Hyperpolarized129 Xe NMR can detect the presence of specific lowconcentration biomolecular analytes by means of the xenon biosensor, which consists of a water-soluble, targeted cryptophane-A cage that encapsulates xenon. In this work we use the prototypical biotinylated xenon biosensor to determine the relationship between the molecular composition of the xenon biosensor and the characteristics of protein-bound resonances.The effects of diastereomer overlap, dipole-dipole coupling, chemical shift anisotropy, xenon exchange, and biosensor conformational exchange on protein-bound biosensor signal were assessed. It was found that optimal protein-bound biosensor signal can be obtained by minimizing the number of biosensor diastereomers and using a flexible linker of appropriate length.Both the linewidth and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length.

Laser-enhanced (LE) 129Xe nuclear magnetic resonance (NMR) is an exceptional tool for sensing extremely small physical and chemical changes; however, the difficult mechanics of bringing polarized xenon and samples of interest together have limited applications, particularly to biological molecules. Here we present a method for accomplishing solution 129Xe biosensing based on flow (bubbling) of LE 129Xe gas through a solution in situ in the NMR probe, with pauses for data acquisition. This overcomes fundamental limitations of conventional solution-state LE 129Xe NMR, e.g., the difficulty in transferring hydrophobic xenon into aqueous environments, and the need to handle the sample to refresh LE 129Xe after an observation pulse depletes polarization. With this new method, we gained a factor of >100 in sensitivity due to improved xenon transfer to the solution and the ability to signal average by renewing the polarized xenon. Polarized xenon in biosensors was detected at very low concentrations,