Nanoemulsion Contrast Agents with Sub-picomolar Sensitivity for Xenon NMR

A new type of contrast agent for Xe NMR based on surfactant-stabilized perfluorocarbon-in-water nanoemulsions has been produced. The contrast agent uses dissolved hyperpolarized xenon gas as a nonperturbing reporting medium, as xenon freely exchanges between aqueous solution and the perfluorocarbon interior of the droplets, which are spectroscopically distinguishable and allow for chemical exchange saturation transfer (CEST) detection of the agent. Nanoemulsions with droplet diameters between 160 and 310 nm were produced and characterized using hyperpolarized ¹²⁹Xe combined with CEST detection. Saturation parameters were varied and data were modeled numerically to determine the xenon exchange dynamics of the system. Nanoemulsion droplets were detected at concentrations as low as 100 fM, corresponding to <1 μL of perfluorocarbon per liter of solution. The straightforward, inexpensive production of these agents will facilitate future development toward molecular imaging and chemical sensing applications

¹³C‑Decoupled <i>J</i>‑Coupling Spectroscopy Using Two-Dimensional Nuclear Magnetic Resonance at Zero-Field

We present a two-dimensional method for obtaining ¹³C-decoupled, ¹H-coupled nuclear magnetic resonance (NMR) spectra in zero magnetic field using coherent spin-decoupling. The result is a spectrum determined only by the proton–proton <i>J</i>-coupling network. Detection of NMR signals in zero magnetic field requires at least two different nuclear spin species, but the proton <i>J</i>-spectrum is independent of isotopomer, thus potentially simplifying spectra and thereby improving the analytical capabilities of zero-field NMR. The protocol does not rely on a difference in Larmor frequency between the coupled nuclei, allowing for the direct determination of <i>J</i>-coupling constants between chemically equivalent spins. We obtain the ¹³C-decoupled zero-field spectrum of [1–¹³C]-propionic acid and identify conserved quantum numbers governing the appearance of cross peaks in the two-dimensional spectrum

High-Resolution Zero-Field NMR <i>J</i>‑Spectroscopy of Aromatic Compounds

We report the acquisition and interpretation of nuclear magnetic resonance (NMR) <i>J</i>-spectra at zero magnetic field for a series of benzene derivatives, demonstrating the analytical capabilities of zero-field NMR. The zeroth-order spectral patterns do not overlap, which allows for straightforward determination of the spin interactions of substituent functional groups. Higher-order effects cause additional line splittings, revealing additional molecular information. We demonstrate resonance linewidths as narrow as 11 mHz, permitting resolution of minute frequency differences and precise determination of long-range <i>J</i>-couplings. The measurement of <i>J</i>-couplings with the high precision offered by zero-field NMR may allow further refinements in the determination of molecular structure and conformation

High-Resolution Zero-Field NMR <i>J</i>‑Spectroscopy of Aromatic Compounds

We report the acquisition and interpretation of nuclear magnetic resonance (NMR) <i>J</i>-spectra at zero magnetic field for a series of benzene derivatives, demonstrating the analytical capabilities of zero-field NMR. The zeroth-order spectral patterns do not overlap, which allows for straightforward determination of the spin interactions of substituent functional groups. Higher-order effects cause additional line splittings, revealing additional molecular information. We demonstrate resonance linewidths as narrow as 11 mHz, permitting resolution of minute frequency differences and precise determination of long-range <i>J</i>-couplings. The measurement of <i>J</i>-couplings with the high precision offered by zero-field NMR may allow further refinements in the determination of molecular structure and conformation

High-Resolution Zero-Field NMR <i>J</i>‑Spectroscopy of Aromatic Compounds

We report the acquisition and interpretation of nuclear magnetic resonance (NMR) <i>J</i>-spectra at zero magnetic field for a series of benzene derivatives, demonstrating the analytical capabilities of zero-field NMR. The zeroth-order spectral patterns do not overlap, which allows for straightforward determination of the spin interactions of substituent functional groups. Higher-order effects cause additional line splittings, revealing additional molecular information. We demonstrate resonance linewidths as narrow as 11 mHz, permitting resolution of minute frequency differences and precise determination of long-range <i>J</i>-couplings. The measurement of <i>J</i>-couplings with the high precision offered by zero-field NMR may allow further refinements in the determination of molecular structure and conformation

