Dual-channel fluorescent probe for the simultaneous monitoring of peroxynitrite and adenosine-5’-triphosphate in cellular applications

The concentrations of ATP and ONOO− have been correlated with the progression a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Here, we report the development of fluorescent probe, ATP-LW, which enables the simultaneous detection of ONOO− and ATP. ONOO− selectively oxidises the boronate pinacol ester of ATP-LW, to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λex = 450 nm, λem = 562 nm or λex = 488 nm, λem = 568 nm). While, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λex = 520 nm, λem = 587 nm). Due to the differences in emission between the ONOO− and ATP products, ATP-LW exhibits the unique ability to image ONOO− levels in the green channel (λex = 488 nm, λem = 500-575 nm) and ATP concentrations using the red channel (λex = 514 nm, λem = 575-650 nm). This was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. The treatment of HL-7702 cell line with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of ATP in the red channel and increase in ONOO− green channel. While, the presence of SIN-1 (an exogenous ONOO− donor) results in an increase of ONOO−, and decrease in ATP. Significantly, when HL-7702 cells were treated with acetaminophen as a biological model for drug-induced liver injury, an increase in ONOO− green and decrease in ATP red channel fluorescence was observed. These results illustrate the utility of ATP-LW as a chemical tool to simultaneously monitor ATP and ONOO− concentrations in cellular-based applications.