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

Due to the fast diffusion, small molecules such as hydronium ions (H₃O⁺) are expected to be homogeneously distributed, even close to the site-of-origin. Given the importance of H₃O⁺ in numerous processes, it is surprising that H₃O⁺ concentration ([H₃O⁺]) has yet to be profiled near its generation site with nanometer resolution. Here, we innovated a single-molecule method to probe [H₃O⁺] in nanometer proximity of individual alkaline phosphatases. We designed a mechanophore with cytosine (C)-C mismatch pairs in a DNA hairpin. Binding of H₃O⁺ 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 [H₃O⁺] 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.