High-Sensitivity Analysis of Native Bacterial Biofilms Using Dynamic Nuclear Polarization-Enhanced Solid-State NMR

Area of Science:

• Biophysics
• Microbiology
• Analytical Chemistry

Background:

• Bacterial biofilms are linked to persistent infections and antimicrobial resistance.
• High-resolution structural data on native biofilms are limited due to methodological challenges.
• Conventional solid-state NMR (ssNMR) has sensitivity limitations for unlabeled, native samples.

Purpose of the Study:

• To apply sensitivity-enhanced ssNMR using Dynamic Nuclear Polarization (DNP) for characterizing native bacterial biofilms.
• To overcome limitations of conventional ssNMR for analyzing complex, unlabeled biological samples.
• To enable rapid, high-resolution structural characterization of native biofilms.

Main Methods:

• Utilized Dynamic Nuclear Polarization (DNP) to enhance sensitivity of solid-state NMR (ssNMR).
• Applied DNP-enhanced ssNMR to native *Pseudomonas fluorescens* colony biofilms.
• Acquired 1D (¹³C, ¹⁵N) and 2D (¹H-¹³C, ¹H-¹⁵N, ¹³C-¹³C) ssNMR spectra.

Main Results:

• Achieved ultrafast structural characterization of native biofilms without isotope-labeling or modification.
• Successfully identified key biofilm components including polysaccharides, proteins, and eDNA.
• Demonstrated the capability of DNP-enhanced ssNMR for analyzing complex biological structures.

Conclusions:

• This study is the first to use ultrasensitive DNP ssNMR for native bacterial biofilm characterization.
• The enhanced ssNMR technique expands the scope for studying *in vitro* and *ex vivo* biofilms.
• This approach can significantly advance structure-guided strategies against biofilm-forming microbes and infections.