RESUMO
The excellent versatility of 5-axis computer numerical control (CNC) micromilling has led to its application for prototyping NMR microcoils tailored to mass-limited samples (reducing development time and cost). However, vibrations during 5-axis milling can hinder the creation of complex 3D volume microcoils (i.e., solenoids and saddle coils). To address these limitations, a high-resolution NSCNC ELARA 4-axis milling machine was developed with the extra precision required for making complex 3D volume microcoils. Upon investigating the performance of resonators made with various copper-coated dielectrics, resonators with poly(methyl methacrylate) (PMMA) provided the best SNR/line shape. Thus, complex 1.7 mm microcoil designs were machined from Cu-coated PMMA. A milled 6.4 mm solenoid also provided 6.6× the total carbon signal for a 13C-labeled broccoli seed compared to a commercial inverse 5 mm NMR probe (demonstrating potential for larger coil designs). However, the manufacture of coils <1.7 mm with copper-coated PMMA rods was challenging as â¼0.5 mm of remaining PMMA was needed to retain their structural integrity. To manufacture smaller microcoils, both a solenoid and saddle coil (both with 1 mm O.D., 0.1 mm thick walls) were etched from Cu-coated glass capillaries using a UV picosecond laser that was mounted onto an NSCNC 5-axis MiRA7L. Both resonators showed excellent signal and identified a wide range of metabolites in a 13C-labeled algae extract, while the solenoid was further tested on two copepod egg sacs (â¼4 µg of total sample). In summary, the flexibility to prototype complex microcoils in-house allows laboratories to tailor microcoils to specific mass-limited samples while avoiding the costs of cleanrooms.
Assuntos
Lasers , Espectroscopia de Ressonância Magnética , Polimetil Metacrilato , Espectroscopia de Ressonância Magnética/instrumentação , Polimetil Metacrilato/química , Cobre/químicaRESUMO
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 (â¼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.
Assuntos
Alumínio , Cobre , Animais , Ligas , Biomassa , DaphniaRESUMO
Comprehensive multiphase-nuclear magnetic resonance (CMP-NMR) is a non-invasive approach designed to observe all phases (solutions, gels, and solids) in intact samples using a single NMR probe. Studies of dead and living organisms are important to understand processes ranging from biological growth to environmental stress. Historically, such studies have utilized 1H-based phase editing for the detection of soluble/swollen components and 1H-detected 2D NMR for metabolite assignments/screening. However, living organisms require slow spinning rates (â¼500 Hz) to increase survivability, but at such low speeds, complications from water sidebands and spectral overlap from the modest chemical shift window (â¼0-10 ppm) make 1H NMR challenging. Here, a novel 13C-optimized E-Free magic angle spinning CMP probe is applied to study all phases in ex vivo and in vivo samples. This probe consists of a two-coil design, with an inner single-tuned 13C coil providing a 113% increase in 13C sensitivity relative to a traditional multichannel single-CMP coil design. For organisms with a large biomass (â¼0.1 g) like the Ganges River sprat (ex vivo), 13C-detected full spectral editing and 13C-detected heteronuclear correlation (HETCOR) can be performed at natural abundance. Unfortunately, for a single living shrimp (â¼2 mg), 13C enrichment was still required, but 13C-detected HETCOR shows superior data relative to heteronuclear single-quantum coherence at low spinning speeds (due to complications from water sidebands in the latter). The probe is equipped with automatic-tuning-matching and is compatible with automated gradient shimmingâa key step toward conducting multiphase screening of dead and living organisms under automation in the near future.
Assuntos
Carbono , Água , Isótopos de Carbono , Espectroscopia de Ressonância MagnéticaRESUMO
Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.
Assuntos
Daphnia , Imageamento por Ressonância Magnética , Animais , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear BiomolecularRESUMO
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 â¼3, 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.
Assuntos
Calefação , Temperatura Alta , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Ondas de RádioRESUMO
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 â¼3 times the volume and â¼2.4 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.
