Efficient Parahydrogen-Induced 13C Hyperpolarization on a Microfluidic Device.

We show the direct production and detection of 13C-hyperpolarized fumarate by parahydrogen-induced polarization (PHIP) in a microfluidic lab-on-a-chip (LoC) device and achieve 8.5% 13C polarization. This is the first demonstration of 13C-hyperpolarization of a metabolite by PHIP in a microfluidic device. LoC technology allows the culture of mammalian cells in a highly controlled environment, providing an important tool for the life sciences. In-situ preparation of hyperpolarized metabolites greatly enhances the ability to quantify metabolic processes in such systems by microfluidic NMR. PHIP of 1H nuclei has been successfully implemented in microfluidic systems, with mass sensitivities in the range of pmol/s. However, metabolic NMR requires high-yield production of hyperpolarized metabolites with longer spin life times than is possible with 1H. This can be achieved by transfer of the polarization onto 13C nuclei, which exhibit much longer T1 relaxation times. We report an improved microfluidic PHIP device, optimized using a finite element model, that enables the direct and efficient production of 13C-hyperpolarized fumarate.

Cross-correlated relaxation in the NMR of near-equivalent spin pairs: Longitudinal relaxation and long-lived singlet order.

The evolution of nuclear spin state populations is investigated for the case of a 13C2-labeled triyne in solution, for which the near-equivalent coupled pairs of 13C nuclei experience cross-correlated relaxation mechanisms. Inversion-recovery experiments reveal different recovery curves for the main peak amplitudes, especially when the conversion of population imbalances to observable coherences is induced by a radio frequency pulse with a small flip angle. Measurements are performed over a range of magnetic fields by using a sample shuttle apparatus. In some cases, the time constant TS for decay of nuclear singlet order is more than 100 times larger than the time constant T1 for the equilibration of longitudinal magnetization. The results are interpreted by a theoretical model incorporating cross-correlated relaxation mechanisms, anisotropic rotational diffusion, and an external random magnetic field. A Lindbladian formalism is used to describe the dissipative dynamics of the spin system in an environment of finite temperature. Good agreement is achieved between theory and experiment.

Rapid hyperpolarization and purification of the metabolite fumarate in aqueous solution.

Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at 13C polarization levels of 30-45%.

Hyperpolarizing Small Molecules using Parahydrogen and Solid-State Spin Diffusion.

Parahydrogen-induced polarization (PHIP) is an inexpensive way to produce hyperpolarized molecules with polarization levels of >10 % in the solution-state, but is strongly limited in generality since it requires chemical reactions/ interactions with H2. Here we report a new method to widen the scope of PHIP hyperpolarization: a source molecule is produced via PHIP with high 13C polarization, and precipitated out of solution together with a target species. Spin diffusion within the solid carries the polarization onto 13C spins of the target, which can then be dissolved for solution-state applications. We name this method PHIP-SSD (PHIP with solid-state spin diffusion) and demonstrate it using PHIP-polarized [1-13C]-fumarate as the source molecule, to polarize different 13C-labelled target molecules. 13C polarizations of between 0.01 and 3 % were measured on [1-13C]-benzoic acid, depending on the molar ratio of fumarate:benzoate in the solid state. We also show that PHIP-SSD does not require specific co-crystallization conditions by grinding dry powders of target molecules together with solid fumarate crystals, and obtain 13C signal enhancements of between 100 and 200 on [13C,15N2]-urea, [1-13C]-pyruvate, and [1-13C]-benzoic acid. This approach appears to be a promising new strategy for facile hyperpolarization based on PHIP.

Direct Production of a Hyperpolarized Metabolite on a Microfluidic Chip.

Microfluidic systems hold great potential for the study of live microscopic cultures of cells, tissue samples, and small organisms. Integration of hyperpolarization would enable quantitative studies of metabolism in such volume limited systems by high-resolution NMR spectroscopy. We demonstrate, for the first time, the integrated generation and detection of a hyperpolarized metabolite on a microfluidic chip. The metabolite [1-13C]fumarate is produced in a nuclear hyperpolarized form by (i) introducing para-enriched hydrogen into the solution by diffusion through a polymer membrane, (ii) reaction with a substrate in the presence of a ruthenium-based catalyst, and (iii) conversion of the singlet-polarized reaction product into a magnetized form by the application of a radiofrequency pulse sequence, all on the same microfluidic chip. The microfluidic device delivers a continuous flow of hyperpolarized material at the 2.5 µL/min scale, with a polarization level of 4%. We demonstrate two methods for mitigating singlet-triplet mixing effects which otherwise reduce the achieved polarization level.

