Cy5 labeled single-stranded DNA-polydopamine nanoparticle conjugate-based FRET assay for reactive oxygen species detection

This work reports on a simple and feasible fluorescence resonance energy transfer (FRET) assay for detecting reactive oxygen species (ROS) both in solution and living cell using polydopamine nanoparticle (PDA NP) as energy acceptor and Cy5 labeled single-stranded DNA (Cy5-ssDNA) as energy donor. The Cy5-ssDNA and PDA NPs form self-assembled conjugates (Cy5-ssDNA-PDA NP conjugates) via π-stacking interactions. In the presence of ROS, the PDA NP adsorbed Cy5-ssDNAs can be effectively cleaved, resulting in the release of Cy5 molecules into solution and recovery of fluorescence emission of Cy5. In order to obtain ROS solution, the glucose oxidase-catalyzed oxidation reaction of glucose with O2 is employed to generate hydrogen peroxide for Fenton-like reaction. The formation of ROS in Fenton-like reaction can be detected as low as glucose oxidase-catalyzed oxidation of 100 pM glucose by the Cy5-ssDNA-PDA NP conjugate-based FRET assay. The recovery ratio of Cy5 fluorescence intensity is increased linearly with logarithm of glucose concentration from 100 pM to 1 μM, demonstrating that the FRET assay has wide dynamic range. In particular, intracellular ROS has been successfully detected in chemical stimulated HepG-2 cells by the Cy5-ssDNA-PDA NP conjugate-based FRET assay with a fluorescence microscopy, indicating that this approach has great potential to monitor ROS in living cells.