TY - JOUR

T1 - Sensing Mechanism and Excited-State Dynamics of a Widely Used Intracellular Fluorescent pH Probe

T2 - pHrodo

AU - Jiang, Simin

AU - He, Yanmei

AU - Brandt, Jonas Højberg

AU - Zhao, Li

AU - Chen, Junsheng

N1 - Funding Information: The authors acknowledge funding support from the Novo Nordisk Foundation (NNF22OC0073582). Y.H. acknowledges the support from the China Scholarship Council (202006150002). Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

U2 - 10.1021/acs.jpclett.3c02653

DO - 10.1021/acs.jpclett.3c02653

M3 - Journal article

C2 - 37967406

AN - SCOPUS:85178545707

VL - 14

SP - 10482

EP - 10488

JO - Journal of Physical Chemistry Letters

JF - Journal of Physical Chemistry Letters

SN - 1948-7185

IS - 46

ER -