Assuntos
Imageamento por Ressonância Magnética , Biomassa , Espectroscopia de Ressonância MagnéticaRESUMO
The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.
Assuntos
Custos e Análise de Custo , Espectroscopia de Ressonância Magnética/economia , Espectroscopia de Ressonância Magnética/instrumentação , Microtecnologia/instrumentação , Animais , Daphnia/química , Desenho de Equipamento , Fenômenos Mecânicos , Fatores de TempoRESUMO
Nuclear Magnetic Resonance (NMR) spectroscopy is a non-invasive analytical technique which allows for the study of intact samples. Comprehensive Multiphase NMR (CMP-NMR) combines techniques and hardware from solution state and solid state NMR to allow for the holistic analysis of all phases (i.e. solutions, gels and solids) in unaltered samples. This study is the first to apply CMP-NMR to deceased, intact organisms and uses 13C enriched Daphnia magna (water fleas) as an example. D. magna are commonly used model organisms for environmental toxicology studies. As primary consumers, they are responsible for the transfer of nutrients across trophic levels, and a decline in their population can potentially impact the entire freshwater aquatic ecosystem. Though in vivo research is the ultimate tool to understand an organism's most biologically relevant state, studies are limited by conditions (i.e. oxygen requirements, limited experiment time and reduced spinning speed) required to keep the organisms alive, which can negatively impact the quality of the data collected. In comparison, ex vivo CMP-NMR is beneficial in that; organisms do not need oxygen (eliminating air holes in rotor caps and subsequent evaporation); samples can be spun faster, leading to improved spectral resolution; more biomass per sample can be analyzed; and experiments can be run for longer. In turn, higher quality ex vivo NMR, can provide more comprehensive NMR assignments, which in many cases could be transferred to better understand less resolved in vivo signals. This manuscript is divided into three sections: 1) multiphase spectral editing techniques, 2) detailed metabolic assignments of 2D NMR of 13C enriched D. magna and 3) multiphase biological changes over different life stages, ages and generations of D. magna. In summary, ex vivo CMP-NMR proves to be a very powerful approach to study whole organisms in a comprehensive manner and should provide very complementary information to in vivo based research.
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The signal to noise ratio of solid state deuteron NMR line shapes can be significantly improved by recording multiple echoes, generated either by a quadrupole Carr-Purcell-Meiboom-Gill pulse train (QCPMG) or by magic angle spinning (MAS). It is shown in this article, theoretically and experimentally, that when these techniques are used to record partially relaxed spectra, the relaxation times of Zeeman order, T(1Z), and quadrupole order, T(1Q), measured for individual side bands in QCPMG experiments preserve relaxation time anisotropy, while rotational side bands in MAS spectra do not. The relaxation times of individual QCPMG sidebands are not identical to those measured at the same frequencies on partially relaxed quadrupole echo powder patterns, and must be computed by explicit simulation.
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Spin-lattice relaxation rates of lead have been measured at 17.6 T (156.9 MHz) as a function of temperature in polycrystalline lead nitrate and lead molybdate. Comparing the results with relaxation rates measured at lower fields, it is found that at high fields and low temperature, chemical shift anisotropy (CSA) makes small but observable contributions to lead relaxation in both materials. At 17.6 T and 200 K, CSA accounts for about 15% of the observed relaxation rate. Above 300 K, the dominant relaxation mechanism even at 17.6 T is an indirect Raman process involving modulation of the (207)Pb spin-rotation tensor, as first proposed by Grutzner et al. [J. Am. Chem. Soc. 123, 7094 (2001)] and later treated theoretically in more detail by Vega et al. [Phys. Rev. B 74, 214420 (2006)]. The improved signal to noise ratio at high fields makes it possible to quantify relaxation time anisotropy by analyzing saturation-recovery functions for individual frequencies on the powder pattern line shape. No orientation dependence is found for the spin-lattice relaxation rate of either material. It is argued from examination of the appropriate theoretical expressions, derived here for the first time, that the lack of observable relaxation time anisotropy is probably a general feature of this indirect Raman mechanism.