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The functional properties of lipid-rich assemblies such as serum lipoproteins, cell membranes, and intracellular lipid droplets are modulated by the fluidity of the hydrocarbon chain environment. Existing methods for monitoring hydrocarbon chain fluidity include fluorescence, electron spin resonance, and nuclear magnetic resonance (NMR) spectroscopy; each possesses advantages and limitations. Here we introduce a new approach based on benchtop time-domain (1)H NMR relaxometry (TD-NMR). Unlike conventional NMR spectroscopy, TD-NMR does not rely on the chemical shift resolution made possible by homogeneous, high-field magnets and Fourier transforms. Rather, it focuses on a multiexponential analysis of the time decay signal. In this study, we investigated a series of single-phase fatty acid oils, which allowed us to correlate (1)H spin-spin relaxation time constants (T2) with experimental measures of sample fluidity, as obtained using a viscometer. Remarkably, benchtop TD-NMR at 40 MHz was able to resolve two to four T2 components in biologically relevant fatty acids, assigned to nanometer-scale domains in different segments of the hydrocarbon chain. The T2 values for each domain were exquisitely sensitive to hydrocarbon chain structure; the largest values were observed for pure fatty acids or mixtures with the highest cis-double bond content. Moreover, the T2 values for each domain exhibited positive linear correlations with fluidity. The TD-NMR T2 and fluidity measurements appear to be monitoring the same underlying phenomenon: variations in hydrocarbon chain packing. The results from this study validate the use of benchtop TD-NMR T2 as a nanofluidity meter and demonstrate its potential for probing nanofluidity in other systems of biological interest.

Nanofluidity of Fatty Acid Hydrocarbon Chains As Monitored by Benchtop Time-Domain Nuclear Magnetic Resonance