Dynamic Nuclear Polarization Method Research Articles

Tailoring solid-state DNP methods to the study of [formula omitted]-synuclein LLPS

Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. DNPjuice comprising of glycerol-d8/D2O/H2O has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the invitro preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at ∼72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for 13C-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.

Fluorescence Quenching for Determination of Catalyst Concentration in the Parahydrogen‐Induced Polarization Method SABRE

AbstractIn recent years, parahydrogen‐induced hyperpolarization has become a focus for future medical applications. Similar to the established dynamic nuclear polarization method, a biocompatible bolus from a hyperpolarized sample can be produced for in vivo studies. However, this requires removing toxic hydrogenation catalysts, which inevitably must be used. Additionally, the ratio between the substrate to be hyperpolarized (e. g. pyruvate) and a mandatory catalyst must also be maintained. Even the smallest differences can lead to a reduction in generated signal amplification. In particular, weighing small amounts of catalysts leads to inaccuracies in sample preparations. Fluorescence spectroscopy provides a rapid and sensitive enough approach to determine catalyst concentration. The Ir‐IMes metal complexes used in SABRE lead to a quenching of the fluorescence of the solvent, dependent on its concentration. This can be used to quickly estimate the actual concentration in a solution with very small quantities of catalysts. Hence, fluorescence spectroscopy offers a rapid and reliable quality control method for the preparation of samples to be hyperpolarized. In addition, it can also be used as a quality control method to assess filtration efficacy before administration of hyperpolarized samples.

Searching for exotic spin-dependent interactions with diamond-based vector magnetometer

We propose a new method to search for exotic spin-spin interactions between electrons and nucleons using a diamond-based vector magnetometer. The vector magnetometer can be constructed from ensembles of nitrogen-vacancy centers along different axes in a diamond. The N14 nuclear spins of nitrogen-vacancy centers in the same diamond can be polarized through the dynamic nuclear polarization method to serve as spin sources. With the vector magnetometer, the sought-after exotic interactions can be distinguished from the magnetic dipole-dipole interaction. For the axion-mediated interaction VPP, new upper bounds of the coupling |gPegPN| are expected within the force range from 10 nm to 100 μm. For the Z′-mediated interaction VAA, new upper bounds of the coupling |gAegAN| are expected within the force range from 10 nm to 1 cm. The new upper bounds for VPP and VAA are both expected to be more than 5 orders of magnitude more stringent than existing constraints at the force range of 1 μm with the total measurement time of 1 day. Published by the American Physical Society 2024

Elementary processes triggered in curcumin molecule by gas-phase resonance electron attachment and by photoexcitation in solution.

Electron-driven processes in isolated curcumin (CUR) molecules are studied by means of dissociative electron attachment (DEA) spectroscopy under gas-phase conditions. Elementary photostimulated reactions initiated in CUR molecules under UV irradiation are studied using the chemically induced dynamic nuclear polarization method in an acetonitrile solvent. Density functional theory is applied to elucidate the energetics of fragmentation of CUR by low-energy (0-15eV) resonance electron attachment and to characterize various CUR radical forms. The adiabatic electron affinity of CUR molecule is experimentally estimated to be about 1eV. An extra electron attachment to the π1* LUMO and π2* molecular orbitals is responsible for the most intense DEA signals observed at thermal electron energy. The most abundant long-lived (hundreds of micro- to milliseconds) molecular negative ions CUR- are detected not only at the thermal energy of incident electrons but also at 0.6eV, which is due to the formation of the π3* and π4* temporary negative ion states predicted to lie around 1eV. Proton-assisted electron transfer between CUR molecules is registered under UV irradiation. The formation of both radical-anions and radical-cations of CUR is found to be more favorable in its enol form. The present findings shed some light on the elementary processes triggered in CUR by electrons and photons and, therefore, can be useful to understand the molecular mechanisms responsible for a variety of biological effects produced by CUR.

Dynamic Nuclear Polarization of Inorganic Halide Perovskites.

The intrinsic low sensitivity of nuclear magnetic resonance (NMR) experiments limits their utility for structure determination of materials. Dynamic nuclear polarization (DNP) under magic angle spinning (MAS) has shown tremendous potential to overcome this key limitation, enabling the acquisition of highly selective and sensitive NMR spectra. However, so far, DNP methods have not been explored in the context of inorganic lead halide perovskites, which are a leading class of semiconductor materials for optoelectronic applications. In this work, we study cesium lead chloride and quantitatively compare DNP methods based on impregnation with a solution of organic biradicals with doping of high-spin metal ions (Mn2+) into the perovskite structure. We find that metal-ion DNP provides the highest bulk sensitivity in this case, while highly surface-selective NMR spectra can be acquired using impregnation DNP. The performance of both methods is explained in terms of the relaxation times, particle size, dopant concentration, and surface wettability. We envisage the future use of DNP NMR approaches in establishing structure-activity relationships in inorganic perovskites, especially for mass-limited samples such as thin films.

