Functionalized xenon as a biosensor

M Spence

S Rubin

I Dimitrov

E Ruiz

^{Department of Chemistry, University of California, Berkeley, CA 94720;}^{Materials Sciences and}^{Physical Biosciences Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;}^{Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104; and}^{Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037}

^{To whom reprint requests should be addressed. E-mail:}ude.yelekreb.mehcc@senip.

^{Present address: Departments of Chemistry and Biological Sciences, National University of Singapore, 3 Science Drive, Singapore 117543.}

Contributed by Alexander Pines

Accepted 2001 Jul 17.

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.

Abstract

Recent biosensor technologies exploit surface plasmon resonance (1), fluorescence polarization (2), and fluorescence resonance energy transfer (3) as detection methods. Although the sensitivity of such techniques is excellent, extending these assays to multiplexing capabilities has proven challenging because of the difficulty in distinguishing signals from different binding events. Although NMR spectroscopy is able to finely resolve signals from different molecules and environments, the spectral complexity and low sensitivity of NMR spectroscopy normally preclude its use as a detector of molecular targets in complex mixtures. Notable successes (4, 5) in the application of NMR to such problems are still limited by long acquisition times or a limited number of detectable analytes. Laser-polarized xenon NMR benefits from good signal-to-noise and spectral simplicity with the added advantage of substantial chemical-shift sensitivity.

Optical pumping (6) has enhanced the use of xenon as a sensitive probe of its molecular environment (7, 8). Laser-polarized xenon is being developed as a diagnostic agent for medical magnetic resonance imaging (9) and spectroscopy (10) and as a probe for the investigation of surfaces and cavities in porous materials and biological systems. Xenon provides information both through direct observation of its NMR spectrum (11–17) and by the transfer of its enhanced polarization to surrounding spins (18, 19). In a protein solution, weak xenon–protein interactions render the chemical shift of xenon dependent on the accessible protein surface and even allow the monitoring of the protein conformation (20). To use xenon as a specific sensor of molecules, it would be valuable to “functionalize” the xenon for the purpose of reporting specific interactions with a molecular target.

In this report, we demonstrate an example of such a functionalized system that exhibits molecular target recognition. Fig. Fig.11 shows the chemical principle used for our initial study, a biosensor molecule designed to bind both xenon and protein. The molecule consists of three parts: the cage, which contains the xenon; the ligand, which directs the functionalized xenon to a specific protein; and the tether, which links the ligand and the cage. The ligand and target can be any two molecules or constructs. In such a molecule, it is expected that the binding of the ligand to the target protein will be reflected in a change of the xenon NMR spectrum.

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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.

Acknowledgments

M.M.S. and S.M.R. acknowledge the National Science Foundation and E.J.R. acknowledges Lucent Technologies/Bell Laboratories for predoctoral fellowships. I.E.D. received support from National Institutes of Health Grant RR02305. This work was supported by the Director, Office of Energy Research, Office of Basic Energy Sciences, Materials Sciences Division, Physical Biosciences Division, of the U.S. Department of Energy under Contract No. DE-AC03–76SF00098. This work was presented at 9th Chianti Workshop on Magnetic Resonance, Tirrenia, Italy, May 26–June 1, 2001.

Acknowledgments

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