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

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.

Asymmetric flow field flow fractionation with light scattering detection - an orthogonal sensitivity analysis.

Asymmetric flow field flow fractionation (AF4) has several instrumental factors that may have a direct effect on separation performance. A sensitivity analysis was applied to ascertain the relative importance of AF4 primary instrument factor settings for the separation of a complex environmental sample. The analysis evaluated the impact of instrumental factors namely, cross flow, ramp time, focus flow, injection volume, and run buffer concentration on the multi-angle light scattering measurement of natural organic matter (NOM) molar mass (MM). A 2(5-1) orthogonal fractional factorial design was used to minimize analysis time while preserving the accuracy and robustness in the determination of the main effects and interactions between any two instrumental factors. By assuming that separations resulting in smaller MM measurements would be more accurate, the analysis produced a ranked list of effects estimates for factors and interactions of factors based on their relative importance in minimizing the MM. The most important and statistically significant AF4 instrumental factors were buffer concentration and cross flow. The least important was ramp time. A parallel 2(5-2) orthogonal fractional factorial design was also employed on five environmental factors for synthetic natural water samples containing silver nanoparticles (NPs), namely: NP concentration, NP size, NOM concentration, specific conductance, and pH. None of the water quality characteristic effects or interactions were found to be significant in minimizing the measured MM; however, the interaction between NP concentration and NP size was an important effect when considering NOM recovery. This work presents a structured approach for the rigorous assessment of AF4 instrument factors and optimal settings for the separation of complex samples utilizing efficient orthogonal factional factorial design and appropriate graphical analysis.

Using light scattering to evaluate the separation of polydisperse nanoparticles.

The analysis of natural and otherwise complex samples is challenging and yields uncertainty about the accuracy and precision of measurements. Here we present a practical tool to assess relative accuracy among separation protocols for techniques using light scattering detection. Due to the highly non-linear relationship between particle size and the intensity of scattered light, a few large particles may obfuscate greater numbers of small particles. Therefore, insufficiently separated mixtures may result in an overestimate of the average measured particle size. Complete separation of complex samples is needed to mitigate this challenge. A separation protocol can be considered improved if the average measured size is smaller than a previous separation protocol. Further, the protocol resulting in the smallest average measured particle size yields the best separation among those explored. If the differential in average measured size between protocols is less than the measurement uncertainty, then the selected protocols are of equivalent precision. As a demonstration, this assessment metric is applied to optimization of cross flow (V(x)) protocols in asymmetric flow field flow fractionation (AF(4)) separation interfaced with online quasi-elastic light scattering (QELS) detection using mixtures of polystyrene beads spanning a large size range. Using this assessment metric, the V(x) parameter was modulated to improve separation until the average measured size of the mixture was in statistical agreement with the calculated average size of particles in the mixture. While we demonstrate this metric by improving AF(4) V(x) protocols, it can be applied to any given separation parameters for separation techniques that employ dynamic light scattering detectors.

Tryptophan-scanning mutagenesis of the ligand binding pocket in Thermotoga maritima arginine-binding protein.

The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9-3.3 µM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.

Polyelectrolyte-linked film assemblies of nanoparticles and nanoshells: growth, stability, and optical properties.

Multi-layer films of nanoparticles and nanoshells featuring various polymeric linkage molecules have been assembled and their optical properties characterized. The growth dynamics, including molecular weight effects, and stability of the various nanoparticle film constructions, using both single polymer as well as combinations of alternating charge polyelectrolytes as linking mechanisms, are presented. The polymeric linkers studied include poly-L-lysine, poly-L-arginine, poly(allylamine hydrochloride), and polyamidoamine dendrimers. Significantly air stable films were achieved with the use of multi-layered polymeric bridges between the nanoparticles and nanoshells. Optical sensitivity normally observed with these nanomaterials in solution was observed for their corresponding film geometries, with the nanoshell films exhibiting a markedly higher ability to report their local dielectric environment.

Stable aqueous nanoparticle film assemblies with covalent and charged polymer linking networks.

The construction of highly stable and efficiently assembled multilayer films of purely water soluble gold nanoparticles is reported. Citrate-stabilized nanoparticles (CS-NPs) of average core diameter of 10 nm are used as templates for stabilization-based exchange reactions with thioctic acid to form more robust aqueous NPs that can be assembled into multilayer films. The thioctic acid stabilized nanoparticles (TAS-NPs) are networked via covalent and electrostatic linking systems, employing dithiols and the cationic polymer poly(L-lysine), respectively. Multilayer films of up to 150 nm in thickness are successfully grown at biological pH with no observable degradation of the NPs within the film. The characteristic surface plasmon band, an optical feature of certain NP film assemblies that can be used to report the local environment and core spacing within the film, is preserved. Growth dynamics and film stability in solution and in the air are examined, with poly(L-lysine) linked films showing no evidence of aggregation for at least 50 days. We believe these films represent a pivotal step toward exploring the potential of aqueous NP film assemblies as a sensing apparatus.