Persistent Luminescence Nanosensors: A Generalized Optode-Based Platform for Autofluorescence-Free Sensing in Biological Systems.

Fluorescent nanosensors have revolutionized diagnostics and our ability to monitor cellular dynamics. Yet, distinguishing sensor signals from autofluorescence remains a challenge. Here, we merged optode-based sensing with near-infrared-emitting ZnGa2O4:Cr3+ persistent luminescence nanoparticles (PLNPs) to create nanocomposites for autofluorescence-free "glow-in-the-dark" sensing. Hydrophobic modification and incorporation of the persistent luminescence nanoparticles into an optode-based nanoparticle core yielded persistent luminescence nanosensors (PLNs) for five analytes (K+, Na+, Ca2+, pH, and O2) via two distinct mechanisms. We demonstrated the viability of the PLNs by quantifying K+ in fetal bovine serum, calibrating the pH PLNs in the same, and ratiometrically monitoring O2 metabolism in cultures of Saccharomyces cerevisiae, all the while overcoming their respective autofluorescence signatures. This highly modular platform allows for facile tuning of the sensing functionality, optical properties, and surface chemistry and promises high signal-to-noise ratios in complex optical environments.

Triplet-Triplet Annihilation Upconversion-Based Oxygen Sensors to Overcome the Limitation of Autofluorescence.

Autofluorescence is one of the many challenges in bioimaging as it can mask the emission from fluorescent probes or markers, a limitation that can be overcome via upconversion. Herein, we have developed a nanosensor that uses triplet-triplet annihilation upconversion to optically report changes in the dissolved oxygen concentration. Using a sensitizer-annihilator dye pairing of platinum(II) octaethylporphyrin and 9,10-diphenylanthracene, we monitored the oxygen consumption (as a proxy for metabolic activity) over time in a biological system─Saccharomyces cerevisiae (brewing yeast). The nanosensor demonstrated good reversibility over multiple cycles and showed good signal and colloidal stability when tested over the course of 7 days, and it was sensitive to dissolved oxygen from 0.00 to 3.17 mg/L O2. Additionally, there was no signal overlap between the nanosensor emission and S. cerevisiae autofluorescence, thus underscoring the utility of upconversion as a facile and economical means of overcoming autofluorescence.

Sensors in a Flash! Oxygen Nanosensors for Microbial Metabolic Monitoring Synthesized by Flash Nanoprecipitation.

Flash nanoprecipitation (FNP) is an efficient and scalable nanoparticle synthesis method that has not previously been applied to nanosensor fabrication. Current nanosensor fabrication methods have traditionally exhibited poor replicability and consistency resulting in high batch-to-batch variability, highlighting the need for a more tunable and efficient method such as FNP. We used FNP to fabricate nanosensors to sense oxygen based on an oxygen-sensitive dye and a reference dye, as a tool for measuring microbial metabolism. We used fluorescence spectroscopy to optimize nanosensor formulations, calibrate the nanosensors for oxygen concentration determination, and measure oxygen concentrations through oxygen-sensitive dye luminescence. FNP provides an effective platform for making sensors capable of responding to oxygen concentration in gas-bubbled solutions as well as in microbial environments. The environments we tested the sensors in arePseudomonas aeruginosa biofilms andSaccharomyces cerevisiae liquid cultures─both settings where oxygen concentration is highly dependent on microbial activity. With FNP now applied to nanosensor fabrication, future nanosensor applications can take advantage of improved product quality through better replicability and consistency while maintaining the original function of the nanosensor.

A near-infrared optical nanosensor for measuring aerobic respiration in microbial systems.

We developed a ratiometric oxygen-sensitive nanosensor and demonstrated application in monitoring metabolic oxygen consumption in microbial samples over time. Based on a near-infrared (NIR) emitting oxygen-quenched luminophore, platinum(II) octaethylporphine ketone (PtOEPK), along with a stable dioctadecyl dicarbocyanine reference dye (DiD), this nanosensor system provides an advantageous approach for overcoming imaging issues in biological systems, such as autofluorescence and optical scattering in the visible wavelength region. The dyes are encapsulated within a polymer-based nanoparticle matrix to maintain them at a constant ratio in biological samples, precluding the need for complex synthetic approaches. With this constant ratio of the two dyes, the nanosensor response can be measured as a ratio of their two signals, accounting for nanosensor concentration artifacts in measurements. The nanosensors are reversible, which enabled us to temporally monitor systems in which dissolved oxygen concentrations both increase and decrease. These sensors were applied for the monitoring of oxygen in samples of Saccharomyces cerevisiae (brewing yeast) in a 96-well optical fluorescence plate reader format over 60 h. By mixing the nanosensors directly into the sample well with the yeast, we were able to dynamically track metabolic activity changes over time due to varying cell concentration and exposure to an antimicrobial agent. This system could be a potential platform for high-throughput screening of various species or variants of microbes with unknown metabolic rates in response to external stimuli (antimicrobials, metabolites, etc.).

Nanodiagnostics to monitor biofilm oxygen metabolism for antibiotic susceptibility testing.

In clinical environments, many serious antibiotic-resistant infections are caused by biofilm-forming species. This presents issues when attempting to determine antimicrobial dosing as traditional antibiotic susceptibility tests (ASTs) are typically designed around planktonic bacteria and thus offer information that is not relevant to the biofilm phenotype present in the patient. Even the popular Calgary biofilm device may provide inaccurate minimum biofilm inhibitory concentrations (MBICs) and can be time- and material-intensive. In this work, we present a method utilizing oxygen-sensitive nanosensor technology to monitor the oxygen consumption dynamics of living biofilms as they are exposed to antibiotics. We incorporated our nanosensors into biofilms grown from P. aeruginosa strains of varying sensitivity to traditional classes of antibiotics. Through measuring nanosensor response under antibiotic administration we determined the concentrations able to cease biofilm metabolism. This method provides information on the MBIC as well as kinetic response information in a manner that requires fewer materials and is more reflective of biofilm behavior than a traditional AST.