Deuteron-Decoupled Singlet NMR in Low Magnetic Fields: Application to the Hyperpolarization of Succinic Acid.

The reaction of unsaturated substrates with hydrogen gas enriched in the para spin isomer leads to products with a high degree of nuclear singlet spin order. This leads to greatly enhanced NMR signals, with important potential applications such as magnetic resonance imaging (MRI) of metabolic processes. Although parahydrogen-induced polarization has the advantage of being cheap, compact, and mobile, especially when performed in ultralow magnetic fields, efficiency is lost when more than a few protons are involved. This strongly restricts the range of compatible substances. We show that these difficulties may be overcome by a combination of deuteration with the application of a sinusoidally modulated longitudinal field as a well as a transverse rotating magnetic field. We demonstrate a six-fold enhancement in the 13 C hyperpolarization of [1-13 C, 2,3-d2 ]-succinic acid, as compared with standard hyperpolarization methods, applied in the same ultralow field regime.

Hyperpolarization read-out through rapidly rotating fields in the zero- and low-field regime.

An integral part of para-hydrogen induced polarization (PHIP) methods is the conversion of nuclear singlet order into observable magnetization. In this study polarization transfer to a heteronucleus is achieved through a selective rotation of the proton singlet-triplet states driven by a combination of a rotating magnetic field and a weak bias field. Surprisingly we find that efficient polarization transfer driven by a STORM (Singlet-Triplet Oscillations through Rotating Magnetic fields) pulse in the presence of sub-µT bias fields requires rotation frequencies on the order of several kHz. The rotation frequencies therefore greatly exceed any of the internal frequencies of typical zero- to ultralow field experiments. We further show that the rotational direction of the rotating field is not arbitrary and greatly influences the final transfer efficiency. Some of these aspects are demonstrated experimentally by considering hyperpolarized (1-13C)fumarate. In contrast to most of the existing methods, the STORM procedure therefore represents a promising candidate for quadrupolar decoupled polarization transfer in PHIP experiments.

31P spin-lattice and singlet order relaxation mechanisms in pyrophosphate studied by isotopic substitution, field shuttling NMR, and molecular dynamics simulation.

Nuclear spin relaxation mechanisms are often difficult to isolate and identify, especially in molecules with internal flexibility. Here we combine experimental work with computation in order to determine the major mechanisms responsible for 31P spin-lattice and singlet order (SO) relaxation in pyrophosphate, a physiologically relevant molecule. Using field-shuttling relaxation measurements (from 2 µT to 9.4 T) and rates calculated from molecular dynamics (MD) trajectories, we identified chemical shift anisotropy (CSA) and spin-rotation as the major mechanisms, with minor contributions from intra- and intermolecular coupling. The significant spin-rotation interaction is a consequence of the relatively rapid rotation of the -PO32- entities around the bridging P-O bonds, and is treated by a combination of MD simulations and quantum chemistry calculations. Spin-lattice relaxation was predicted well without adjustable parameters, and for SO relaxation one parameter was extracted from the comparison between experiment and computation (a correlation coefficient between the rotational motion of the groups).

Nuclear hyperpolarization of (1-13C)-pyruvate in aqueous solution by proton-relayed side-arm hydrogenation.

We employ Parahydrogen Induced Polarization with Side-Arm Hydrogenation (PHIP-SAH) to polarize (1-13C)-pyruvate. We introduce a new method called proton-relayed side-arm hydrogenation (PR-SAH) in which an intermediate proton is used to transfer polarization from the side-arm to the 13C-labelled site of the pyruvate before hydrolysis. This significantly reduces the cost and effort needed to prepare the precursor for radio-frequency transfer experiments while still maintaining acceptable polarization transfer efficiency. Experimentally we have attained on average 4.33% 13C polarization in an aqueous solution of (1-13C)-pyruvate after about 10 seconds of cleavage and extraction. PR-SAH is a promising pulsed NMR method for hyperpolarizing 13C-labelled metabolites in solution, conducted entirely in high magnetic field.

