Using single-molecule spectroscopic approaches, the researchers analyzed the interactions of ATTO655 with two supramolecular hosts, cucurbit[8]uril (CB8) and p-sulfonatocalix[4]arene (sCX4). While ATTO655 forms a standard inclusion complex with CB8, its interaction with sCX4 revealed a previously unrecognized binding mechanism. Instead of occupying the host cavity, the dye binds externally to the aromatic surface of sCX4, forming the dim exclusion complexes while leaving the cavity available for additional guest molecules.
Furthermore, the study demonstrates the capabilities of single-molecule spectroscopy to resolve the heterogeneity properties, binding stoichiometry, kinetic processes, and photophysical dynamics that are often hidden inside bulk measurements. More broadly, the findings provide new insights about the design of responsive supramolecular systems and may facilitate to the development of advanced bioimaging probes, molecular sensors, and functional host-guest assemblies.