title: 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 H2O2and caspase 3, the respective markers of ROS and apoptosis. The probe contains a H2O2fluorescence 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 H2O2induces 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 H2O2in apoptotic cells. Moreover, Pep4-NP1 shows excellent selectivity towards H2O2and caspase 3 on their respective reaction sites. Therefore, pep4-NP1 can distinguish endogenously generated H2O2between living cells and apoptotic cells with different fluorescence wavelengths, providing additional information on the ROS production pathways.
authors.affiliation: Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China.
authors.forename: Fengfeng
authors.initials: F
authors.lastname: Xue
authors.affiliation: Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China; College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, Hubei, China.
authors.forename: Haichuang
authors.initials: H
authors.lastname: Lan
authors.affiliation: Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Li
authors.affiliation: College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, Hubei, China. Electronic address: shuzhangxiao@ctgu.edu.cn.
authors.forename: Shuzhang
authors.initials: S
authors.lastname: Xiao
authors.affiliation: Department of Chemistry and Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, Shanghai 200433, China. Electronic address: yitao@fudan.edu.cn.
authors.forename: Tao
authors.initials: T
authors.lastname: Yi
mesh_terms: D000818:Animals; D017209:Apoptosis; D015374:Biosensing Techniques / Q000379:methods; D002232:Carbocyanines / Q000737:chemistry*; D053148:Caspase 3 / Q000032:analysis; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis*; D051379:Mice; D061848:Optical Imaging / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D000067996:RAW 264.7 Cells; D017382:Reactive Oxygen Species / Q000032:analysis
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D010455:Peptides; D017382:Reactive Oxygen Species; C085321:cyanine dye 5; D006861:Hydrogen Peroxide; D053148:Caspase 3
keywords: Apoptotic cells; Caspase 3; Fluorescent probe; Hydrogen peroxide; Living cells; Multicolor imaging