Off-the-Shelf Gd(NO3)3 as an Efficient High-Spin Metal Ion Polarizing Agent for Magic Angle Spinning Dynamic Nuclear Polarization

Magic angle spinning nuclear magnetic resonance spectroscopy experiments are widely employed in the characterization of solid media. The approach is incredibly versatile but deleteriously suffers from low sensitivity, which may be alleviated by adopting dynamic nuclear polarization methods, resulting in large signal enhancements. Paramagnetic metal ions such as Gd3+ have recently shown promising results as polarizing agents for 1H, 13C, and 15N nuclear spins. We demonstrate that the widely available and inexpensive chemical agent Gd(NO3)3 achieves significant signal enhancements for the 13C and 15N nuclear sites of [2-13C,15N]glycine at 9.4 T and ∼105 K. Analysis of the signal enhancement profiles at two magnetic fields, in conjunction with electron paramagnetic resonance data, reveals the solid effect to be the dominant signal enhancement mechanism. The signal amplification obtained paves the way for efficient dynamic nuclear polarization without the need for challenging synthesis of Gd3+ polarizing agents.

Dynamic Nuclear Polarization Enables NMR of Surface Passivating Agents on Hybrid Perovskite Thin Films.

Surface and bulk molecular modulators are the key to improving the efficiency and stability of hybrid perovskite solar cells. However, due to their low concentration, heterogeneous environments, and low sample mass, it remains challenging to characterize their structure and dynamics at the atomic level, as required to establish structure–activity relationships. Nuclear magnetic resonance (NMR) spectroscopy has revealed a wealth of information on the atomic-level structure of hybrid perovskites, but the inherent insensitivity of NMR severely limits its utility to characterize thin-film samples. Dynamic nuclear polarization (DNP) can enhance NMR sensitivity by orders of magnitude, but DNP methods for perovskite materials have so far been limited. Here, we determined the factors that limit the efficiency of DNP NMR for perovskite samples by systematically studying layered hybrid perovskite analogues. We find that the fast-relaxing dynamic cation is the major impediment to higher DNP efficiency, while microwave absorption and particle morphology play a secondary role. We then show that the former can be mitigated by deuteration, enabling 1H DNP enhancement factors of up to 100, which can be harnessed to enhance signals from dopants or additives present in very low concentrations. Specifically, using this new DNP methodology at a high magnetic field and with small sample volumes, we have recorded the NMR spectrum of the 20 nm (6 μg) passivating layer on a single perovskite thin film, revealing a two-dimensional (2D) layered perovskite structure at the surface that resembles the n = 1 homologue but which has greater disorder than in bulk layered perovskites.

Structure of In Vitro-Synthesized Cellulose Fibrils Viewed by Cryo-Electron Tomography and 13C Natural-Abundance Dynamic Nuclear Polarization Solid-State NMR.

Cellulose, the most abundant biopolymer, is a central source for renewable energy and functionalized materials. In vitro synthesis of cellulose microfibrils (CMFs) has become possible using purified cellulose synthase (CESA) isoforms from Physcomitrium patens and hybrid aspen. The exact nature of these in vitro fibrils remains unknown. Here, we characterize in vitro-synthesized fibers made by CESAs present in membrane fractions of P. patens over-expressing CESA5 by cryo-electron tomography and dynamic nuclear polarization (DNP) solid-state NMR. DNP enabled measuring two-dimensional 13C–13C correlation spectra without isotope-labeling of the fibers. Results show structural similarity between in vitro fibrils and native CMF in plant cell walls. Intensity quantifications agree with the 18-chain structural model for plant CMF and indicate limited fibrillar bundling. The in vitro system thus reveals insights into cell wall synthesis and may contribute to novel cellulosic materials. The integrated DNP and cryo-electron tomography methods are also applicable to structural studies of other carbohydrate-based biomaterials.

