Simultaneous acquisition of multi-nuclei enhanced NMR/MRI by solution-state dynamic nuclear polarization

Dynamic nuclear polarisation (DNP) is a process that transfers electron spin polarisation to nuclei by applying resonant microwave radiation, and has been widely used to improve the sensitivity of nuclear magnetic resonance (NMR). Here we demonstrate new levels of performance for static cross-effect proton DNP using high peak power chirped inversion pulses at 94 GHz to create a strong polarisation gradient across the inhomogeneously broadened line of the mono-radical 4-amino TEMPO. Enhancements of up to 340 are achieved at an average power of a few hundred mW, with fast build-up times (3 s). Experiments are performed using a home-built wideband kW pulsed electron paramagnetic resonance (EPR) spectrometer operating at 94 GHz, integrated with an NMR detection system. Simultaneous DNP and EPR characterisation of other mono-radicals and biradicals, as a function of temperature, leads to additional insights into limiting relaxation mechanisms and give further motivation for the development of wideband pulsed amplifiers for DNP at higher frequencies.

Nuclear Magnetic Resonance (NMR) spectroscopy would be a method of choice to follow biochemical events in cells because it can analyze molecules in complex environments. However, the intrinsically low sensitivity of NMR makes in-cell measurements challenging. Dynamic Nuclear Polarization (DNP) has emerged as a method to circumvent this limitation, but most polarizing agents developed for DNP are unstable in reducing cellular environments. Here, we introduce the use of Gd(III)-based DNP polarizing agents for in-cell NMR spectroscopy. Specifically, we show their persistent stability in cellular formulations, and we investigate the DNP performance of the Gd(III)-based complex [Gd(tpatcn)] in human embryonic kidney cell lysates and intact cells. For cell lysates, DNP enhancements up to -27 are obtained on the cellular signals, reproducible even after storage at room temperature for days. Mixing the [Gd(tpatcn)] solution with intact cells enables the observation of cellular signals with DNP, and DNP enhancement factors of about -40 are achieved.

The inherently low sensitivity of nuclear magnetic resonance(NMR)spectroscopy is the major limiting factor for its application to elucidatestructure and dynamics in solids. In the solid state, nuclear spinhyperpolarization methods based on microwave-induced dynamic nuclearpolarization (DNP) provide a versatile platform to enhance the bulkNMR signal of many different sample formulations, leading to significantsensitivity improvements. Here we show that 1H NMR hyperpolarizationcan also be generated in solids at high magnetic fields by opticalirradiation of the sample. We achieved this by exploiting a donor–chromophore–acceptormolecule with an excited state electron–electron interactionsimilar to the nuclear Larmor frequency, enabling solid-state 1H photochemically induced DNP (photo-CIDNP) at high magneticfields. Through hyperpolarization relay, we obtained bulk NMR signalenhancements εH by factors of ∼100 at both9.4 and 21.1 T for the 1H signal of o-terphenylin magic angle spinning (MAS) NMR experiments at 100 K. These findingsopen a pathway toward a general light-induced hyperpolarization approachfor dye-sensitized high-field NMR in solids.

Dynamic nuclear polarization (DNP) has significantly enhanced the sensitivity of nuclear magnetic resonance (NMR) experiments. While Overhauser DNP has a well-established nuclear counterpart, the nuclear Overhauser effect (NOE), the cross-effect DNP has traditionally been considered as an exclusively electron-mediated process. Here, we present the first evidence of a nuclear counterpart to the cross effect, termed the nuclear cross effect, which mediates polarization transfer in a three-spin nuclear system. The effect was experimentally verified using single-crystal 13C-15N-labeled glycine, with results consistent with both analytical theory and numerical simulations. The experimental realization of the pulsed nuclear cross-effect in this work provides new insights into its theoretical foundations, addressing challenges posed by hardware limitations and the absence of a suitable model system for pulsed cross-effect DNP. Furthermore, the pulsed cross-effect mechanism has the potential to advance enhanced NMR applications in various fields.

Review: 194 refs.

