] increases in the nanometer vicinity of an active alkaline phosphatase, which supports that the proximity effect is the cause for increased rates in cascade enzymatic reactions.