PhD defense of Mariah S. Harris

With the global health crisis of both antimicrobial resistant (AMR) bacteria and cancer on the rise, there is an urgent need for improved early detection methods. Optical fluorescent biosensors with fluorescent nanoparticles (FNPs) offer the ease of surface functionalization, biocompatibility, sensitivity, and selectivity. Previous studies have demonstrated polymeric fluorescent nanoparticles coupled with a pH sensor can function as ratiometric optical sensors for the detection of Escherichia coli (E. coli). In this work, we investigate polymeric green fluorescent nanoparticles (G-FNPs) consisting of a green BODIPY fluorophore core and hydrophilic polymer shells composed of poly (ethylene oxide) and poly (acrylic acid) with a 4-dibenzocyclooctynol (DIBO) moiety. The attachment of a X-rhodamine pH-sensitive fluorophore, Ruby, by strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry, allowed for the creation of a ratiometric fluorescent biosensor.

In the first part, the photophysical properties of the bare G-FNPs, free Ruby, and the Ruby grafted onto the G-FNPs (dubbed “grafted particles – GPs) were investigated using steady-state and time-resolved fluorescence measurements. In ultrapure water (pH ~6.4-7.4), the photophysical properties of G-FNPs and Ruby were retained after grafting. In citrate-phosphate buffers (pH 4-8), the GPs displayed a ratiometric pH behavior with a higher fluorescence ratio in acidic conditions and an apparent pKa of 4.9 ± 0.2. The GPs also displayed pH reversibility in basic to acidic conditions. Time-resolved measurements revealed quenching of the G-FNPs lifetime in the presence of Ruby, indicating the potential of FRET between the two components.

In the second part, the GPs were investigated in the presence of bacterial cells and eukaryotic cells. GPs and G-FNPs showed no toxic against Gram-positive and Gram-negative bacteria. Furthermore, GPs detected increased acid production of E. coli in the presence of glucose by higher fluorescence ratios and showed sensitivity to bacteria growth within a cuvette during the first two hours. In eukaryotic cells, passive cellular uptake of the GPs was studied at different incubation times, and G-FNPs and GPs displayed no toxic effect when tested against non-cancerous (COS-7) and cancerous (HeLa) cell lines by measuring the activation of caspase-3/7 by fluorescence microscopy. Then, the intracellular detection of the GPs with the COS-7 and HeLa cell lines was investigated with and without saponin. The third part focuses on fabricating luminescent surfaces by immobilizing the G-FNPs and Ruby on silanized glass. These surfaces were studied in citrate-phosphate buffers for pH sensing, but the irreversible photobleaching precluded reliable pH calibration. After, the immobilization strategy was studied by utilizing SPAAC click chemistry for grafting a range of azide-functionalized dyes from different fluorophore families, producing luminescent surfaces with emission spanning the visible spectrum. Among these, ROX and BDP-TR displayed the best potential for FRET with the G-FNPs.

Lastly, the fourth part introduces preliminary work on novel pH-sensitive fluorophores, such as a pH-sensitive BODIPY fluorophore and PEGylated H-Ruby for the development of other optical biosensors with various pH sensors.

Overall, this work establishes a modular fluorescent nanoparticle platform adaptable to diverse fluorophores, paving the way for future biosensing applications.