Low-field, not low quality: 1D simplification, selective detection, and heteronuclear 2D experiments for improving low-field NMR spectroscopy of environmental and biological samples.

Understanding environmental change is challenging and requires molecular-level tools to explain the physicochemical phenomena behind complex processes. Nuclear magnetic resonance (NMR) spectroscopy is a key tool that provides information on both molecular structures and interactions but is underutilized in environmental research because standard "high-field" NMR is financially and physically inaccessible for many and can be overwhelming to those outside of disciplines that routinely use NMR. "Low-field" NMR is an accessible alternative but has reduced sensitivity and increased spectral overlap, which is especially problematic for natural, heterogeneous samples. Therefore, the goal of this study is to investigate and apply innovative experiments that could minimize these challenges and improve low-field NMR analysis of environmental and biological samples. Spectral simplification (JRES, PSYCHE, singlet-only, multiple quantum filters), selective detection (GEMSTONE, DREAMTIME), and heteronuclear (reverse and CH3/CH2/CH-only HSQCs) NMR experiments are tested on samples of increasing complexity (amino acids, spruce resin, and intact water fleas) at-high field (500 MHz) and at low-field (80 MHz). A novel experiment called Doubly Selective HSQC is also introduced, wherein 1H signals are selectively detected based on the 1H and 13C chemical shifts of 1H-13C J-coupled pairs. The most promising approaches identified are the selective techniques (namely for monitoring), and the reverse and CH3-only HSQCs. Findings ultimately demonstrate that low-field NMR holds great potential for biological and environmental research. The multitude of NMR experiments available makes NMR tailorable to nearly any research need, and low-field NMR is therefore anticipated to become a valuable and widely used analytical tool moving forward.

Cutting without a Knife: A Slice-Selective 2D 1H-13C HSQC NMR Sequence for the Analysis of Inhomogeneous Samples.

Nuclear magnetic resonance (NMR) is a powerful technique with applications ranging from small molecule structure elucidation to metabolomics studies of living organisms. Typically, solution-state NMR requires a homogeneous liquid, and the whole sample is analyzed as a single entity. While adequate for homogeneous samples, such an approach is limited if the composition varies as would be the case in samples that are naturally heterogeneous or layered. In complex samples such as living organisms, magnetic susceptibility distortions lead to broad 1H line shapes, and thus, the additional spectral dispersion afforded by 2D heteronuclear experiments is often required for metabolite discrimination. Here, a novel, slice-selective 2D, 1H-13C heteronuclear single quantum coherence (HSQC) sequence was developed that exclusively employs shaped pulses such that only spins in the desired volume are perturbed. In turn, this permits multiple volumes in the tube to be studied during a single relaxation delay, increasing sensitivity and throughput. The approach is first demonstrated on standards and then used to isolate specific sample/sensor elements from a microcoil array and finally study slices within a living earthworm, allowing metabolite changes to be discerned with feeding. Overall, slice-selective NMR is demonstrated to have significant potential for the study of layered and other inhomogeneous samples of varying complexity. In particular, its ability to select subelements is an important step toward developing microcoil receive-only arrays to study environmental toxicity in tiny eggs, cells, and neonates, whereas localization in larger living species could help better correlate toxin-induced biochemical responses to the physical localities or organs involved.

Application of 15N-Edited 1H-13C Correlation NMR Spectroscopy─Toward Fragment-Based Metabolite Identification and Screening via HCN Constructs.

Many key building blocks of life contain nitrogen moieties. Despite the prevalence of nitrogen-containing metabolites in nature, 15N nuclei are seldom used in NMR-based metabolite assignment due to their low natural abundance and lack of comprehensive chemical shift databases. However, with advancements in isotope labeling strategies, 13C and 15N enriched metabolites are becoming more common in metabolomic studies. Simple multidimensional nuclear magnetic resonance (NMR) experiments that correlate 1H and 15N via single bond 1JNH or multiple bond 2-3JNH couplings using heteronuclear single quantum coherence (HSQC) or heteronuclear multiple bond coherence are well established and routinely applied for structure elucidation. However, a 1H-15N correlation spectrum of a metabolite mixture can be difficult to deconvolute, due to the lack of a 15N specific database. In order to bridge this gap, we present here a broadband 15N-edited 1H-13C HSQC NMR experiment that targets metabolites containing 15N moieties. Through this approach, nitrogen-containing metabolites, such as amino acids, nucleotide bases, and nucleosides, are identified based on their 13C, 1H, and 15N chemical shift information. This approach was tested and validated using a [15N, 13C] enriched Daphnia magna (water flea) metabolite extract, where the number of clearly resolved 15N-containing peaks increased from only 11 in a standard HSQC to 51 in the 15N-edited HSQC, and the number of obscured peaks decreased from 59 to just 7. The approach complements the current repertoire of NMR techniques for mixture deconvolution and holds considerable potential for targeted metabolite NMR in 15N, 13C enriched systems.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Exploring Proton-Only NMR Experiments and Filters for Daphnia In Vivo: Potential and Limitations.

