A dual-mechanism strategy to design a wide-range pH probe with multicolor fluorescence

Abstract

A multicolor fluorescent probe BNO for measuring intracellular pH is designed and synthesized, which is the first pH probe with a dual response mechanism combining an excited state intramolecular proton transfer (ESIPT) process and an intramolecular charge transfer (ICT) process. The probe shows negligible fluorescence at neutral and weak alkaline pH due to fluorescence quenching caused by ESIPT, and a sharp fluorescence enhancement in both acidic and strong alkaline environments. Meanwhile, it responds to pH changes with different emissions (green at acidic range, cyan at alkaline range). BNO is able to indicate pH variations in a wide range of 3.9–13.0. It is also capable of distinguishing intracellular pH values with high sensitivity. Furthermore, the probe exhibits favorable two-photon photophysics and high photostability, thus it is successfully applied to trace the dynamic change of intracellular pH with two-photon microscopy (TPM). The dual-mechanism strategy may be expanded to design other fluorescent probes.