Sample volume effects in optical overhauser dynamic nuclear polarization

The optical dynamic nuclear polarization (DNP) method has been proposed as an alternative to microwave pumping as a hyperpolarization method for solution-state NMR studies. Using continuous laser illumination to photogenerate triplet states in the presence of a persistent radical produces chemically-induced dynamic electron polarization (CIDEP) via the radical-triplet pair mechanism (RTPM), with cross-relaxation transferring this to nuclear hyperpolarization via an Overhauser mechanism. Numerical simulations have previously indicated that reducing the sample volume while maintaining a constant optical density can significantly increase the NMR signal enhancement, due to the larger steady-state concentration of triplets obtained. Here we provide the first experimental confirmation of these effects, producing a nearly five-fold increase in the optical DNP enhancement factor just by reducing the sample volume with optimal dye and radical concentrations adjusted for each optical path length. The results are supported with an in depth analysis of volume effects in the numerical model, with which they are in good qualitative agreement. These important observations will impact on the future development of the technique, with particular significance for attempts to apply DNP methods to increase sensitivity for volume-limited biological samples.

DNPSOUP: A simulation software package for dynamic nuclear polarization

Dynamic Nuclear Polarization Simulation Optimized with a Unified Propagator (DNPSOUP) is an open-source numerical software program that models spin dynamics for dynamic nuclear polarization (DNP). The software package utilizes a direct numerical approach using the inhomogeneous master equation to treat the time evolution of the spin density operator under coherent Hamiltonians and stochastic relaxation effects. Here we present the details of the theory behind the software, starting from the master equation, and arriving at characteristic operators for any section of density operator time-evolution. We then provide an overview of the DNPSOUP software architecture. The efficacy of the program is demonstrated by simulating DNP field profiles on small spin systems exploiting both continuous wave and time-domain DNP mechanisms. Examples include Zeeman field profiles for the solid effect, Overhauser effect, and cross effect, and microwave field profiles for NOVEL, off-resonance NOVEL, the integrated solid effect, the stretched solid effect, and TOP-DNP. The software should facilitate a better understanding of the DNP process, aid in the design of optimized DNP polarizing agents, and allow us to examine new pulsed DNP methods at conditions that are not currently experimentally accessible, especially at high magnetic fields with high-power microwave pulses.

Spin Temperature and Dynamic Nuclear Polarization. From the History of Researches (1953 – 1983)

An attempt is proposed in the most concise form to provide the reader with a history of research and applications of dynamic nuclear polarization (DNP). The main attention is paid to the first three decades of DNP research, and the history of the discovery and development of multiparticle DNP and its relationship with the spin temperature approximation are outlined in some detail. The article emphasizes the role of such researchers as Anatol Abraham, Maurice Goldman, Michel Borghini, Thomas Wenckebach, Vadim Atsarkin, Boris Provotorov, Maya Rodak, Mortko Kozhushner, Levan Buishvili, Givi Khutsishvili. As far as possible, the contributions of many other scientists are considered. The establishment of a uniform temperature for nuclear spins due to the effect of spin diffusion was first proposed by Nicholas Blombergen in 1949. The content of the article is based on the bibliography available in the public domains, in particular on the memoirs of the research participants, and first of all on the materials of Atsarkin's 1978 review in Sov. Phys. Uspekhi and on the oral history of the development of the multiparticle concept of DNP effects, recorded from the speeches of the participants of the Moscow seminar "Problems of Magnetic Resonance" in 2001. A simplified description of the effects of DNP and a summary of the history of their discovery is given in section “Introduction”. The shortest biographical data and portraits of participants in the DNP study are given in Appendix 1, and a selected bibliography on the problems of DNP and spin temperatures is given in Appendix 2. The bibliography divided into four sections according to the time and type of publication (I - historical research, memoirs; II – monographs, reviews; III - original publications 1953 - 1983; IV – some original publications of a later time, mainly during the transformation of DNP into an method for the implementation of nuclear magnetic spectroscopy and tomography in the interests of chemistry, biochemistry and medicine). The widespread use of DNP methods is evidenced, for example, by the fact, that by now company Bruker BioSpin has installed about 50 gyrotron based spectrometers for DNP operating upto 593 GHz worldwide to date.

Tumor Microenvironment Biosensors for Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopy.

Hyperpolarization (HP) of a carbon-13 molecule via dynamic nuclear polarization (DNP) involves polarization at low temperature, followed by a dissolution procedure producing a solution with highly polarized spins at room temperature. This dissolution DNP method significantly increases the signal-to-noise ratio (SNR) of nuclear magnetic resonance (NMR) over 10,000-fold and facilitates the use of magnetic resonance spectroscopy (MRS) to image not only metabolism but also the extracellular microenvironment. The extracellular tumor microenvironment (TME) closely interacts with tumor cells and stimulates their growth and metastasis. Thus, the ability to detect pathological changes in the TME is pivotal for the detection and study of cancers. This review highlights the potential use of MRS to study features of the TME-elevated export of lactate, reduced interstitial pH, imbalanced redox equilibrium, and altered metal homeostasis. The promising outcomes of both in vitro and in vivo assays suggest that DNP-MRS may be a useful technique to study aspects of the TME. With continued improvements, this tool has the potential to study the TME and provide guidance for accurate patient stratification and precise personal therapy. Graphical Abstract.