Environmental metabolomics provides insight into how anthropogenic activities have an impact on the health of an organism at the molecular level. Within this field, in vivo NMR stands out as a powerful tool for monitoring real-time changes in an organism's metabolome. Typically, these studies use 2D 13C-1H experiments on 13C-enriched organisms. Daphnia are the most studied species, given their widespread use in toxicity testing. However, with COVID-19 and other geopolitical factors, the cost of isotope enrichment increased ~6-7 fold over the last two years, making 13C-enriched cultures difficult to maintain. Thus, it is essential to revisit proton-only in vivo NMR and ask, "Can any metabolic information be obtained from Daphnia using proton-only experiments?". Two samples are considered here: living and whole reswollen organisms. A range of filters are tested, including relaxation, lipid suppression, multiple-quantum, J-coupling suppression, 2D 1H-1H experiments, selective experiments, and those exploiting intermolecular single-quantum coherence. While most filters improve the ex vivo spectra, only the most complex filters succeed in vivo. If non-enriched organisms must be used, then, DREAMTIME is recommended for targeted monitoring, while IP-iSQC was the only experiment that allowed non-targeted metabolite identification in vivo. This paper is critically important as it documents not just the experiments that succeed in vivo but also those that fail and demonstrates first-hand the difficulties associated with proton-only in vivo NMR.

Exploring the Potential of Broadband Complementary Metal Oxide Semiconductor Micro-Coil Nuclear Magnetic Resonance for Environmental Research.

With sensitivity being the Achilles' heel of nuclear magnetic resonance (NMR), the superior mass sensitivity offered by micro-coils can be an excellent choice for tiny, mass limited samples such as eggs and small organisms. Recently, complementary metal oxide semiconductor (CMOS)-based micro-coil transceivers have been reported and demonstrate excellent mass sensitivity. However, the ability of broadband CMOS micro-coils to study heteronuclei has yet to be investigated, and here their potential is explored within the lens of environmental research. Eleven nuclei including 7Li, 19F, 31P and, 205Tl were studied and detection limits in the low to mid picomole range were found for an extended experiment. Further, two environmentally relevant samples (a sprouting broccoli seed and a D. magna egg) were successfully studied using the CMOS micro-coil system. 13C NMR was used to help resolve broad signals in the 1H spectrum of the 13C enriched broccoli seed, and steady state free precession was used to improve the signal-to-noise ratio by a factor of six. 19F NMR was used to track fluorinated contaminants in a single D. magna egg, showing potential for studying egg-pollutant interactions. Overall, CMOS micro-coil NMR demonstrates significant promise in environmental research, especially when the future potential to scale to multiple coil arrays (greatly improving throughput) is considered.

NMR assignment of the in vivo daphnia magna metabolome.

Daphnia (freshwater fleas) are among the most widely used organisms in regulatory aquatic toxicology/ecology, while their recent listing as an NIH model organism is stimulating research for understanding human diseases and processes. Daphnia are small enough to fit inside high field NMR spectrometers and can be kept alive indefinitely using flow systems that deliver food and oxygen. As such, in vivo NMR holds the potential to monitor when/if environmental stress is occurring, understand "why" chemicals are toxic (biochemical pathways impacted and toxic-mode-of-action), and differentiate between a temporary flux response (i.e. return to homeostasis) and a permanent change in biochemistry (likely a precursor to disease). At present however, such studies are limited as the in vivo NMR data of Daphnia are highly complex and the lack of spectral assignments makes extracting metabolic information difficult. In this study, Daphnia are 13C enriched to >97% 13C and numerous 1H and 13C 1D, 2D, and 3D NMR approaches are combined to provide, as complete as possible, an assignment of the Daphnia magna metabolome in vivo. Assignments are transferred (where possible) back to line narrowed susceptibility suppressed 1H 1D NMR spectra in order to permit the maximum amount of information to be gained in the future without the need for 13C enrichment. To our knowledge, this work represents the first time a comprehensive metabolic assignment of any small living organism has been performed using high field flow-based NMR.

A simple 1H (12C/13C) filtered experiment to quantify and trace isotope enrichment in complex environmental and biological samples.