Nuclear magnetic resonance (NMR) has been broadly investigated and applied in many industries (e.g., medical treatment, food safety and materials) as a powerful approach to explore the internal structure and composition of substances. However, the need for more sensitivity has been one of the most severe problems hindering the practical application of NMR. This paper suggests various approaches to deal with this defect, from adjusting factors based on the theoretical principle to the progressive hyperpolarization method. Classified by polarization sources, several representative sub-fields of hyperpolarization are further interpreted and analyzed, including Dynamic Nuclear Polarization (DNP), Optical Pumping (OP) and Parahydrogen Induced Polarization (PHIP). Based on the analysis and prospect of the aforementioned feasible methods, the main factors that restrict the sensitivity of NMR will be identified. Moreover, the prospects and new technologies brought by the methods based on different theoretical foundations have been considered to improve these factors and make NMR apply to a broader range of fields. Overall, these results not only indicate the current research status but also shed light on guiding further exploration of methods of enhancing NMR sensitivity and usability.

Dynamic nuclear polarization with photo-excited triplet electrons (triplet-DNP) has the potential to enhance the sensitivity of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) at a moderate temperature. While many efforts have been devoted to achieving a large nuclear polarization based on triplet-DNP, the application of triplet-DNP has been limited to nuclear physics experiments. The recent introduction of materials chemistry into the field of triplet-DNP has achieved air-stable and water-soluble polarizing agents as well as the hyperpolarization of nanomaterials with a large surface area such as nanoporous metal-organic frameworks (MOFs) and nanocrystal dispersion in water. This Feature Article overviews the recently-emerged materials chemistry of triplet-DNP that paves new paths towards unprecedented biological and medical applications.

Gyrotrons have been developed as terahertz wave sources in dynamic nuclear polarisation (DNP) system to increase the sensitivity of nuclear magnetic resonance (NMR) in the high-magnetic field regime. However, the transmission of the Gaussian beam from the gyrotron output window to the NMR system proves to be a critical challenge. In this study, a terahertz transmission system, composed of a rectangular corrugated waveguide and a back-to-back rectangular corrugated horn for Gaussian mode filtering, is proposed. The attenuation in the optimised 0.33 THz corrugated waveguide remains lower than 0.01 dB/m in a broad frequency range from 280 GHz to 450 GHz. Besides, the coupling coefficient from the electric field in the 0.33 THz mode-filtering horn to the fundamental HE11 mode in the rectangular corrugated waveguide is obtained nearly 99.5%. This terahertz transmission system is potential to be applied in DNP-NMR spectrometer based on continuous wave gyrotrons.

Solid-state nuclear magnetic resonance (NMR) spectroscopy is an atomic-level method used to determine the chemical structure, three-dimensional structure, and dynamics of solids and semi-solids. This Primer summarizes the basic principles of NMR as applied to the wide range of solid systems. The fundamental nuclear spin interactions and the effects of magnetic fields and radiofrequency pulses on nuclear spins are the same as in liquid-state NMR. However, because of the anisotropy of the interactions in the solid state, the majority of high-resolution solid-state NMR spectra is measured under magic-angle spinning (MAS), which has profound effects on the types of radiofrequency pulse sequences required to extract structural and dynamical information. We describe the most common MAS NMR experiments and data analysis approaches for investigating biological macromolecules, organic materials, and inorganic solids. Continuing development of sensitivity-enhancement approaches, including 1H-detected fast MAS experiments, dynamic nuclear polarization, and experiments tailored to ultrahigh magnetic fields, is described. We highlight recent applications of solid-state NMR to biological and materials chemistry. The Primer ends with a discussion of current limitations of NMR to study solids, and points to future avenues of development to further enhance the capabilities of this sophisticated spectroscopy for new applications.

Solid Effect DNP in a Rapid-melt setup

Outlining biological processes and disease mechanisms requires a real-time understanding of cellular metabolism. Mass spectrometry (MS) and Nuclear magnetic resonance (NMR) serve as potent analytical methods for metabolomics, leveraging their advantages in the identification and quantification of metabolites. In this review we have discussed MS and NMR based techniques, such as matrix-assisted laser desorption/ionization (MALDI), secondary ionization mass spectrometry (SIMS), desorption electrospray ionization (DESI), direct analysis in real time (DART), and nano electrospray ionization (nano ESI), to develop and implement a MS based technique in its live state for single cell and NMR strategies for thorough, real-time analysis of cell metabolism at bulk cellular level. Single-cell metabolomic investigations using mass spectrometry is a valuable tool for understanding cellular heterogeneity and cell-to-cell variation. However, they can reveal hidden processes and heterogeneity across cells that are often missed by bulk cell analysis. Overcoming the inherently low sensitivity of NMR is crucial for omics studies. This review examines hyperpolarization techniques, including dynamic nuclear polarization (DNP), parahydrogen-induced polarization, sample amplification by reversible exchange (SABER), high-resolution magic angle spinning (HRMAS) NMR, and in vivo magnetic resonance spectroscopy (MRS) for the analysis of live bulk cells. Our discussion encompasses the technological platforms and recent applications of these techniques, utilizing both NMR and MS, along with an overview of metabolomics data analysis tools, such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA).

