Digital fluorescent pH probes: polymer-based design, fluorescence response, mechanism, functional tuning and...

We report fluorescent digital pH probes based on a polymeric design comprising thermoresponsive, proton-binding and environment-sensitive fluorescent units. At selected temperatures, fluorescence switching was nearly complete, with a slight pH variation of <1 unit. The fluorescence-switching mechanism involved a proton-induced three-dimensional structural change in the polymer, which altered local hydrophobicity near the environment-sensitive fluorescent units. Based on the change in local hydrophobicity during operation, extremely sharp responses were produced because of the variation in proton-binding abilities of the probes. The polymeric design ensured functional flexibility, including a pH-responsive region and a direction of fluorescence switching. As an application of fluorescent digital pH probes, intracellular logic operations were performed by varying the pH and temperature. By distinguishing the minute pH and temperature difference inside mammalian cells, a specific condition-high pH and temperature-generated strong fluorescence with an extended fluorescence lifetime, as confirmed via fluorescence-lifetime imaging microscopy.