Morphology-Dependent Sensitivity of Chitosan-Based Sensing Materials for the Colorimetric Detection of β-Glucuronidase From Pathogenic E. coli.

To address the global threat of increasing antimicrobial resistance, the investigation and development of rapid identification and detection methods for bacteria and infection sensing approaches have become major areas of research. Expanding on earlier work on enzyme-sensitive biopolymer-based autonomous sensing materials, the impact of different morphologies and concomitantly surface-to-volume ratio on the sensitivity of the detection of the enzyme β-glucuronidase (β-Gus) from pathogenic and non-pathogenic E. coli was unraveled. The enzymatic hydrolysis of microbeads and freeze-dried scaffolds was compared to that of neat films equipped by post-fabrication modification with 4-methylumbelliferyl-β-D-glucuronide hydrate (MUG) as a β-Gus reporting functionality. Fluorescence spectroscopy and in vitro tests with planktonic MACH-1 and NCTC strains revealed that the scaffolds outperformed the films with a close to ten-fold increased sensitivity, while the beads showed an enhanced rate of only a factor of 2.4. For an observation time of 60 min, the scaffold, beads, and film exhibited a limit of detection (LOD) of 3.3, 13.4, and 17.5 nm, respectively. These results support the hypothesis that increased surface-to-volume ratios enhance the sensitivity, but also point to the role of surface porosity and accessibility of enzyme-labile sites in swollen materials, which may be compromised due to the fabrication process.