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

23 July 2021, Version 1


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.


Reactive Oxygen Species
Reactive Nitrogen Species

Supplementary materials

Supplementary Material for Dual-channel fluorescent probe for the simultaneous monitoring of peroxynitrite and adenosine-5’-triphosphate in cellular applications
Supplementary Material Including: 1. Materials and instruments 2. Mechanism of APAP-induced toxicity 3. Synthesis of probe ATP-LW 4. Generation of various ROS/RNS 5. UV-Vis and fluorescence analysis 6. Theoretical calculations to confirm no FRET 7. Mass spectroscopic analysis 8. Protocols for cell culture 9. MTT assay and fluorescence imaging in live cells 10. NMR spectra 11. Author contributions 12. References


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