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Researchers developed a method to precisely measure how molecules move through simplified biofilm models using quantum dots as tracers and high-speed microscopy. They created DNA nanostar hydrogels that mimic the protective matrix surrounding bacterial biofilms and found that diffusion rates decreased significantly as the hydrogel concentration increased, dropping from 94.9 μm²/s in a glycerol control to 8.3 μm²/s in the densest hydrogel. The study established optimized conditions for tracking individual particles without trajectory overlap, enabling quantitative measurement of molecular transport through biofilm-like structures.
Why it matters
Understanding how molecules move through biofilms is critical for developing more effective treatments for chronic bacterial infections, as antibiotics must penetrate the protective biofilm matrix to reach bacteria. This modular experimental framework allows researchers to systematically test how individual biofilm components affect drug delivery, potentially leading to better antimicrobial strategies.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
Chronic infections persist in large part thanks to protection that biofilms afford their bacterial creators. The extracellular polymeric substance of biofilms is a hydrated matrix of DNA, polysaccharides, and structural proteins, amongst other components, through which nutrients, signalling molecules, and antimicrobial agents must diffuse to reach the bacteria within. Quantitative measurement of transport on the nanoscale within in vivo biofilms remains challenging due to optical heterogeneity, autofluorescence, active remodelling of biofilms and the ambiguity in trajectory reconstruction during single-particle tracking (SPT). Here, we present a methodological framework for measuring molecular transport in defined minimal extracellular matrix models using quantum dots as fluorescent nanoscale probes imaged with high-speed SlimVar microscopy. To establish conditions in which high-diffusivity particle trajectories can be reliably reconstructed, upper limits to quantum dot concentrations were estimated from Brownian motion. The 99th-percentile inter-frame jump distance was estimated from the three-dimensional Brownian jump distance distribution and used to define a target average nearest neighbour distance, and therefore a per-particle volume, used for calculating a concentration which minimises the probability of trajectory collision during data acquisition. Quantum dot movement was imaged at sub-millisecond frame rates and diffusion coefficients were calculated in a 20% glycerol control and in DNA nanostar hydrogels modelling minimal extracellular matrix scaffolds assembled at 250 M and 500 M. Median diffusion coefficients decreased from 94.9 m2*s-1 in glycerol to 15.9 m2*s-1 and 8.3 m2*s-1 in the 250 M and 500 M hydrogels, respectively. More broadly, this work establishes a workflow for quantitative SPT in minimal biofilm models. Rather than attempting to reproduce the full biological complexity of native biofilms, this approach provides the basis of a modular experimental framework in which individual extracellular matrix components can be incorporated sequentially and their effects on molecular transport quantified.
Source: A Bottom-Up Platform for Quantitative Single-Molecule Tracking Through Bacterial Biofilm Mimics