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20 February 2009Activatable optical imaging probes with various fluorophore-quencher combinations
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, 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