Grantee Research Project Results
Title of Talk:
Transformations of Biologically-Conjugated CdSe Quantum Dots Released into Water and Biofilms
Abstract of Talk:
Semiconductor nanocrystals (quantum dots) differ in important ways from bulk semiconductor materials. Their increased band gap means that they function as strong oxidizing and/or reducing agents, and their small size allows them to pass into living cells. Conjugation of biomolecules to the crystal surface can alter any or all of these properties. In preliminary experiments, we have observed that nucleobase-conjugated CdSe quantum dots were actively taken up by soil and aquatic bacteria (for example, Bacillus subtilis and Escherichia coli). Effects on microbial viability attributed to the presence of the quantum dots included slower doubling times, heavy metal sequestration, and “blebbing” of metals into the environment. In this project we will quantify these effects using a variety of biologically-conjugated quantum dots and an assortment of microbial species, monitoring the process of quantum dot uptake and breakdown and characterizing the breakdown products that result from bacterial metabolism of these particles. Possible hazards to microbial populations with extrapolation to humans through contamination of soil and water with quantum dot breakdown products will be analyzed and quantified.
Bare, core-shell and biologically-conjugated quantum dots (QDs) will be studied. Abiotic breakdown kinetics and products in aqueous environments will be determined by inductively coupled plasma (ICP) spectrometry for QDs as a function of exposure to light, pH, and oxidizing or reducing conditions. In preliminary experiments, biologically-conjugated QDs are easily taken up by B. subtilis, but the process is light and pH dependent. Some breakdown occurs inside and outside of cells. Working with Pseudomonas aeruginosa and Staphylococcus aureus to represent Cd-sensitive and Cd-resistant strains, we will quantify population growth and fluorescence for pure liquid cultures previously exposed to QDs. Conventional methods (shake flask, viable and direct counting over time) will be used to assess the effects of labeling on bacterial growth rates under high and low nutrient conditions. QD fluorescence will be monitored throughout, adjusting final results for the dilution effect of growing populations. Concentrations of Cd and Se will be assessed inside and outside cells, and membrane associations of whole QDs and breakdown products will be quantified. The relationship of QD release and breakdown to cell viability will be assessed. DNA damage in bacteria will be assessed by quantifying 8-oxoguanine, a product of oxidative DNA damage, by microscopy and a commercially-available fluorescent label. These experiments will provide basic insight into cellular interactions with QDs. The potential for single base pair damage from whole QDs and breakdown products will be assessed using time-correlated single photon counting techniques.
Because most bacteria exist as biofilms in nature, we will also culture mono- and dual-species bacterial biofilms under continuous flow conditions in a commercially-available flow cell. Using digital photomicroscopy and computerized image analysis, we will assess the effects of QD labeling on biofilm growth. Unsaturated biofilms will also be cultured on membranes to assess the effects of QD labeling on development under soil-like conditions, and as a function of nutrient and water availability. Cryo-environmental scanning electron microscopy (ESEM) coupled with energy dispersive spectrometry (EDS) will be used to visualize ultrastructural QD associations. Biofilms cultured in the absence of QDs will be exposed under a range of experimental conditions and assessed over time for viability and QD content. For all biofilm experiments, QD effects on exopolymeric substances (EPS) can be quantified by GC-MS of derivatized glycosyl residues, and DNA and protein content determined by standard fluorometric and colorimetric methods, respectively. Finally, column studies, using packed porous media under saturated and unsaturated conditions, will be conducted to assess QD and Cd mobility as a function of bacterial colonization. Because EPS is expected to chelate Cd, we will quantify whole QDs, Cd, Se and biopolymers in breakthrough experiments, followed by sacrificial characterization of residual analytes.
Overall, this project is intended to provide a comprehensive investigation
into bacterial-QD interactions, which is needed to understand the impact
and fates of these nanoparticles in the environment.