Luminescent Nanosensors for Ratiometric Monitoring of Three-Dimensional Oxygen Gradients in Laboratory and Clinical Pseudomonas aeruginosa Biofilms

Abstract

Bacterial biofilms can form persistent infections on wounds and implanted medical devices and are associated with many chronic diseases, such as cystic fibrosis. These infections are medically difficult to treat, as biofilms are more resistant to antibiotic attack than their planktonic counterparts. An understanding of the spatial and temporal variation in the metabolism of biofilms is a critical component toward improved biofilm treatments. To this end, we developed oxygen-sensitive luminescent nanosensors to measure three-dimensional (3D) oxygen gradients, an application of which is demonstrated here with Pseudomonas aeruginosa biofilms. The method was applied here and improves on traditional one-dimensional (1D) methods of measuring oxygen profiles by investigating the spatial and temporal variation of oxygen concentration when biofilms are challenged with antibiotic attack. We observed an increased oxygenation of biofilms that was consistent with cell death from comparisons with antibiotic kill curves for PAO1. Due to the spatial and temporal nature of our approach, we also identified spatial and temporal inhomogeneities in the biofilm metabolism that are consistent with previous observations. Clinical strains of P. aeruginosa subjected to similar interrogation showed variations in resistance to colistin and tobramycin, which are two antibiotics commonly used to treat P. aeruginosa infections in cystic fibrosis patients. IMPORTANCE Biofilm infections are more difficult to treat than planktonic infections for a variety of reasons, such as decreased antibiotic penetration. Their complex structure makes biofilms challenging to study without disruption. To address this limitation, we developed and demonstrated oxygen-sensitive luminescent nanosensors that can be incorporated into biofilms for studying oxygen penetration, distribution, and antibiotic efficacy—demonstrated here with our sensors monitoring antibiotic impacts on metabolism in biofilms formed from clinical isolates. The significance of our research is in demonstrating not only a nondisruptive method for imaging and measuring oxygen in biofilms but also that this nanoparticle-based sensing platform can be modified to measure many different ions and small molecule analytes.

LipiSensors: Exploiting Lipid Nanoemulsions to Fabricate Ionophore-Based Nanosensors

“…To expand on the functionality of the LipiSensor design, we also developed LipiSensors for sensing oxygen concentration based on collisional quenching of a metal centered porphyrin dye, a commonly used approach in optical oxygen sensors and reviewed extensively in [ 40 , 41 , 42 ]. Here, we used the medium-sized LipiSensor formulation with Platinum tetra-fluorophenyl porphyrin (PtTFPP) as the oxygen-responsive dye and DiA as an oxygen-insensitive reference dye to enable ratiometric measurements—as we have done before in oxygen sensing [ 4 , 18 ]. When oxygen concentrations increase, the fluorescence of the PtTFPP at 650 nm decreases through quenching while the DiA at 585 nm is unaffected ( Figure 6 a).…”

“…(PtTFPP) as the oxygen-responsive dye and DiA as an oxygen-insensitive reference dye to enable ratiometric measurements-as we have done before in oxygen sensing [4,18]. When oxygen concentrations increase, the fluorescence of the PtTFPP at 650 nm decreases through quenching while the DiA at 585 nm is unaffected (Figure 6a).…”

“…A complementary technology to molecular probes, ionophore-based nanosensors (IBNS), can be employed as sensing units, as they are easy to fabricate [ 13 ], have a highly tunable response [ 14 , 15 ] and are larger than traditional molecular probes which makes it much easier for in vivo [ 16 , 17 ] and extracellular applications [ 4 , 18 ]. IBNS are typically fabricated from a plasticized polymer phase encased in a polyethylene glycol (PEG) surfactant layer to form nanoparticles that encase the necessary sensing moieties inside the hydrophobic polymer phase.…”

Nanoparticle-Based Liquid–Liquid Extraction for the Determination of Metal Ions

“…By adopting the structure from polymeric nanoparticle sensors (PNS), our extraction approach not only benefits from a faster response time but also adds the potential application in systems where LLE and its variations are not feasible. Nanoparticle sensors with this structure have been used to characterize complex biological systems such as clinically grown biofilms of Pseudomonas aeruginosa, eukaryotic intracellular environments, and animal models by probing for a range of diverse analytes. − A large factor to consider when applying analytical techniques to live models is the biocompatibility of the reagents used as well as the assay’s procedure itself . The reagents used in classic LLE and variations thereof are often toxic to live samples.…”

Triplet-Triplet Annihilation Upconversion Based Nanosensors for Fluorescence Detection of Potassium

“…The nanosensors were then concentrated ∼10× via ultrafiltration. Biofilm growth procedures were adapted from methods by Kirchner et al and Jewell et al Briefly, Pseudomonas aeruginosa strain PAO1 (ATCC 15692) was plated onto Luria Bertani (LB) agar (Sigma-Aldrich, St. Louis, MO, USA) from frozen stocks and incubated at 37 °C for 24 h. One colony was dispersed in 1 mL of LB broth and incubated for 24 h at 37 °C. Liquid culture was diluted to an optical density (OD 600 ) of 0.05.…”