Exploring the Applications of Carbon-Detected NMR in Living and Dead Organisms Using a 13C-Optimized Comprehensive Multiphase NMR Probe.

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 (∼500 Hz) to increase survivability, but at such low speeds, complications from water sidebands and spectral overlap from the modest chemical shift window (∼0-10 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 (∼0.1 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 (∼2 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 shimming─a key step toward conducting multiphase screening of dead and living organisms under automation in the near future.