Sensing Mechanism and Excited-State Dynamics of a Widely Used Intracellular Fluorescent pH Probe: pHrodo

Abstract

The pHrodo with an “off–on” response to the changes of pH has been widely used as a fluorescent pH probe for bioimaging. The fluorescence off–on mechanism is fundamentally important for its application and further development. Herein, the sensing mechanism, especially the relevant excited-state dynamics, of pHrodo is investigated by steady-state and time-resolved spectroscopy as well as quantum chemical calculations, showing that pHrodo is best understood using the bichromophore model. Its first excited state (S1) is a charge transfer state between two chromophores. From S1, pHrodo relaxes to its ground state (S0) via an ultrafast nonradiative process (∼0.5 ps), which causes its fluorescence to be “off”. After protonation, S1 becomes a localized excited state, which accounts for the fluorescence being turned “on”. Our work provides photophysical insight into the sensing mechanism of pHrodo and indicates the bichromophore model might be relevant to a wide range of fluorescent probes.

Excited-state dynamics of 3-hydroxychromone in gas phase

“…We determined the lifetime of S 2 to be 118 fs, which is within the typical timescale of the internal conversion process. 8,55,56 We then applied bootstrapping to extract 10 000 sets of data, each containing 1701 values. Subsequently, we fitted each set and obtained a 95% confidence interval of [118.90685135, 119.00291629], which is consistent with our fitting results.…”