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

Abstract 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 H 2 O 2 and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H 2 O 2 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 H 2 O 2 , which was verified by molecular calculation and in vitro spectral studies. In the absent of caspase 3, the accumulation of H 2 O 2 induces red fluorescence of pep4-NP1 centered at 663 nm 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 (555 nm) predominated when labelling production of H 2 O 2 in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H 2 O 2 and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H 2 O 2 between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.