Multiplexed Bioorthogonal Imaging through Affinity Hierarchies in a Supramolecular Host

Bioorthogonal chemistry has transformed molecular imaging by enabling highly selective synthetic labeling of biomolecules in their native habitats. Despite its remarkable success, the chemical space available for multiplexed bioorthogonal imaging remains fundamentally constrained by the limited repertoire of mutually orthogonal reactions. Herein, we expand this chemical space by introducing affinity encoding, a bioorthogonal multiplexing strategy in which molecular identity is encoded through programmable differences in binding affinity rather than reaction orthogonality. Using the synthetic host cucurbit[7]uril (CB[7]), we establish a single-host, multiguest framework that enables dynamic and reconfigurable multiplexed imaging through affinity-programmed host–guest exchange. By exploiting the hierarchical binding affinities of structurally distinct guest molecules, we achieve selective fluorophore displacement, enabling fluorescence erasure from lower-affinity targets while preserving higher-affinity host–guest assemblies intact. Subsequent recomplexation of the liberated lower-affinity binding sites with a spectrally distinct CB[7]–fluorophore conjugate enables programmable signal reassignment for multiplexed imaging in cells as well as in live tissues. Moreover, affinity encoding operates orthogonally to established covalent bioorthogonal chemistries, enabling four-color multiplexed imaging by combining affinity- and reaction-based encoding. Collectively, our results establish affinity encoding as a new scaling dimension for bioorthogonal multiplexing, providing a dynamically reconfigurable framework for high-dimensional fluorescence imaging.