Fluorescent tools for imaging oxidative stress in biology

Abstract

Oxidative stress has been implicated in a myriad of pathological conditions, but measuring it and deciphering the underlying the mechanisms has been a long-standing challenge. Most of the existing redox probes are reaction-based and irreversible. In contrast, reversible redox probes with biologically-tuned redox potential can distinguish between transient and chronic elevations in oxidative capacity. In this work, a series of novel fluorescent redox probes based on flavins and nicotinamides have been developed and tested. The first redox-responsive probe, NpFR1, localises in the cytoplasm and exhibits robust reversibility of oxidation and reduction and a clear fluorescence increase upon oxidation. Based on these promising results, attention turned to developing analogues of NpFR1, with two main aims: to send the probe to the specific sub-cellular organelles, and to develop a ratiometric probe, in which a change in redox state was signalled by a change in colour rather than fluorescence intensity. To this end, six further probes were developed. A mitochondrially-localising derivative NpFR2 was developed by incorporating a lipophilic cationic tag. The design of the emission-ratiometric redox probe FCR1 whose fluorescence emission changes from blue to green upon oxidation, is based on the FRET process between coumarin donor and flavin acceptor. Furthermore, a set of excitation-ratiometric FRET probes FRR1 and FRR2 were developed, in which mitochondrial targeting was achieved by employing rhodamine B with inherent mitochondrial accumulation properties. Finally, nicotinamide-based redox probes NCR3 and NCR4 demonstrate the use of ICT as a strategy to attain ratiometric fluorescence properties. Following the successful utilisation of ratiometric and targeting strategies, the developed probes were applied in a variety of biological investigations, whether in cellulo, ex vivo or non-mammalian systems. The probes demonstrated excellent abilities to report on the differences in oxidative capacities under different conditions within each system. This work therefore demonstrates that reversible redox probes based on flavins and nicotinamides exhibit suitable properties for use as cellular redox probes. The developed probes can be modulated to give ratiometric output and targeted to specific sub-cellular compartments. These probes therefore exhibit potential to aid in deciphering the role of oxidative stress in pathogenesis and disease progression.