Comprehensive Multiphase NMR Probehead with Reduced Radiofrequency Heating Improves the Analysis of Living Organisms and Heat-Sensitive Samples.

Comprehensive multiphase (CMP) NMR, first described in 2012, combines all of the hardware components necessary to analyze all phases (solid, gel, and solution) in samples in their natural state. In combination with spectral editing experiments, it can fully differentiate phases and study the transfer of chemical species across and between phases, providing unprecedented molecular-level information in unaltered natural systems. However, many natural samples, such as swollen soils, plants, and small organisms, contain water, salts, and ionic compounds, making them electrically lossy and susceptible to RF heating, especially when using high-strength RF fields required to select the solid domains. While dedicated reduced-heating probes have been developed for solid-state NMR, to date, all CMP-NMR probes have been based on solenoid designs, which can lead to problematic sample heating. Here, a new prototype CMP probe was developed, incorporating a loop gap resonator (LGR) for decoupling. Temperature increases are monitored in salt solutions analogous to those in small aquatic organisms and then tested in vivo on Hyalella azteca (freshwater shrimp). In the standard CMP probe (solenoid), 80% of organisms died within 4 h under high-power decoupling, while in the LGR design, all organisms survived the entire test period of 12 h. The LGR design reduced heating by a factor of ∼3, which allowed 100 kHz decoupling to be applied to salty samples with generally ≤10 °C sample heating. In addition to expanding the potential for in vivo research, the ability to apply uncompromised high-power decoupling could be beneficial for multiphase samples containing true crystalline solids that require the strongest possible decoupling fields for optimal detection.

Expanding current applications and permitting the analysis of larger intact samples by means of a 7 mm CMP-NMR probe.

Comprehensive multiphase NMR combines the ability to study and differentiate all phases (solids, gels, and liquids) using a single NMR probe. The general goal of CMP-NMR is to study intact environmental and biological samples to better understand conformation, organization, association, and transfer between and across phases/interfaces that may be lost with conventional sample preparation such as drying or solubilization. To date, all CMP-NMR studies have used 4 mm probes and rotors. Here, a larger 7 mm probehead is introduced which provides ∼3 times the volume and ∼2.4 times the signal over a 4 mm version. This offers two main advantages: (1) the additional biomass reduces experiment time, making 13C detection at natural abundance more feasible; (2) it allows the analysis of larger samples that cannot fit within a 4 mm rotor. Chicken heart tissue and Hyalella azteca (freshwater shrimp) are used to demonstrate that phase-based spectral editing works with 7 mm rotors and that the additional biomass from the larger volumes allows detection with 13C at natural abundance. Additionally, a whole pomegranate seed berry (aril) and an intact softgel capsule of hydroxyzine hydrochloride are used to demonstrate the analysis of samples too large to fit inside a conventional 4 mm CMP probe. The 7 mm version introduced here extends the range of applications and sample types that can be studied and is recommended when 4 mm CMP probes cannot provide adequate signal-to-noise (S/N), or intact samples are simply too big for 4 mm rotors.

Digital microfluidics and nuclear magnetic resonance spectroscopy for in situ diffusion measurements and reaction monitoring.

In recent years microcoils and related structures have been developed to increase the mass sensitivity of nuclear magnetic resonance spectroscopy, allowing this extremely powerful analytical technique to be extended to small sample volumes (<5 µl). In general, microchannels have been used to deliver the samples of interest to these microcoils; however, these systems tend to have large dead volumes and require more complex fluidic connections. Here, we introduce a two-plate digital microfluidic (DMF) strategy to interface small-volume samples with NMR microcoils. In this system, a planar microcoil is surrounded by a copper plane that serves as the counter-electrode for the digital microfluidic device, allowing for precise control of droplet position and shape. This feature allows for the user-determination of the orientation of droplets relative to the main axes of the shim stack, permitting improved shimming and a more homogeneous magnetic field inside the droplet below the microcoil, which leads to improved spectral lineshape. This, along with high-fidelity droplet actuation, allows for rapid shimming strategies (developed over decades for vertically oriented NMR tubes) to be employed, permitting the determination of reaction-product diffusion coefficients as well as quantitative monitoring of reactive intermediates. We propose that this system paves the way for new and exciting applications for in situ analysis of small samples by NMR spectroscopy.