Low-frequency excitation of singlet-triplet transitions. Application to nuclear hyperpolarization.

Coupled pairs of nuclear spin-1/2 support one singlet state and three triplet states. Transitions between the singlet state and one of the triplet states may be driven by an oscillating low-frequency magnetic field, in the presence of couplings to a third nuclear spin, and a weak bias magnetic field. The oscillating field is in the same direction as the bias field and is called a WOLF (Weak Oscillating Low Field) pulse. Application of a WOLF pulse allows for the generation of strong nuclear hyperpolarization of 13C nuclei, starting from the nuclear singlet polarization of a 1H spin pair, associated with the enriched para-spin isomer of hydrogen gas. Hyperpolarization is demonstrated for two molecular systems.

Nuclear singlet relaxation by chemical exchange.

The population imbalance between nuclear singlet states and triplet states of strongly coupled spin-1/2 pairs, also known as nuclear singlet order, is well protected against several common relaxation mechanisms. We study the nuclear singlet relaxation of 13C pairs in aqueous solutions of 1,2-13C2 squarate over a range of pH values. The 13C singlet order is accessed by introducing 18O nuclei in order to break the chemical equivalence. The squarate dianion is in chemical equilibrium with hydrogen-squarate (SqH-) and squaric acid (SqH2) characterized by the dissociation constants pK1 = 1.5 and pK2 = 3.4. Surprisingly, we observe a striking increase in the singlet decay time constants TS when the pH of the solution exceeds ∼10, which is far above the acid-base equilibrium points. We derive general rate expressions for chemical-exchange-induced nuclear singlet relaxation and provide a qualitative explanation of the TS behavior of the squarate dianion. We identify a kinetic contribution to the singlet relaxation rate constant, which explicitly depends on kinetic rate constants. Qualitative agreement is achieved between the theory and the experimental data. This study shows that infrequent chemical events may have a strong effect on the relaxation of nuclear singlet order.

15 N hyperpolarisation of the antiprotozoal drug ornidazole by Signal Amplification By Reversible Exchange in aqueous medium.

Signal amplification by reversible exchange (SABRE) offers a cost-effective route to boost nuclear magnetic resonance (NMR) signal by several orders of magnitude by employing readily available para-hydrogen as a source of hyperpolarisation. Although 1 H spins have been the natural choice of SABRE hyperpolarisation since its inception due to its simplicity and accessibility, limited spin lifetimes of 1 H makes it harder to employ them in a range of time-dependent NMR experiments. Heteronuclear spins, for example, 13 C and 15 N, in general have much longer T1 lifetimes and thereby are found to be more suitable for hyperpolarised biological applications as demonstrated previously by para-hydrogen induced polarisation (PHIP) and dynamic nuclear polarisation (DNP). In this study we demonstrate a simple procedure to enhance 15 N signal of an antibiotic drug ornidazole by up to 71,000-folds with net 15 N polarisation reaching ~23%. Further, the effect of co-ligand strategy is studied in conjunction with the optimum field transfer protocols and consequently achieving 15 N hyperpolarised spin lifetime of >3 min at low field. Finally, we present a convenient route to harness the hyperpolarised solution in aqueous medium free from catalyst contamination leading to a strong 15 N signal detection for an extended duration of time.

CP MAS kinetics in soft matter: Spin diffusion, local disorder and thermal equilibration in poly(2-hydroxyethyl methacrylate).

The 1H-13C cross-polarization magic angle spinning kinetics was studied in poly(2-hydroxyethyl methacrylate) (pHEMA), i.e. a soft material with high degrees of internal freedom and molecular disorder, having the purpose to track the influence of increasing local incoherent contributions to the effects of coherent nature in the open quantum spin systems. The experimental CP MAS kinetic curves were analyzed in the frame of the models of isotropic and anisotropic spin diffusion with thermal equilibration. The rates of spin diffusion and spin-lattice relaxation as well as the profiles of distribution of dipolar coupling, the parameters accounting the effective size of spin clusters and the local order parameters were determined. The intensities of the peaks of periodic quasi-equilibrium origin gradually decrease with increasing disorder, i.e. going from most ordered to more disordered sites in the polymer. Assuming that the thermal motion induced by the temperature gradients is much faster than the equilibration driven by spin diffusion due the difference in spin temperatures, it was deduced that the thermal equilibration in pHEMA occurs in the time scale of 10-4 s. This is one order of magnitude faster than the spectral spin diffusion, which occurs between spins having different resonance frequencies. The thermal equilibration in the case of remote spin clusters was described by the 'stretched exponent' decay. This led to the fractal dimension Dp ≈ 1.65 for both carbon sites (quaternary and carbonyl). The obtained Dp value corresponds to the aggregates, which images are very similar to those for pHEMA and some other related polymer structures are usually conceived.

