Next, we used 1 H NMR titration to investigate the pH sensing mechanism of Mito-pH2 (Scheme 2). We studied the structural changes of Mito-pH2 in DMSO-d6 alone or after adding base (trimethylamine) followed by acid (hydrochloric acid). Figure shows a signal at 9.9 ppm (phenolic hydroxyl group) that is seen in DMSO-d6 alone, is no longer observed on the addition of base. However, when the solvent mixture was adjusted to an acidic environment with aqueous HCl, the phenol hydroxyl proton peak reappeared (Figure ). This suggests the pH response Mito-pH2 as seen in absorption and fluorescence changes arises from the deprotonation of phenolic hydroxyl group (Scheme 2).