Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 4, Pages 1268-1274Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac102095j
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Funding
- National Natural Science Foundation of China [21075032, 21005026]
- National Grand Program on Key Infectious Disease [2009ZX10004-312]
- Hunan Province Key Project of Scientific and Technical Programs [2010TP4013-1]
- Hunan National Science Foundation [10JJ7002]
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Despite that considerable efforts have been devoted to the design of various fluorogenic enzyme substrates, they are single-enzyme assay approaches that cannot afford detection of multienzyme activity. In this study, we set out our first attempt to design a new probe that could measure intracellular beta-D-glucosidase and phosphodiesterase I activities. Unlike the commonly used fluorogenic enzyme substrates that contain one recognition site and signaling reporter, the new probe molecule possesses two cleavage sites, specificly corresponding to beta-D-glucosidase and phosphodiesterase I, and three fluorescent reporters, 7-beta-D-glucopyranosyloxycoumarin, 7-hydroxycoumarin, and meso-tetraphenylporphyrin. On the basis of intramolecular photoinduced electron transfer and fluorescence resonance energy transfer mechanisms, interaction of the probe with the two enzymes, whether only one or both, produces different signal readouts with high sensitivity. Remarkably, the probe is chemically stable in complex biological fluids. Fluorescence outputs are not significantly affected by biologically related metal ions, anions, amino acids, and proteins. Furthermore, fluorescence microscopy confirmed that the probe is an excellent candidate for intracellular delivery and can be accumulated intensively in cells. We demonstrated the applicability for the simultaneous images of intracellular beta-D-glucosidase and phosphodiesterase I activities using the different optical imaging modes.
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