Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R-2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 x 10(-11) to 2.80 x 10(-3)*s(-1) per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Automated summary

A novel method involving biofilm EPS-functionalized SWCNT probes was developed for rapid screening of hydrolases on EPS, with specific studies conducted on six enzymes in CF environments. Extraction of relevant parameters and confirmation of experimental results were achieved through model fitting.