Surface functionalization-dependent localization and affinity of SiO2 nanoparticles within the biofilm EPS matrix

The contribution of the biofilm extracellular polymeric substance (EPS) matrix to reduced antimicrobial sus-ceptibility in biofilms is widely recognised. As such, the direct targeting of the EPS matrix is a promising biofilmcontrol strategy that allows for the disruption of the matrix, thereby allowing a subsequent increase in suscep-tibility to antimicrobial agents. To this end, surface-functionalized nanoparticles (NPs) have received considerableattention. However, the fundamental understanding of the interactions occurring between engineered NPs andthe biofilm EPS matrix has not yet been fully elucidated. An insight into the underlying mechanisms involvedwhen a NP interacts with the EPS matrix will aid in the design of more efficient NPs for biofilm control. Here wedemonstrate the use of highly specificfluorescent probes in confocal laser scanning microscopy (CLSM) toillustrate the distribution of EPS macromolecules within the biofilm. Thereafter, a three-dimensional (3D)colocalization analysis was used to assess the affinity of differently functionalized silica NPs (SiNPs) and EPSmacromolecules fromPseudomonasfluorescensbiofilms. Results show that both the charge and surface functionalgroups of SiNPs dramatically affected the extent to which SiNPs interacted and localized with EPS macromole-cules, including proteins, polysaccharides and DNA. Hypotheses are also presented about the possible physico-chemical interactions which may be dominant in EPS matrix-NP interactions. This research not only develops aninnovative CLSM-based methodology for elucidating biofilm-nanoparticle interactions but also provides a plat-form on which to build more efficient NP systems for biofilm control.