15 N-carnitine, a novel endogenous hyperpolarized MRI probe with long signal lifetime.

The purpose of this study was to investigate hyperpolarization and in vivo imaging of [15 N]carnitine, a novel endogenous MRI probe with long signal lifetime. L-[15 N]carnitine-d9 was hyperpolarized by the method of dynamic nuclear polarization followed by rapid dissolution. The T1 signal lifetimes were estimated in aqueous solution and in vivo following intravenous injection in rats, using a custom-built dual-tuned 15 N/1 H RF coil at 4.7 T. 15 N chemical shift imaging and 15 N fast spin-echo images of rat abdomen were acquired 3 minutes after [15 N]carnitine injection. Estimated T1 times of [15 N]carnitine at 4.7 T were 210 seconds (in H2 O) and 160 seconds (in vivo), with an estimated polarization level of 10%. Remarkably, the [15 N]carnitine coherence was detectable in rat abdomen for 5 minutes after injection for the nonlocalized acquisition. No downstream metabolites were detected on localized or nonlocalized 15 N spectra. Diffuse liver enhancement was detected on 15 N fast spin-echo imaging 3 minutes after injection, with mean hepatic SNR of 18 ± 5 at a spatial resolution of 4 × 4 mm. This study showed the feasibility of hyperpolarizing and imaging the biodistribution of HP [15 N]carnitine.

High-Field Dynamic Nuclear Polarization.

Dynamic nuclear polarization (DNP) is one of the most prominent methods of sensitivity enhancement in nuclear magnetic resonance (NMR). Even though solid-state DNP under magic-angle spinning (MAS) has left the proof-of-concept phase and has become an important tool for structural investigations of biomolecules as well as materials, it is still far from mainstream applicability because of the potentially overwhelming combination of unique instrumentation, complex sample preparation, and a multitude of different mechanisms and methods available. In this review, I introduce the diverse field and history of DNP, combining aspects of NMR and electron paramagnetic resonance. I then explain the general concepts and detailed mechanisms relevant at high magnetic field, including solution-state methods based on Overhauser DNP but with a greater focus on the more established MAS DNP methods. Finally, I review practical considerations and fields of application and discuss future developments.

Room temperature "optical nanodiamond hyperpolarizer": Physics, design, and operation.

Dynamic Nuclear Polarization (DNP) is a powerful suite of techniques that deliver multifold signal enhancements in nuclear magnetic resonance (NMR) and MRI. The generated athermal spin states can also be exploited for quantum sensing and as probes for many-body physics. Typical DNP methods require the use of cryogens, large magnetic fields, and high power microwave excitation, which are expensive and unwieldy. Nanodiamond particles, rich in Nitrogen-Vacancy (NV) centers, have attracted attention as alternative DNP agents because they can potentially be optically hyperpolarized at room temperature. Here, unraveling new physics underlying an optical DNP mechanism first introduced by Ajoy et al. [Sci. Adv. 4, eaar5492 (2018)], we report the realization of a miniature "optical nanodiamond hyperpolarizer," where 13C nuclei within the diamond particles are hyperpolarized via the NV centers. The device occupies a compact footprint and operates at room temperature. Instrumental requirements are very modest: low polarizing fields, low optical and microwave irradiation powers, and convenient frequency ranges that enable miniaturization. We obtain the best reported optical 13C hyperpolarization in diamond particles exceeding 720 times of the thermal 7 T value (0.86% bulk polarization), corresponding to a ten-million-fold gain in averaging time to detect them by NMR. In addition, the hyperpolarization signal can be background-suppressed by over two-orders of magnitude, retained for multiple-minute long periods at low fields, and deployed efficiently even to 13C enriched particles. Besides applications in quantum sensing and bright-contrast MRI imaging, this work opens possibilities for low-cost room-temperature DNP platforms that relay the 13C polarization to liquids in contact with the high surface-area particles.

Optically-generated Overhauser dynamic nuclear polarization: A numerical analysis.

