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20 February 2009 Activatable optical imaging probes with various fluorophore-quencher combinations
Mikako Ogawa; Nobuyuki Kosaka; Yasuteru Urano; Peter L. Choyke; Hisataka Kobayashi
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Proceedings Volume 7190, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications; 71900Z (2009) https://doi.org/10.1117/12.808326
Event: SPIE BiOS, 2009, San Jose, California, United States
Abstract
Molecular imaging probes rely on high target-to-background ratios (TBR) to achieve maximum sensitivity and specificity. We utilized "quenchers" to turn off the background signal from the unbound probe and investigated the ability of specific fluorophore-quencher pairs to activate at target tissues. Both fluorophore and quencher were conjugated to a single cancer targeting molecule, either avidin or antibody. Fluorescence signal from these targeting molecules was "turned off" by the quencher in the unbound state, but was "turned on" only when the molecules bound to the cell surface target and was internalized. We tested the following fluorophore-quencher combinations based on fluorescence resonance energy transfer (FRET) pairs; OregonG-BHQ1, RhodG-BHQ1/ATTO540Q, TAMRA-QSY7/QSY21, TexRed-QSY21, Alexa647-QSY21, Cy5.5-QSY21/BHQ3 and Alexa680-QSY21/BHQ3. Among these, only RhodGATTO540Q and TAMRA-QSY7/21 pair showed activation upon cell binding/internalization. Among these combinations, TAMRA-QSY7 pair showed the highest activation (40-fold and 13-fold for avidin and antibody conjugate, respectively) as measured with an in vitro dissociation assay. The activation was dependent on the method used to conjugate fluorophores and quenchers to the targeting molecule. In vitro microscopic studies with TAMRA-QSY7 pair conjugated to avidin or antibody showed high fluorescent signal inside the target cancer cells, indicating activation after internalization. In vivo imaging studies in tumor bearing mice demonstrated that tumors could be clearly detected with low background. Although the precise quenching mechanism remains to be determined, this activation system can achieve high TBR in vivo molecular imaging.
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Mikako Ogawa, Nobuyuki Kosaka, Yasuteru Urano, Peter L. Choyke, and Hisataka Kobayashi "Activatable optical imaging probes with various fluorophore-quencher combinations", Proc. SPIE 7190, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications, 71900Z (20 February 2009); https://doi.org/10.1117/12.808326
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