Quasi-Equilibria and Polarization Transfer Between Adjacent and Remote Spins: 1H-13C CP MAS Kinetics in Glycine.

The 1H-13C CP MAS kinetic curves were measured in glycine powder sample at the MAS rates of 7, 10, and 12 kHz. Each experimental curve contained up to 1000 equidistant points over the whole contact time range of 10 µs - 10 ms. The CP kinetic data for CH2 group, i.e., for the system containing adjacent 1H-13C spin pairs with a definite dominant dipolar coupling can be described in the frame of the isotropic spin-diffusion approach. The local order parameter ⟨ S⟩ ≈ 1.0, determined as the ratio of the measured dipolar 1H-13C coupling constant and the calculated static dipolar coupling constant, is very close to the values deduced in series of other amino acids. The strong narrow peaks observed in the spin coupling spectrum at multiples of the MAS frequency can be considered as the confirmation that the periodic quasi-equilibrium state can appear also in the powder samples. The anisotropic spin-diffusion approach improved by the introducing of the thermal equilibration in the proton bath is the most proper model to describe the CP kinetics in the system containing remote spins. Very realistic values of the spin-cluster size ( N) have been obtained without any constraint on the flow of the nonlinear curve fitting. The finite values of N ≤ 4 means that CP transfer is located within one glycine molecule.

NMR and XRD Study of Hydrogen Bonding in Picolinic Acid N-Oxide in Crystalline State and Solutions: Media and Temperature Effects on Potential Energy Surface.

Solvent and temperature effects on H-bonding in crystalline picolinic acid N-oxide (PANO) and in solutions were studied by NMR (1H MAS and 1H-13C CP/MAS) and X-ray diffraction (XRD) methods. The single-crystal XRD experiments on ß-polymorph were carried out at 105 and 299 K. 13C chemical shifts of PANO pyridine ring carbons were chosen as an effective diagnostic tool for the H-bond sensing. The crystal field in PANO forces the proton displacement from donor to acceptor atoms much stronger than the solvent reaction field, including that created by the most polar solvents. NMR and XRD data for crystalline PANO do not confirm any H-bond geometry changes in the studied temperature range. On the contrary, a considerable contraction of r(O-H) bond was observed for PANO in acetonitrile (ACN) solution upon heating. The relative contraction of r(O-H) bond with respect to R(O···O) perfectly fits the global dielectric scheme deduced for a vast set of common solvents and the dependence of the dielectric permittivity of ACN on temperature. The subtle H-bond changes can be explained by the temperature dependence of the shape of potential energy surface in the liquid state. Both factors, temperature and dielectric permittivity, are comparable in triggering this effect.

Towards a unified picture of polarization transfer - pulsed DNP and chemically equivalent PHIP.

Nuclear spin hyperpolarization techniques, such as dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP), have revolutionized nuclear magnetic resonance and magnetic resonance imaging. In these methods, a readily available source of high spin order, either electron spins in DNP or singlet states in hydrogen for PHIP, is brought into close proximity with nuclear spin targets, enabling efficient transfer of spin order under external quantum control. Despite vast disparities in energy scales and interaction mechanisms between electron spins in DNP and nuclear singlet states in PHIP, a pseudo-spin formalism allows us to establish an intriguing equivalence. As a result, the important low-field polarization transfer regime of PHIP can be mapped onto an analogous system equivalent to pulsed-DNP. This establishes a correspondence between key polarization transfer sequences in PHIP and DNP, facilitating the transfer of sequence development concepts. This promises fresh insights and significant cross-pollination between DNP and PHIP polarization sequence developers.

Robust parahydrogen-induced polarization at high concentrations.

