Synthesis, Characterization, and Investigation of Metal Ion Quenching in Fluorescent Carbon Dot Surrogates for Particulate Matter Black Carbon and Evaluation of Cellular Health Effects Due to the Surrogate Materials
Doctor of Philosophy (PhD)
Black carbon (BC) is an environmental pollutant of particular concern to many international organizations for both its health effects and environmental effects. Probing health effects of BC as produced in the environment is difficult due to the complex nature of environmental pollutants found in their naturally occurring state. Fluorescent carbon dots (FCDs) were chosen to be used as a surrogate for BC. In the process of examining FCDs and their behavior as surrogates, information was gained on the health effects and behavior of FCDs as a class of nanomaterials for cell and tissue studies, which is outlined in this dissertation. The synthesis conditions were explored to optimize the yields and efficacy of the FCDs. Upon synthesis optimization, characterization of the FCDs occurred with the use of dynamic light scattering (DLS), low-resolution transmission electron microscopy (LR-TEM), high-resolution transmission electron microscopy (HR-TEM), infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), and electron-paramagnetic spectroscopy (EPR). Evaluation of the optical properties were studied using fluorescence spectroscopy. Evaluation of the photostability, chemical stability, and physical stability was performed as well. The FCDs produced in this research ranged in size from 10-50 nm, exhibiting a graphene oxide structure with suggestions of hydroxide functional groups on the surface and semiquinone-type radicals. The FCDs exhibited significant stability in all testing methodologies, confirming their efficacy as fluorescent probes. The FCDs were designed to be a turn-on fluorescent probe, so fluorescence quenching was tested. Multiple metal ions were tested for their ability to quench the fluorescence of the FCDs, followed by a testing of fluorescence recovery due to a reducing agent. Ferric ions were determined to be the optimal quencher, effectively quenching 100% of the fluorescence. Upon evaluating fluorescence recovery through reduction of the ferric ions by various reducing agents, only limited fluorescence recovery occurred (~ 25%). It was determined that fluorescence recovery by reduction of the ferric ions could not occur through proving that ferrous ions quench the fluorescence as well. Through characterization of the FCDs before and after fluorescence quenching revealed information used to generate a proposed quenching mechanism. The proposed mechanism, involving the stabilization of a semiquinone radical by the ferric ions is the first of its kind reported in literature to this point. Cellular studies regarding the health effects of the FCDs, and therefore BC, was performed on lung epithelial cells (BEAS-2B). Uptake was evaluated by confocal fluorescence imaging and LR-TEM. The uptake was confirmed due to visualization of FCDs within the cells, with an active transport mechanism rather than a passive transport process suggested to be occurring. Cytotoxicity was evaluated by trypan blue assay, MTT assay, and MTS assay. Results reveal cytotoxic effects when FCDs are exposed to cells for longer time periods (8 hours), even at concentrations as low as 0.5 mg/mL. When exposed to higher concentrations of FCDs (8 mg/mL), time periods as short as 30 min begin to show cytotoxic effects. Any cytotoxic effects due to the metal ions present in the quenched FCDs was also evaluated and it was confirmed that the metal ion quencher increases the overall toxicity of the FCDs. Through an evaluation of the GSH:GSSG ratio, no cellular oxidative stress was witnessed at short time periods, though further study is warranted. The cell studies of FCDs in this dissertation are more comprehensive regarding time period and FCD concentration than any found in literature.
Sumner, Christopher Lee Jr, "Synthesis, Characterization, and Investigation of Metal Ion Quenching in Fluorescent Carbon Dot Surrogates for Particulate Matter Black Carbon and Evaluation of Cellular Health Effects Due to the Surrogate Materials" (2020). LSU Doctoral Dissertations. 5255.