Synthesis of a highly HOCl-selective fluorescent probe and its use for imaging HOCl in cells and organisms.
During infection, nicotinamide adenine dinucleotide phosphate-oxidase of innate immune cells generates important microbicidal reactive oxygen species (ROS) such as hypochlorous acid (HOCl) to kill the invading pathogens. However, excess amounts of HOCl induce oxidative damage of functional biomolecules such as DNA and proteins, which may cause chronic inflammatory diseases. Herein, we outline protocols for the preparation of a rhodamine-based HOCl probe, as well as applications thereof, with which to detect HOCl in living cells and organisms. The probe (R19S) can be prepared from a commercially available rhodamine, rhodamine 6G, in two steps. When R19S is treated with HOCl, the sulfur atom is replaced by an oxygen atom, resulting in opening of the lactone ring; thus, nonfluorescent R19S is converted to highly fluorescent rhodamine 19 (R19). R19S exhibits high selectivity for HOCl over other ROS and high sensitivity in a weakly acidic environment. In addition, we describe fluorescence imaging assays of HOCl in mouse neutrophils and Drosophila targeted using this probe. The approximate amount of time required to synthesize the probe is 2-3 d, after which it can be used for up to 5 h in the bioimaging of living cells.
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, South Korea.|Department of Bioinspired Science (WCU), Ewha Womans University, Seoul, South Korea.|State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
Xiaoqiang
X
Chen
National Creative Research Initiative Center for Hologenomics, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.|School of Biological Science, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Kyung-Ah
KA
Lee
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing, People's Republic of China.
Xintong
X
Ren
Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul, South Korea.|Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea.|BK 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Jae-Chan
JC
Ryu
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, South Korea.|Department of Bioinspired Science (WCU), Ewha Womans University, Seoul, South Korea.
Gyungmi
G
Kim
Research Center for Human Natural Defense System, Yonsei University College of Medicine, Seoul, South Korea.|Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea.|BK 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Ji-Hwan
JH
Ryu
National Creative Research Initiative Center for Hologenomics, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.|School of Biological Science, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea.
Won-Jae
WJ
Lee
Department of Chemistry and Nano Science, Ewha Womans University, Seoul, South Korea.|Department of Bioinspired Science (WCU), Ewha Womans University, Seoul, South Korea.
Juyoung
J
Yoon
D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila / Q000737:chemistry / Q000382:microbiology; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006997:Hypochlorous Acid / Q000032:analysis*; D007422:Intestines / Q000737:chemistry / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D009504:Neutrophils / Q000737:chemistry / Q000166:cytology; D061848:Optical Imaging / Q000379:methods*; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry*
D005456:Fluorescent Dyes; D012235:Rhodamines; C026188:rhodamine 6G; D006997:Hypochlorous Acid