Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where
chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of
single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in
solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens
from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple
fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at
sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra,
which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of
chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a
protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background.
Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein
traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical
scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently
offers expanded opportunities for cellular protein discovery.