Measuring Binding Constants of Cucurbituril-Based Host–Guest Interactions at the Single-Molecule Level with Nanopores

Cucurbiturils are one type of widely used macrocyclic host compound in supramolecular chemistry. Their peculiar properties have led to applications in a wide variety of research areas such as fluorescence spectroscopy, drug delivery, catalysis, and nanotechnology. However, the solubilities of cucurbiturils are rather poor in water and many organic solvents, which may cause accuracy problems when measuring binding constants with traditional methods. In this report, we aim to develop an approach to measure the binding constants of cucurbituril-based host–guest interactions at the single-molecule level. First, we covalently attach different guest compounds to the side-chain of DNA molecules. Then, excess cucurbiturils are incubated with DNA probes to form the host–guest complexes. Next, the modified DNA hybrids are threaded through α-hemolysin nanopore to generate highly characteristic current events. Finally, statistical analyses of the obtained events afford the binding constants of cucurbiturils with various molecules. This new approach provides a simple and straightforward method to compare binding strength of different host–guest complexes and may find applications for quantifying other macrocycle-based host–guest interactions.