Rapid Catalyst Capture Enables Metal-Free <i>para</i>-Hydrogen-Based Hyperpolarized Contrast Agents

Hyperpolarization techniques based on the use of <i>para</i>-hydrogen provide orders of magnitude signal enhancement for magnetic resonance spectroscopy and imaging. The main drawback limiting widespread applicability of <i>para</i>-hydrogen-based techniques in biomedicine is the presence of organometallic compounds (the polarization transfer catalysts) in solution with hyperpolarized contrast agents. These catalysts are typically complexes of platinum-group metals, and their administration in vivo should be avoided. Herein, we show how extraction of a hyperpolarized compound from an organic phase to an aqueous phase combined with a rapid (less than 10 s) Ir-based catalyst capture by metal scavenging agents can produce pure <i>para</i>-hydrogen-based hyperpolarized contrast agents, as demonstrated by high-resolution nuclear magnetic resonance (NMR) spectroscopy and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The presented methodology enables fast and efficient means of producing pure hyperpolarized aqueous solutions for biomedical and other uses

Targeted Molecular Imaging of Cancer Cells Using MS2-Based ¹²⁹Xe NMR

We have synthesized targeted, selective, and highly sensitive ¹²⁹Xe NMR nanoscale biosensors using a spherical MS2 viral capsid, Cryptophane A molecules, and DNA aptamers. The biosensors showed strong binding specificity toward targeted lymphoma cells (Ramos line). Hyperpolarized ¹²⁹Xe NMR signal contrast and hyper-CEST ¹²⁹Xe MRI image contrast indicated its promise as highly sensitive hyperpolarized ¹²⁹Xe NMR nanoscale biosensor for future applications in cancer detection in vivo

Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes

Nuclear magnetic resonance (NMR) can reveal the chemical constituents of a complex mixture without resorting to chemical modification, separation, or other perturbation. Recently, we and others have developed magnetic resonance agents that report on the presence of dilute analytes by proportionately altering the response of a more abundant or easily detected species, a form of amplification. One example of such a sensing medium is xenon gas, which is chemically inert and can be optically hyperpolarized, a process that enhances its NMR signal by up to 5 orders of magnitude. Here, we use a combinatorial synthetic approach to produce xenon magnetic resonance sensors that respond to small molecule analytes. The sensor responds to the ligand by producing a small chemical shift change in the Xe NMR spectrum. We demonstrate this technique for the dye, Rhodamine 6G, for which we have an independent optical assay to verify binding. We thus demonstrate that specific binding of a small molecule can produce a xenon chemical shift change, suggesting a general approach to the production of xenon sensors targeted to small molecule analytes for <i>in vitro</i> assays or molecular imaging <i>in vivo</i>

¹²⁹Xe NMR Relaxation-Based Macromolecular Sensing

We report a ¹²⁹Xe NMR relaxation-based sensing approach that exploits changes in the bulk xenon relaxation rate induced by slowed tumbling of a cryptophane-based sensor upon target binding. The amplification afforded by detection of the bulk dissolved xenon allows sensitive detection of targets. The sensor comprises a xenon-binding cryptophane cage, a target interaction element, and a metal chelating agent. Xenon associated with the target-bound cryptophane cage is rapidly relaxed and then detected after exchange with the bulk. Here we show that large macromolecular targets increase the rotational correlation time of xenon, increasing its relaxation rate. Upon binding of a biotin-containing sensor to avidin at 1.5 μM concentration, the free xenon T₂ is reduced by a factor of 4

Zero-Field NMR Enhanced by Parahydrogen in Reversible Exchange

We have recently demonstrated that sensitive and chemically specific NMR spectra can be recorded in the absence of a magnetic field using hydrogenative parahydrogen induced polarization (PHIP)− and detection with an optical atomic magnetometer. Here, we show that non-hydrogenative parahydrogen-induced polarization− (NH-PHIP) can also dramatically enhance the sensitivity of zero-field NMR. We demonstrate the detection of pyridine, at concentrations as low as 6 mM in a sample volume of 250 μL, with sufficient sensitivity to resolve all identifying spectral features, as supported by numerical simulations. Because the NH-PHIP mechanism is nonreactive, operates in situ, and eliminates the need for a prepolarizing magnet, its combination with optical atomic magnetometry will greatly broaden the analytical capabilities of zero-field and low-field NMR