Parahydrogen-induced polarization (PHIP) is a potent technique for generating target molecules with high nuclear spin polarization. The PHIP process involves a chemical reaction between parahydrogen and a target molecule, followed by the transformation of nuclear singlet spin order into magnetization of a designated target nucleus through magnetic field manipulations. Although the singlet-to-magnetization polarization transfer process works effectively at moderate concentrations, it is observed to become much less efficient at high molar polarization, defined as the product of polarization and concentration. This strong dependence on the molar polarization is attributed to interference due to the field produced by the sample magnetization during polarization transfer, which leads to complex dynamics and can severely affect the scalability of the technique. We address this challenge with a pulse sequence that suppresses the influence of the distant dipolar field, while simultaneously achieving singlet-to-magnetization polarization transfer to the desired target spins, free from restrictions on the molar polarization.

Over 20% Carbon-13 Polarization of Perdeuterated Pyruvate Using Reversible Exchange with Parahydrogen and Spin-Lock Induced Crossing at 50 µT.

Carbon-13 hyperpolarized pyruvate is about to become the next-generation contrast agent for molecular magnetic resonance imaging of cancer and other diseases. Here, efficient and rapid pyruvate hyperpolarization is achieved via signal amplification by reversible exchange (SABRE) with parahydrogen through synergistic use of substrate deuteration, alternating, and static microtesla magnetic fields. Up to 22 and 6% long-lasting 13C polarization (T1 = 3.7 ± 0.25 and 1.7 ± 0.1 min) is demonstrated for the C1 and C2 nuclear sites, respectively. The remarkable polarization levels become possible as a result of favorable relaxation dynamics at the microtesla fields. The ultralong polarization lifetimes will be conducive to yielding high polarization after purification, quality assurance, and injection of the hyperpolarized molecular imaging probes. These results pave the way to future in vivo translation of carbon-13 hyperpolarized molecular imaging probes prepared by this approach.

Solid-State NMR and Impedance Spectroscopy Study of Spin Dynamics in Proton-Conducting Polymers: An Application of Anisotropic Relaxing Model.

The 1H-13C cross-polarization (CP) kinetics in poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMETAC) was studied under moderate (10 kHz) magic-angle spinning (MAS). To elucidate the role of adsorbed water in spin diffusion and proton conductivity, PMETAC was degassed under vacuum. The CP MAS results were processed by applying the anisotropic Naito and McDowell spin dynamics model, which includes the complete scheme of the rotating frame spin-lattice relaxation pathways. Some earlier studied proton-conducting and nonconducting polymers were added to the analysis in order to prove the capability of the used approach and to get more general conclusions. The spin-diffusion rate constant, which describes the damping of the coherences, was found to be strongly depending on the dipolar I-S coupling constant (DIS). The spin diffusion, associated with the incoherent thermal equilibration with the bath, was found to be most probably independent of DIS. It was deduced that the drying scarcely influences the spin-diffusion rates; however, it significantly (1 order of magnitude) reduces the rotating frame spin-lattice relaxation times. The drying causes the polymer hardening that reflects the changes of the local order parameters. The impedance spectroscopy was applied to study proton conductivity. The activation energies for dielectric relaxation and proton conductivity were determined, and the vehicle-type conductivity mechanism was accepted. The spin-diffusion processes occur on the microsecond scale and are one order faster than the dielectric relaxation. The possibility to determine the proton location in the H-bonded structures in powders using CP MAS technique is discussed.

Robust transformation of singlet order into heteronuclear magnetisation over an extended coupling range.

Several important NMR procedures involve the conversion of nuclear singlet order into heteronuclear magnetisation, including some experiments involving long-lived spin states and parahydrogen-induced hyperpolarisation. However most existing sequences suffer from a limited range of validity or a lack of robustness against experimental imperfections. We present a new radio-frequency scheme for the transformation of the singlet order of a chemically-equivalent homonuclear spin pair into the magnetisation of a heteronuclear coupling partner. The proposed radio-frequency (RF) scheme is called gS2hM (generalized singlet-to-heteronuclear magnetisation) and has good compensation for common experimental errors such as RF and static field inhomogeneities. The sequence retains its robustness for homonuclear spin pairs in the intermediate coupling regime, characterised by the in-pair coupling being of the same order of magnitude as the difference between the out-of-pair couplings. This is a substantial improvement to the validity range of existing sequences. Analytical solutions for the pulse sequence parameters are provided. Experimental results are shown for two test cases.