Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Flavin-based fluorescent ROS sensors for high-throughput screening (#463)

Aedena-Raquel REMY 1 2 , Elizabeth New 1 2
  1. The University of Sydney, School of Chemistry and Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, Sydney, NSW, Australia
  2. University of Sydney, Sydney/Camperdown, NSW, Australia

Cells undergo constant ROS fluctuations either due to normal function of the cells or as a response to various types of stress.1 However, most of the current redox probes react with ROS irreversibly making the development of reversible probes attractive for monitoring dynamic changes within the cells. The flavin moiety is a good candidate for ROS sensing as it can reversibly cycle between its oxidised or reduced form, enhancing or quenching the fluorescence intensity respectively.2 With its origin in biomolecules, it is also notable for its biological relevance as the reduction potential of flavin lies within the biological range.3

Several flavin-based redox fluorescent sensors have already been synthesised and applied across a range of organisms and disease models.4-7 However, their photophysical properties are limited to green emission and, for that reason, we decided to synthesise various flavin-based probes to develop a bright, ratiometric and red-shifted probe. A variety of fluorescent flavin analogues sensing probes were synthesized chemically and their photophysical properties were evaluated by testing their responses to changes in different reductants or oxidant concentration.

In addition, we implemented flavin-based probes in high throughput studies. In recent studies, it has been shown that brain cells can withstand up to 3 mM of copper without showing any oxidative stress markers.8 We therefore used two flavin-based probes, NpFR14 and NpFR26, to monitor the copper-induced cellular redox buffering capacity. Imaging studies were performed on neuroblastoma cells using an Opera Phenix high throughput microscope.

We noticed that that the cells have the capacity to buffer any copper-induced redox activity at low concentrations, but this is overcome at higher concentrations. Our results demonstrate that an exposure of 3mM of copper sulfate induces cytotoxic effect and enhance the oxidative stress in vitro.

In conclusion, the synthesis of new reversible fluorescent ROS-sensing probes and the development of a new microscopy high throughput method will significantly help unravelling the oxidative stress within cells.Moreover, this method can also be used for broader applications, like screening for peptide activity, and identifying conditions that induce redox stress or perturb the redox buffering capacity of the cell.

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