Reversible Sequential‐Binding Probe Receptor–Ligand Interactions in Single Cells

With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association–dissociation kinetics. We chose the nicotinic‐acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition‐binding assays using fluorescent derivatives of the antagonist α‐conotoxin GI ( α ‐CnTx). Thereby, we determined the binding constants of unlabeled α‐CnTx and d‐tubocurarine. The high selectivity of α‐CnTx for muscle‐type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand‐binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq‐binding assays even at low receptor‐expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells.