Programming A Molecular Relay for Ultrasensitive Biodetection through 129Xe NMR

A supramolecular strategy for detecting specific proteins in complex media by using hyperpolarized ¹²⁹Xe NMR is reported. A cucurbit6]uril (CB6])‐based molecular relay was programmed for three sequential equilibrium conditions by designing a two‐faced guest (TFG) that initially binds CB6] and blocks the CB6]–Xe interaction. The protein analyte recruits the TFG and frees CB6] for Xe binding. TFGs containing CB6]‐ and carbonic anhydrase II (CAII)‐binding domains were synthesized in one or two steps. X‐ray crystallography confirmed TFG binding to Zn²⁺ in the deep CAII active‐site cleft, which precludes simultaneous CB6] binding. The molecular relay was reprogrammed to detect avidin by using a different TFG. Finally, Xe binding by CB6] was detected in buffer and in E. coli cultures expressing CAII through ultrasensitive ¹²⁹Xe NMR spectroscopy.