Oral Presentation Royal Australian Chemical Institute National Congress 2026

Towards fluorescent multiplexing : Engineering multi-color and targeted reversible redox sensors (136589)

Aedena REMY 1 2 , William Ryder 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 redox fluctuations either due to the normal function of the cells or as a response to various types of stress1.  Oxidative events are not necessarily harmful if they are transient, but a chronic increase leads to oxidative stress, which is known to be a key factor in chronic diseases like diabetes, cardiovascular disease, cancer, aging and many other diseases2. While reversible probes are essential for tracking the dynamic fluctuations of ROS in live imaging, most of the current redox sensors are irreversible, limiting their application for the transient nature of oxidative signalling. In addition, there is a limited availability of ROS sensors targeted to certain organelles for site-specific sensing3.

For our probes, we use a moiety called flavin, which is a good candidate for its biocompatibility and its general ROS sensing, as it can reversibly cycle between its oxidised or reduced form, enhancing or quenching the fluorescence intensity respectively.4 Several flavin-based redox fluorescent sensors have already been synthesised and applied across a range of organisms and disease models.5-8 However, their photophysical properties are limited to green emission and cytoplasmic or mitochondrial subcellular targeting.

To address these challenges, we synthesised a wide range of flavin-based redox probes that differ in their chemical structures, targeting capabilities and colours. We engineered a library of sensors using different fluorophores and targeting moieties, followed by a systematic characterization of their physicochemical properties. This evaluation included the determination of redox potentials and the assessment of their responsiveness to oxidative and reductive environments in solution. Subsequently, the probes were imaged via confocal microscopy for biological validation.

Our ultimate goal is to develop a fluorescent multiplexing platform. By modulating the emission and the targeting capability, these probes would allow for the simultaneous, real-time monitoring of ROS fluctuations across multiple subcellular compartments. This approach allows site-specific oxidative stress measurement in a single dish, providing a fast mapping of subcellular stress.

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