Soil Organic Matter in Its Native State: Unravelling the Most Complex Biomaterial on Earth.

Since the isolation of soil organic matter in 1786, tens of thousands of publications have searched for its structure. Nuclear magnetic resonance (NMR) spectroscopy has played a critical role in defining soil organic matter but traditional approaches remove key information such as the distribution of components at the soil-water interface and conformational information. Here a novel form of NMR with capabilities to study all physical phases termed Comprehensive Multiphase NMR, is applied to analyze soil in its natural swollen-state. The key structural components in soil organic matter are identified to be largely composed of macromolecular inputs from degrading biomass. Polar lipid heads and carbohydrates dominate the soil-water interface while lignin and microbes are arranged in a more hydrophobic interior. Lignin domains cannot be penetrated by aqueous solvents even at extreme pH indicating they are the most hydrophobic environment in soil and are ideal for sequestering hydrophobic contaminants. Here, for the first time, a complete range of physical states of a whole soil can be studied. This provides a more detailed understanding of soil organic matter at the molecular level itself key to develop the most efficient soil remediation and agricultural techniques, and better predict carbon sequestration and climate change.

Comprehensive multiphase NMR applied to a living organism.

Comprehensive multiphase (CMP) NMR is a novel technology that integrates all the hardware from solution-, gel- and solid-state into a single NMR probe, permitting all phases to be studied in intact samples. Here comprehensive multiphase (CMP) NMR is used to study all components in a living organism for the first time. This work describes 4 new scientific accomplishments summarized as: (1) CMP-NMR is applied to a living animal, (2) an effective method to deliver oxygen to the organisms is described which permits longer studies essential for in-depth NMR analysis in general, (3) a range of spectral editing approaches are applied to fully differentiate the various phases solutions (metabolites) through to solids (shell) (4) 13C isotopic labelling and multidimensional NMR are combined to provide detailed assignment of metabolites and structural components in vivo. While not explicitly studied here the multiphase capabilities of the technique offer future possibilities to study kinetic transfer between phases (e.g. nutrient assimilation, contaminant sequestration), molecular binding at interfaces (e.g. drug or contaminant binding) and bonding across and between phases (e.g. muscle to bone) in vivo. Future work will need to focus on decreasing the spinning speed to reduce organism stress during analysis.

From Spill to Sequestration: The Molecular Journey of Contamination via Comprehensive Multiphase NMR.

Comprehensive multiphase NMR is a novel NMR technique that permits all components (solutions, gels, and solids) to be studied in unaltered natural samples. In this study a wide range of CMP-NMR interaction and editing-based experiments are combined to follow contaminants (pentafluorophenol (PFP) and perfluorooctanoic acid (PFOA)) from the solution state (after a spill) through the gel-state and finally into the true solid-state (sequestered) in an intact water-swollen soil. Kinetics experiments monitoring each phase illustrate PFOA rapidly transfers from solution to the solid phase while for PFP the process is slower with longer residence times in the solution and gel phase. Interaction-based experiments reveal that PFOA enters the soil via its hydrophobic tails and selectively binds to soil microbial protein. PFP sorption shows less specificity exhibiting interactions with a range of gel and solid soil components with a preference toward aromatics (mainly lignin). The results indicate that in addition to more traditional measurements such as Koc, other factors including the influence of the contaminant on the soil-water interface, specific biological interactions, soil composition (content of lignin, protein, etc.) and physical accessibility/swellability of soil organic components will likely be central to better explaining and predicting the true behavior of contaminants in soil.

Comprehensive multiphase NMR: a promising technology to study plants in their native state.

Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)-NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid-state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild-type Arabidopsis thaliana and the cellulose-deficient mutant ectopic lignification1 (eli1) as examples. Using CMP-NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP-NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT.

Comprehensive multiphase NMR spectroscopy of intact ¹³C-labeled seeds.

Seeds are complex entities composed of liquids, gels, and solids. NMR spectroscopy is a powerful tool for studying molecular structure but has evolved into two fields, solution and solid state. Comprehensive multiphase (CMP) NMR spectroscopy is capable of liquid-, gel-, and solid-state experiments for studying intact samples where all organic components are studied and differentiated in situ. Herein, intact (13)C-labeled seeds were studied by a variety of 1D/2D (1)H/(13)C experiments. In the mobile phase, an assortment of metabolites in a single (13)C-labeled wheat seed were identified; the gel phase was dominated by triacylglycerides; the semisolid phase was composed largely of carbohydrate biopolymers, and the solid phase was greatly influenced by starchy endosperm signals. Subsequently, the seeds were compared and relative similarities and differences between seed types discussed. This study represents the first application of CMP-NMR to food chemistry and demonstrates its general utility and feasibility for studying intact heterogeneous samples.

In-situ molecular-level elucidation of organofluorine binding sites in a whole peat soil.

The chemical nature of xenobiotic binding sites in soils is of vital importance to environmental biogeochemistry. Interactions between xenobiotics and the naturally occurring organic constituents of soils are strongly correlated to environmental persistence, bioaccessibility, and ecotoxicity. Nevertheless, because of the complex structural and chemical heterogeneity of soils, studies of these interactions are most commonly performed indirectly, using correlative methods, fractionation, or chemical modification. Here we identify the organic components of an unmodified peat soil where some organofluorine xenobiotic compounds interact using direct molecular-level methods. Using (19)F→(1)H cross-polarization magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectroscopy, the (19)F nuclei of organofluorine compounds are used to induce observable transverse magnetization in the (1)H nuclei of organic components of the soil with which they interact after sorption. The observed (19)F→(1)H CP-MAS spectra and dynamics are compared to those produced using model soil organic compounds, lignin and albumin. It is found that lignin-like components can account for the interactions observed in this soil for heptafluoronaphthol (HFNap) while protein structures can account for the interactions observed for perfluorooctanoic acid (PFOA). This study employs novel comprehensive multi-phase (CMP) NMR technology that permits the application of solution-, gel-, and solid-state NMR experiments on intact soil samples in their swollen state.

Comprehensive multiphase NMR spectroscopy: basic experimental approaches to differentiate phases in heterogeneous samples.

Heterogeneous samples, such as soils, sediments, plants, tissues, foods and organisms, often contain liquid-, gel- and solid-like phases and it is the synergism between these phases that determine their environmental and biological properties. Studying each phase separately can perturb the sample, removing important structural information such as chemical interactions at the gel-solid interface, kinetics across boundaries and conformation in the natural state. In order to overcome these limitations a Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) probe has been developed, and is introduced here, that permits all bonds in all phases to be studied and differentiated in whole unaltered natural samples. The CMP-NMR probe is built with high power circuitry, Magic Angle Spinning (MAS), is fitted with a lock channel, pulse field gradients, and is fully susceptibility matched. Consequently, this novel NMR probe has to cover all HR-MAS aspects without compromising power handling to permit the full range of solution-, gel- and solid-state experiments available today. Using this technology, both structures and interactions can be studied independently in each phase as well as transfer/interactions between phases within a heterogeneous sample. This paper outlines some basic experimental approaches using a model heterogeneous multiphase sample containing liquid-, gel- and solid-like components in water, yielding separate (1)H and (13)C spectra for the different phases. In addition, (19)F performance is also addressed. To illustrate the capability of (19)F NMR soil samples, containing two different contaminants, are used, demonstrating a preliminary, but real-world application of this technology. This novel NMR approach possesses a great potential for the in situ study of natural samples in their native state.