Nuclear magnetic resonance (NMR) based 13C tracing has broad applications across medical and environmental research. As many biological and environmental samples are heterogeneous, they experience considerable spectral overlap and relatively low signal. Here a 1D 1H-12C/13C is introduced that uses "in-phase/opposite-phase" encoding to simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan. Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, this approach creates separate sub-spectra for the 12C and 13C bound protons. These spectra can be used for both quantitative and qualitative analysis of complex samples with significant spectral overlap. Due to the presence of the 13C dipole, faster relaxation of the 1H-13C pairs results in slight underestimation compared to the 1H-12C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of 13C can be calculated by difference. When combined with the result, 12C and 13C percent enrichment in both 1H-12C and 1H-13C fractions are obtained. As the approach uses isotope filtered 1H NMR for detection, it retains nearly the same sensitivity as a standard 1H spectrum. Here, a proof-of-concept is performed using simple mixtures of 12C and 13C glucose, followed by suspended algal cells with varying 12C /13C ratios representing a complex mixture. The results consistently return 12C/13C ratios that deviate less than 1 % on average from the expected. Finally, the sequence was used to monitor and quantify 13C% enrichment in Daphnia magna neonates which were fed a 13C diet over 1 week. The approach helped reveal how the organisms utilized the 12C lipids they are born with vs. the 13C lipids they assimilate from their diet during growth. Given the experiments simplicity, versatility, and sensitivity, we anticipate it should find broad application in a wide range of tracer studies, such as fluxomics, with applications spanning various disciplines.

In vivo singlet state filtered nuclear magnetic resonance: towards monitoring toxic responses inside living organisms.

In line with recent paradigm shifts in toxicity testing, in vivo nuclear magnetic resonance (NMR) is a powerful tool for studying the biological impacts and perturbations caused by toxicants in living organisms. However, despite the excellent molecular insights that can be obtained through this technique, in vivo NMR applications are hampered by considerable experimental challenges such as poor line shape and spectral overlap. Here, we demonstrate the application of singlet-filtered NMR to target specific metabolites and facilitate the study of metabolite fluxes in living Daphnia magna, an aquatic keystone species and model organism. Informed by mathematical simulations and experiments on ex vivo organisms, singlet state NMR is used to monitor the flux of metabolites such as d-glucose and serine in living D. magna, during the environmentally relevant processes of anoxic stress and reduced food availability. Overall, singlet state NMR is shown to have significant future potential for studying metabolic processes in vivo.

NMR spectroscopy of wastewater: A review, case study, and future potential.

NMR spectroscopy is arguably the most powerful tool for the study of molecular structures and interactions, and is increasingly being applied to environmental research, such as the study of wastewater. With over 97% of the planet's water being saltwater, and two thirds of freshwater being frozen in the ice caps and glaciers, there is a significant need to maintain and reuse the remaining 1%, which is a precious resource, critical to the sustainability of most life on Earth. Sanitation and reutilization of wastewater is an important method of water conservation, especially in arid regions, making the understanding of wastewater itself, and of its treatment processes, a highly relevant area of environmental research. Here, the benefits, challenges and subtleties of using NMR spectroscopy for the analysis of wastewater are considered. First, the techniques available to overcome the specific challenges arising from the nature of wastewater (which is a complex and dilute matrix), including an examination of sample preparation and NMR techniques (such as solvent suppression), in both the solid and solution states, are discussed. Then, the arsenal of available NMR techniques for both structure elucidation (e.g., heteronuclear, multidimensional NMR, homonuclear scalar coupling-based experiments) and the study of intermolecular interactions (e.g., diffusion, nuclear Overhauser and saturation transfer-based techniques) in wastewater are examined. Examples of wastewater NMR studies from the literature are reviewed and potential areas for future research are identified. Organized by nucleus, this review includes the common heteronuclei (13C, 15N, 19F, 31P, 29Si) as well as other environmentally relevant nuclei and metals such as 27Al, 51V, 207Pb and 113Cd, among others. Further, the potential of additional NMR methods such as comprehensive multiphase NMR, NMR microscopy and hyphenated techniques (for example, LC-SPE-NMR-MS) for advancing the current understanding of wastewater are discussed. In addition, a case study that combines natural abundance (i.e. non-concentrated), targeted and non-targeted NMR to characterize wastewater, along with in vivo based NMR to understand its toxicity, is included. The study demonstrates that, when applied comprehensively, NMR can provide unique insights into not just the structure, but also potential impacts, of wastewater and wastewater treatment processes. Finally, low-field NMR, which holds considerable future potential for on-site wastewater monitoring, is briefly discussed. In summary, NMR spectroscopy is one of the most versatile tools in modern science, with abilities to study all phases (gases, liquids, gels and solids), chemical structures, interactions, interfaces, toxicity and much more. The authors hope this review will inspire more scientists to embrace NMR, given its huge potential for both wastewater analysis in particular and environmental research in general.