Construction of a ratiometric two-photon ER-targeting fluorescent probe for the imaging of peroxynitrite in living systems

Peroxynitrite (ONOO-) could deplete ER-associated Ca2+ and cause ER stress, and thereby precise and in situ detection of ONOO- at ER is highly important for the study of its biological roles. Herein, we present a ratiometric two-photon ER-targeting fluorescent probe (NAB) for the detection of ONOO- in living cells and brain tissues. NAB utilized 4-bromomethylphenyl borate as a recognition site for ONOO-, and utilized p-toluenesulfonamide as ER-targeting group. After the response of NAB to ONOO-, the 4-bromomethylphenyl borate converted to hydroxyl, resulting in the change of the fluorescence color from blue to yellow. The probe NAB could show highly sensitive response to ONOO- within 6 s, and exhibited excellent selectivity to ONOO-. The probe NAB mainly located at ER and showed excellent ER-targeting property. Ratiometric two-photon imaging demonstrated that the probe NAB could selectively detect exogenous ONOO- at ER, and also could be applied to image metformin-induced ONOO- in living HepG2 cells. Furthermore, NAB can effectively detect the changes of ONOO- level in brain tissues, and revealed that the endogenous ONOO- level at ER showed increase in hippocampus of the depressive mouse.