Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 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.

Keywords: 13C enriched mass limited biological samples; heteronuclear 2D NMR; metabolites; surface microcoils.