Functionalized xenon as a biosensor

Abstract

The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon "functionalized" by a biotin-modified supramolecular cage to detect biotin-avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes.

Figure 1

Structure of a biosensor molecule designed to bind xenon to a protein with high affinity and specificity. The cage binding xenon, cryptophane-A, is shown in black; the ligand, in this case biotin, is shown in red; and the tether connecting them is shown in purple. The schematic representation of this structure is shown below.

Figure 2

Xenon-129 NMR spectra monitoring the binding of biotin-functionalized xenon to avidin. The spectrum of functionalized xenon in the absence of protein is shown in full, with the peak at 193 ppm corresponding to xenon in water, and peaks at 70 ppm corresponding to cryptophane-bound xenon. Inset shows only the cryptophane-bound peaks. a shows the functionalized xenon before the addition of avidin, with the more intense peak corresponding to functionalized xenon and the smaller peak corresponding to xenon in the cage without linker and ligand, serving as both a chemical shift and signal intensity reference. b shows the spectrum on the addition of ≈80 nmol of avidin monomer. A third peak, corresponding to functionalized xenon bound to avidin, has appeared, and the unbound functionalized xenon peak has decreased in intensity. All chemical shifts are referenced to that of xenon gas.

Figure 3

Effect of cage structure on xenon chemical shift. a shows the chemical shift of xenon in cryptophane-A. b shows the chemical shift of xenon in a similar cage, cryptophane-E, a 30-ppm difference from that in cryptophane-A. The line widths for cryptophanes A and E are broadened by the exchange of xenon between the cage and tetrachloroethane, the organic solvent used.

Figure 4

Schematic diagram of one possible multiplexing scheme envisioned for functionalized xenon biosensors. The top spectrum shows the three distinct functionalized xenon peaks, corresponding to different cages linked to three ligands. The bottom spectrum shows the effect of adding the functionalized xenon to an unknown solution. On addition to the unknown solution, the leftmost peak shifts entirely, representing the case in which all functionalized xenon is bound to its corresponding protein. The central peak decreases in intensity, and a peak corresponding to the protein-bound functionalized xenon appears. The rightmost peak remains unaffected, indicating the absence of the corresponding protein target. The color of each xenon atom reflects its chemical shift in accordance with the color bar shown beneath the spectra.