Novel luminescent nanosensors and their applications in quantifying and monitoring analyte gradients in bacterial biofilms

Abstract

Bacterial biofilms are complex, heterogeneous communities of bacteria encased in an extracellular matrix of polysaccharides, DNA, protein, and other biopolymers. Biofilms are ubiquitous across bacterial species and are believed to be the default mode of growth for many species. Opportunistic pathogens such as Pseudomonas aeruginosa form biofilm infections in the airways of cystic fibrosis patients and in wounds. Because of reasons such as this, investigating the ways different metabolites are utilized is crucial to our understanding, use, and control of these complex microbial communities. In this work, polymeric oxygen nanosensors are presented as both a research tool for quantifying oxygen dynamics with enhanced resolution as well as a potential clinical tool for evaluating antibiotic efficacy in vitro. These nanosensors can determine 3-dimensional oxygen variation through both space and time and monitor metabolic changes from administration of antibiotics. Potassium-selective nanosensors that utilize the unique photomechanism of triplet-triplet annihilation upconversion are also detailed as a potential solution to overcome issues of signal crosstalk in imaging-based monitoring. These upconversion sensors have a potassium-specific response with a response midpoint of 0.82 mM K+ and no measurable crosstalk with typical downconversion probes (demonstrated with a GFP-producing bacterial biofilm). The development of these nanosensors provides new tools and methods to investigate the biochemical dynamics within bacterial biofilms. The oxygen nanosensors let us monitor 4D dynamics for oxygen whereas prior approaches have been limited in spatial and temporal resolution, and the upconversion sensors allow for monitoring of ion dynamics without interference from traditional fluorescent reporters in these biological systems.