A fast microfluidic mixer enabling rapid preparation of homogeneous PEG and bicelle media for RDC in NMR analysis
Residual dipolar couplings (RDCs) are widely used in NMR for the investigation of protein structure and dynamics. However, commonly used RDC media ( e.g. , C 12 E 5 /Hexanol/H 2 O or PEG system, lipid bilayer or bicelle system) are unstable viscous liquids, making it challenging to obtain stable and homogeneous NMR samples by laborious manual operation. In this paper, we report a unique fishhook-shaped micromixer (FS-mixer) that enables fast and thorough mixing of high-viscosity PEG and bicelle media for RDC analysis. Numerical simulations and experimental characterizations proved that the micromixer could achieve a complete mixing of viscous liquids in 382.4 μs, improved significantly over current mixers. In compared to manually prepared RDC samples, the on-chip prepared samples have a better uniformity and alignment efficiency, and show more stable peak split in the 2 H spectrum. As a proof of concept, we prepared a RDC sample for INSM1 ZF1-2 protein by using this FS-mixer. The IPAP-HSQC spectra demonstrated that this strategy can efficiently prevent the generation of low resolution and distorted spectra, which could facilitate further refinement of the NMR structure. This micromixer-assisted sampling method greatly simplifies the procedure, increases the media uniformity and stability, and shortens the time in RDC analysis. We believe it could serve as a useful tool in RDC-based NMR analysis.
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