Development and application of electrochemical dithiothreitol (DTT) assay for analysis of particulate matter
Particulate matter (PM) in air pollution, known to have a negative impact on biological systems, is regulated in many countries across the globe. The generation of PM from energy and mining industries is monitored in an effort to minimize its contributions to diminished human health. And although quantifying total PM generation (mass and number) and exposure can help track health risks, ultimately there exists a need to develop rapid, efficient, accurate methods for analyzing PM composition and health effects. Leading hypotheses over the last decade have theorized that PM, once absorbed into bodily tissues, generate reactive oxygen species (ROS), leading to oxidative stress, thus catalyzing cellular damage. A recently developed analytical electrochemical protocol has shown great potential for investigating the potential for PM to cause oxidative stress. The work discussed within this thesis focuses on the development of the electrochemical assay and its application to real world PM samples. Herein, the development of an electrochemical version of the well-studied dithiothreitol (DTT) absorbance spectroscopy assay is presented as a platform for the analysis of PM in air samples. Flow injection amperometry was used as the primary electrochemical method. Amperometry was performed using a CH Instruments potentiostat and commercially available DropSens modified carbon screen printed electrodes (SPE's) with a DropSens impinging jet flow cell, granting the assay flexibility to be performed in any lab with commercially available components. Previous examples used homemade components restricting accessibility to the field. The ability to use inexpensive, purchasable components eases trouble shooting, training, and allows for the potential to make the assay more mobile. The use of a flow cell also allows for the possibility for linking the assay to other analytical methods to further analyze PM which may not be reactive in the assay. Assay development focused on optimizing the assay temporally, as well as investigating its precision, detection limits, fluid dynamics, reproducibility, and relative accuracy. The electrochemical assay uses shorter reaction times and avoids the need for additional chemical quenching agents used in the absorbance assay, allowing for batch processing. Assay performance was compared to literature with a model oxidant quinone, 1,4-naphthoquinone, and trace metal, Cu(II). The assay was then applied to real exposure samples collected in Fresno, California and Honduras. Data from these samples were correlated against data obtained by the traditional DTT assay to investigate accuracy. Analyses of the Honduras samples will be correlated against health data as part of the Honduras Cookstove Project, moving one step closer to directly connecting PM reactivity to health effects. Although the traditional absorbance DTT assay is the standard in assessing PM reactivity in air samples, and has been used for the last 15 years, the electrochemical assay is a robust, quick, and precise alternative method that can be readily performed using readily available components.
flow injection amperometry, quinone, Honduras, dithiothreitol assay
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