Multicolor imaging of hydrogen peroxide level in living and apoptotic cells by a single fluorescent probe

To understand the entangled relationship between reactive oxygen species (ROS) and apoptosis, there is urgent need for simultaneous dynamic monitoring of these two important biological events. In this study, we have developed a fluorescent probe, pep4-NP1, which can simultaneously detect H₂O₂ and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H₂O₂ fluorescence reporter (NP1) and Cy5 fluorescent chromophore connected by a caspase 3 specific recognition peptide. The detecting strategy was realized through a controllable fluorescence resonance energy transfer (FRET) process between NP1 and Cy5 of pep4-NP1, after reaction with H₂O₂, which was verified by molecular calculation and in vitro spectral studies. In the absent of caspase 3, the accumulation of H₂O₂ induces red fluorescence of pep4-NP1 centered at 663nm in living cells due to the existence of FRET. In contrast, FRET is inhibited in apoptotic cells due to cleavage of the peptide spacer of pep4-NP1 by over-expressed caspase 3. Consequently, green fluorescence (555nm) predominated when labelling production of H₂O₂ in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H₂O₂ and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H₂O₂ between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.