NIR two-photon fluorescent probe for biothiol detection and imaging of living cells in vivo

A fluorescence probe, Cz-BDP-NBD, for detecting biothiols with two photon excited fluorescence has been designed and used under irradiation from sapphire pulsed lasers at 800 nm. Upon addition of biothiols, NBD was removed through a SNAr reaction and the fluorophore Carbazyl BODIPY (Cz-BDP) was released simultaneously. A two photon excited fluorescence was turned on and accompanied by a strong red emission at 660 nm, the two-photon absorption (TPA) cross section of the Cz-BDP reached a value of 198.5 GM at 800 nm. Moreover, Hcy/Cys and GSH could be distinguished between by a two emission channel, which resulted from the different mechanisms. For Hcy and Cys, N-NBD-Hcy and N-NBD-Cys were formed through a 5/6-membered cyclic transition, they displayed distinct fluorescent turn-on signal changes at 540 nm (Φ = 0.28, Φ = 0.24) and 670 nm, while GSH only has a single emission channel at 670 nm. Therefore, the Cz-BDP-NBD can distinguish GSH, Hcy and Cys with a fast response time (within one minute), high selectivity and sensitivity. In addition, it has been successfully applied for the detection of Cys/Hcy and GSH in living cells and ratio imaging. The Cz-BDP-NBD probe could be used for discriminative sensing of intracellular Cys, Hcy and GSH, and has the potential for use in biological applications.