Correlation of Disinfection A ction of Silver Nanoparticles to Silver Ion Release and Reactive Oxygen Species Generation Characteristics

Silver nanotechnology is the most prevalent use of nanotechnology within consumer products. It is most commonly used as an antimicrobial agent in products such as cleaning sprays, no-odor clothing, and filters for drinking water. While nanosilver has been shown to be an effective disinfectant, the mechanism of antibacterial disinfection is currently an area of intense investigation. Current research suggests two separate pathways of disinfection: 1) the release of silver ions from the nanoparticles and 2) the generation of reactive oxygen species (ROS). An improved understanding of these mechanisms is required in order to increase the efficiency and effectiveness of nanosilver enabled products, as well as understanding environmental impact of such products. In this dissertation, the antimicrobial action of nanosilver for disinfection of drinking water is quantified. Specifically, this research focuses on quantifying the impact of nanoparticle properties (size, functionalization, immobilization) and water chemistries on antibacterial disinfection of silver nanoparicles. Particle functionalization, size, and concentration are likely to alter the production of silver ions and the generation of reactive oxygen species from silver nanoparticles. To quantify the effect of these properties, the production rates of silver ions and reactive oxygen species were measured for citrate, starch, and proteinate functionalized silver nanoparticles ranging in diameter from 10 to 100 nm. The concentration of silver ions released from each of these particles was continuously recorded at 1Hz for 24 hours using a silver-ion specific probe. The quantification of reactive oxygen species is a major challenge, because these species are highly reactive and short lived. As a result, it is thought that ROS species predominantly exist within a small layer of fluid surrounding the particle. An experimental method based on a dihydrorhodamine 6G fluorescence assay was developed as a part of this work to detect the generation of ROS at concentrations ranging from 10 to 100 nM. Reactive oxygen species were continuously measured using this method as a function of time at 1 minute intervals. Initial results show that ion release rates between 0.01 and 4.78 µmol /hr *m 2 for air saturated solutions and ROS generation rates v between 0.25 and 1.91 µmol /hr *m 2 . Upon removal of oxygen from the experimental system, it was found that both the silver ion release and the ROS generation rates decreased to zero. The generation of ROS was found to be approximately 30 times greater than the generation of silver ions for citrate capped nanoparticles. The ratio of ROS generation to silver ion release was found to be significantly affected by surface functionalization. Large surface capping agents favored the release of silver ions while small surface capping agents favored the release of reactive oxygen species. Additionally, the mechanism of silver ion and ROS release was found to be second order in proton concentration, independent of surface functionalization. Experiments were conducted to measure the rates of disinfection of E.coli by silver nanoparticles. Fluorescent microscopy was used in conjunction with the LIVE/DEAD® Baclight™ assay to determine cell viability over time. The solution consists of two fluorescent dyes, SYTO® 9 and propidium iodide, which are commonly used for the detection of cellular membrane integrity. Disinfection rates over time were determined by measuring the ratio of the intensity of the dyes. Experimental results suggest a power law relationship between the rate of disinfection of nanoparticles and nanoparticle concentration. The coefficient of dilution was experimentally found to be approximately 0.5. Experimental results also indicate that disinfection rates of silver nanoparticles cannot be accounted solely based on release of silver ions. Nanoparticle disinfection was found to be proportional to the total mass flux of antibacterial agents (silver ions + ROS) released. For citrate capped nanoparticles, ROS was found to be the dominant antibacterial agent, while disinfection by the citrate and starch capped particles seem to be dominated by silver ions. Water chemistry also influenced the disinfection action of the particles. Salt solutions were found to reduce disinfection effectiveness in all particle types, while NOM solutions preferentially impaired citrate capped particles. As a result, optimal nanoparticle disinfection will require that the surface functionalization be selected based on the environmental conditions of the target application.

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