Dual-Channel Fluorescent Probe for the Simultaneous Monitoring of Peroxynitrite and Adenosine-5′-triphosphate in Cellular Applications

Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO-) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO- and ATP. ONOO- selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (lambda(ex) = 450 nm, lambda(em) = 562 nm or lambda(ex) = 488 lambda(em) = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (lambda(ex )= 520 nm, lambda(em) = 587 nm). Due to the differences in emission between the ONOO- and ATP products, ATP-LW allows ONOO- levels to be monitored in the green channel (lambda(ex) = 488 nm, lambda(em) = 500-575 nm) and ATP concentrations in the red channel (lambda(ex) = 514 nm, lambda(em) = 575-650 nm). The use of ATP-LW as a combined ONOO- and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO- donor).

Automated summary

The fluorescent probe ATP-LW was developed for simultaneous detection of ONOO- and ATP levels. By utilizing different emission properties of the products, ONOO- levels can be monitored in the green channel and ATP concentrations in the red channel. The probe was successfully used in hepatocytes to demonstrate changes in signal intensity in response to ATP synthase inhibition and exogenous ONOO- exposure.