Recently, an alternative approach to dynamic nuclear polarization (DNP) in the liquid state was introduced using optical illumination instead of microwave pumping. By exciting a suitable dye to the triplet state which undergoes a diffusive encounter with a persistent radical forming a quartet-doublet pair in the encounter complex, dynamic electron polarization (DEP) is generated via the radical-triplet pair mechanism. Subsequent cross-relaxation generates nuclear polarization without the need for microwave saturation of the electronic transitions. Here, we present a theoretical justification for the initial experimental results by means of numerical simulations. These allow investigation of the effects of various experimental parameters, such as radical and dye concentrations, sample geometry, and laser power, on the DNP enhancement factors, providing targets for experimental optimization. It is predicted that reducing the sample volume will result in larger enhancements by permitting a higher concentration of triplets in a sample of increased optical density. We also explore the effects of the pulsed laser rather than continuous-wave illumination, rationalizing the failure to observe the optical DNP effect under illumination conditions common to DEP experiments. Examining the influence of the illumination duty cycle, the conditions necessary to permit the use of pulsed illumination without compromising signal enhancement are determined, which may reduce undesirable laser heating effects. This first simulation of the optical DNP method therefore underpins the further development of the technology.

X-nuclei hyperpolarization for studying molecular dynamics by DNP-FFC.

Dynamic Nuclear Polarization methods are used for improving the quality of the NMR data, opening new possibilities by increasing both the sensitivity and the selectivity in NMR relaxation experiments. Recently, Fast Field Cycling relaxometry combined with DNP was introduced, demonstrating that molecular dynamics studies in the presence of natural or artificial radicals are indeed feasible under conditions where the signal-to-noise ratio is frequently critical. In this work, the extension of NMR relaxation dispersion beyond 1H NMR, by hyperpolarization of X-nuclei, is demonstrated. Overhauser effect via nitroxide radicals in simple (low viscous) liquids and saline solutions was observed for 2H, 7Li and 13C nuclei at ambient temperature. Substantial NMR signal enhancement up to several hundred was achieved for the studied samples. An advanced approach for reconstructing of the original relaxation dispersion of pure substances is used to eliminate the effect of the additional radical relaxivity of the X-nuclei.

One- and Two-Dimensional High-Resolution NMR from Flat Surfaces.

Determining atomic-level characteristics of molecules on two-dimensional surfaces is one of the fundamental challenges in chemistry. High-resolution nuclear magnetic resonance (NMR) could deliver rich structural information, but its application to two-dimensional materials has been prevented by intrinsically low sensitivity. Here we obtain high-resolution one- and two-dimensional 31P NMR spectra from as little as 160 picomoles of oligonucleotide functionalities deposited onto silicate glass and sapphire wafers. This is enabled by a factor >105 improvement in sensitivity compared to typical NMR approaches from combining dynamic nuclear polarization methods, multiple-echo acquisition, and optimized sample formulation. We demonstrate that, with this ultrahigh NMR sensitivity, 31P NMR can be used to observe DNA bound to miRNA, to sense conformational changes due to ion binding, and to follow photochemical degradation reactions.

A novel broad-band neutron spin filter based on dynamically polarized protons using photo-excited triplet states

The use of polarized protons as a broad-band neutron spin filter is an attractive alternative to the well-established neutron polarization techniques, namely polarized 3He gas and super mirrors, since the spin-dependent neutron proton scattering cross-section is large in a broad wavelength range. We have developed a novel neutron spin filter where we create the necessary large proton polarization in a solid with a recent method of dynamic nuclear polarization (DNP) that uses photo-excited triplet states. This requires only moderate experimental means and allows a compact design. In order to quantify the efficiency of the spin filter, we have measured the relevant spin-dependent and spin-independent terms of the neutron scattering cross-section of a naphthalene single crystal. The data allows to estimate the triplet spin filter performance over a broad wavelength range. With the recently achieved proton polarization of 80% the triplet filter compares well with a state of the art 3He filter.

Hyperpolarized MRI for Studying Tumor Metabolism.

Hyperpolarized magnetic resonance imaging (MRI) can be used to detect real-time in vivo tumor metabolism. Dissolution dynamic nuclear polarization method increases polarization of 13C-labeled molecules, typically [1-13C]pyruvate, which can be injected into an animal during MRI scanning. Increased polarization leads to a higher observed signal, which allows for the detection and imaging of the transfer of 13C-label between the injected marker molecule, pyruvate, and its metabolic products, most importantly lactate. This information can be used to assess the metabolic status of the tumor, for example, during therapy. Here, the basic methodology and data analysis for a preclinical hyperpolarized pyruvate experiment are described.