Metabolism is an interconnecting network of metabolite consumption and creation. Metabolomics has focused on metabolite concentrations in metabolic networks. Fluxomics is also required in the study of metabolism and quantifies the flux of substrate through each reaction step or a series of reaction steps (i.e., metabolic pathway or cycle), and ultimately is required for energy balance equations of the system. The primary noninvasive method of quantifying fluxes in living systems is by in vivo 13C nuclear magnetic resonance (NMR) spectroscopy. The present state of noninvasive in vivo NMR technology allows for just four simultaneous flux measurements of metabolic pathways: gluconeogenesis, glycogen synthesis, glycolysis, and citric acid cycle. Since the liver is the gatekeeper and metabolic center for the animal, in vivo fluxomics of liver is extensively reviewed. Additionally, other organ systems studies are discussed demonstrating interorgan cycles, such as the Cori and Randall cycles. This review discusses the basics of in vivo fluxomics focusing on the general details of the NMR experimental protocol and required hardware/software needed to analyze the data.Currently, there are two general methods for determining multiple flux rates. The dynamic method entails acquiring serial time points, whereas the static method is a single measurement in which flux through metabolic pathways is quantified by isotopomer (i.e., isotope isomers) analysis. The flux data are analyzed by mathematical models to calculate the global flux measurement (in silico fluxomics), and create a mass balance of the biosystem. Models are especially useful for inferring various metabolic states of the system, which are affected by drugs, toxicants, or pathology. As with all in silico models, increasing the number of empirically derived concentrations and fluxes into the model greatly increases the accuracy and utility of the model. NMR spectroscopy (NMRS) is inherently insensitive compared to other analytical modalities, limiting the temporal resolution of the dynamic in vivo measurements. To address the NMR sensitivity, several technological advances have been made. First, magnets are now at higher magnetic field strengths. Second, the technique of dynamic nuclear polarization (DNP) of substrates increases the signal for 13C up to five orders of magnitude.In vivo fluxomics requires a broad knowledge of biochemistry, in vivo NMRS, and metabolic modeling. Therefore, this chapter is intended as a handbook for upper division undergraduate students and graduate students in biochemistry or engineering and relates the basics of electrical and biochemical engineering and animal handling. The chapter is intended for use in an introductory graduate course on NMR-based fluxomics for physical scientist.

Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

While dynamic nuclear polarization using photo-excited triplet electrons (triplet-DNP) can improve the sensitivity of nuclear magnetic resonance at room temperature, it has not been carried out in water. Here, we report the first example of triplet-DNP in water by downsizing the conventional bulk crystals to nanocrystals.

The coherent transfer of electron spin polarization to nuclei by means of a microwave pulse sequence is a promising new approach to enhancing the sensitivity of solid-state nuclear magnetic resonance (NMR). The development of pulse sequences for dynamic nuclear polarization (DNP) of bulk nuclei is far from complete, as is the understanding of what makes a good DNP sequence. In this context, we introduce a new sequence, termed Two-Pulse Phase Modulation (TPPM) DNP. We provide a general theoretical description for electron-proton polarization transfer by periodic DNP pulse sequences and find it in excellent agreement with numerical simulations. In experiments at 1.2T, TPPM DNP generates a higher gain in sensitivity than existing sequences XiX (X-inverse-X) and TOP (Time-Optimized Pulsed) DNP but does so at relatively high nutation frequencies. In contrast, we find that the XiX sequence performs very well at nutation frequencies as low as 7MHz. A combination of theoretical analysis and experimental investigation makes clear that fast electron-proton polarization transfer, due to a well-preserved dipolar coupling in the effective Hamiltonian, correlates with a short build-up time of the dynamic nuclear polarization of the bulk. Experiments further show that the performances of XiX and TOP DNP are affected differently by the concentration of the polarizing agent. These results constitute important reference points for the development of new and better DNP sequences.