Thomas J. Lowery

Optimization of Xenon Biosensors for Detection of Protein Interactions

Hyperpolarized 129Xe NMR spectroscopy can detect the presence of specific low‐concentration biomolecular analytes by means of a xenon biosensor that consists of a water‐soluble, targeted cryptophane‐A cage that encapsulates the 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 the protein‐bound biosensor signal were assessed. It was found that an 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 line width and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length.

Single-Coil, Multisample, Proton Relaxation Method for Magnetic Relaxation Switch Assays

Nanoparticle based magnetic relaxation switch (MRSw) biosensors offer the opportunity to develop magnetic resonance based in vitro diagnostics. Critical attributes for point of care in vitro diagnostic products include simple instrumentation and ease of use. To this end, high-resolution biexponential analysis was used to permit measurement and assignment of two samples with a single radio frequency detection coil. This approach was used to calibrate and expand the dynamic range of MRSw biosensors in a single step. The potential for easy-to-use MRSw-based diagnostics was demonstrated by combining the method for single-step measurement of two samples with a disposable, plastic cartridge and dried MRSw reagents to obtain a calibrated reading after only two steps: mix and read. Taken together, our results suggest the feasibility of developing magnetic resonance based in vitro diagnostics that offer extreme ease of use and simple instrumentation.

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR

The chemical shift of the 129Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the 129Xe shift can sense more subtle changes: magnesium binding, BeF3− activation, and peptide binding by the Escherichia coli chemotaxis Y protein. 1H‐15N correlation spectroscopy and X‐ray crystallography were used to identify two xenon‐binding cavities in CheY that are primarily responsible for the shift changes. One site is near the active site, and the other is near the peptide binding site.

Relaxivity of gadolinium complexes detected by atomic magnetometry

Laser atomic magnetometry is a portable and low‐cost yet highly sensitive method for low magnetic field detection. In this work, the atomic magnetometer was used in a remote‐detection geometry to measure the relaxivity of aqueous gadolinium‐diethylenetriamine pentaacetic acid Gd(DTPA) at the Earths magnetic field (40 μT). The measured relaxivity of 9.7 ± 2.0 s−1 mM−1 is consistent with field‐cycling experiments measured at slightly higher magnetic fields, but no cryogens or strong and homogeneous magnetic field were required for this experiment. The field‐independent sensitivity of 80 fT Hz–1/2 allowed an in vitro detection limit of ∼ 10 μM Gd(DTPA) to be measured in aqueous buffer solution. The low detection limit and enhanced relaxivity of Gd‐containing complexes at Earths field motivate continued development of atomic magnetometry toward medical applications. Magn Reson Med 66:603–606, 2011.