Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small ¹³C enriched biological samples using ¹H‐¹³C 2D heteronuclear correlation NMR spectroscopy

Abstract

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.

Comparing the Potential of Helmholtz and Planar NMR Microcoils for Analysis of Intact Biological Samples

“…As the need to study mass-limited samples continues in the medical and environmental fields, microcoil NMR can be best improved by double tuning the microcoils to study multiple nuclei, such as 1 H and 13 C, allowing 2D 1 H− 13 C experiments to be performed. 27 In summary, the Helmholtz microcoil outperformed the planar coil in more complex pulse sequences, and as such for experiments that are normally required for biological analysis, volume coils such as the Helmholtz hold much greater practicability for the study of biological systems in general.…”

“…Microcoil NMR is a more economical and accessible approach for very small samples, which can increase mass sensitivity by thousands of times over a 5 mm room temperature probe, − and thus holds great potential for the study of mass limited complex biological samples. ,, Due to the small coil diameter, only small amounts of sample are required, decreasing costs and improving the filling factor. Furthermore, the increase in mass sensitivity potentially permits the study of single organisms/eggs or considerable reductions in tissue sample requirements.…”

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics

“…38 1 H- 13 C HMQCs (phase sensitive using echo/antiecho gradient selection with decoupling) were collected using an adiabatic shape pulse (Crp60, 0.5, 20.1, 500 μs), 2048-time domain points, 64 increments, a relaxation delay of 0.25 s, 64 scans (in vivo experiments), 1536 scans (ex vivo experiment), and 4 scans (microcoil comparison experiments). The version of HMQC used here was chosen as it was previously found to be optimal (amongst a wide range of HSQC and HMQC variants tested) for 2D NMR on double-tuned single-sided planar coils with inhomogeneous B 1 profiles, 39 and the 90°p ulse was calculated for each experiment. For coil comparison experiments, samples of 13 C-D-glucose (500 mM) described above were used to compare the signal to noise ratio (SNR), line shape, and nutation curves to determine the best signal response.…”

Modern Manufacturing Enables Magnetic Field Cycling Experiments and Parahydrogen-Induced Hyperpolarization with a Benchtop NMR

“…MR spectroscopy, for example, allows us to obtain biochemical information non-invasively and in vivo but is (typically) limited to a chemical, temporal, and spatial resolution of mM, minutes, and cubic centimeters, respectively, in the clinical routine . Hardware: lab on a chip with microcoils, − remote detection and cryo-coils, NMR sequences, , and post-processing procedures are being developed to improve the SNR.…”

“…3D rendering of the MFC setup (a), magnification (b,c), and top view (d). The setup was mounted on a btNMR spectrometer (1, 3D model provided by Magritek), consisting of a resistive polarization coil (2), a stepper motor with a spur gear (3), a gear rack (4), a gas tube guide(5), and a tube carrier (6) holding an NMR tube(7). The aluminum rod at the center served to hold the polarization coil (2) and to guide the NMR tube carrier(6).…”