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.R ecent 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). Laserpolarized 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)(12)(13)(14)(15)(16)(17) and by the transfer of its enhanced polarization to surrounding spins (18,19). In a protein solution, weak xenonprotein 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. 1 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 su...

Development of a Functionalized Xenon Biosensor

“…51 We have now extended the experiments to a complete titration of the free biosensor with avidin ( Figure 4) showing that increasing amounts of avidin lead to corresponding …”

“…[60][61][62][63] Previous experimental studies, which are now being understood with theoretical models by Sears and Jameson, 64 have shown that direct changes to the basic structure of the cryptophane-A cage perturb the chemical shift of the cryptophane-encapsulated xenon. For example, a 30 ppm change in chemical shift was noted when the length of the bridges between the cyclotriveratrylene caps of cryptophane-A was increased by one methylene group, 51 and even deuteration of a single methoxy group on a cryptophane-A cage has been shown to produce a noticeable shift (~0.1 ppm). 65 In this work, we show that significant xenon chemical shift differences also result from derivatizations of cryptophane-A that are not local to the encapsulated xenon.…”

“…By combining different chemical changes in and around the cryptophane it will be possible to tune the chemical shift of the xenon in the cage, making it possible to follow binding events of different ligand-cage combinations in parallel. 51 …”

“…Recently, as a proof-of-principle case, we reported a molecule designed to be an NMR biosensor that targeted the biotin-binding protein avidin. 51 The sensor consists of a modified cryptophane-A cage, to which xenon binds as the NMR reporter, connected via a tether to the ligand biotin ( Figure 1). The Xe NMR signal demonstrated a response to Implementation of high-sensitivity fluorescence-based systems in a direct multiplexing assay is challenging due to spectral overlap, despite the high wavelength tunability of some systems such as quantum-dot-tagged microbeads.…”

Temperature‐Controlled Molecular Depolarization Gates in Nuclear Magnetic Resonance

“…Second, the use of low-bandwidth pulses is an important step for reading out different sensor signals in the same system (multiplexing, (7,10)). …”