Transparent carbon ultramicroelectrode arrays for the in vitro detection of biogenic species
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Extracellular communication relies on signaling molecules for cellular survival, division, differentiation, and even organized cell death, or apoptosis. The types of molecules that are used for cellular communication are highly varied in size and type, ranging from large proteins to small diffuse gaseous molecules. The focus of the research discussed in this dissertation is on the development of an electroanalytical sensing platform for the in vitro detection of biogenic reactive oxygen species (ROS) and various cellular metabolites. The biogenic species of analytical interest are hydrogen peroxide (H₂O₂), nitric oxide (NO•), and pyocyanin. H₂O₂ is a product of a wide variety of metabolic processes and is a crucial signal transducer in cellular processes. NO• is a major immunological defense mechanism and its production is upregulated in response to infections caused by pathogenic species. Pyocyanin is a bacterial warfare toxin that is secreted by pathogenic bacteria in order to damage host/immune cells. Although these molecules are known to play key roles in cellular function as well as pathophysiological mechanisms, little is known about how their concentrations fluctuate and direct cellular behavior. The primary explanation as to why this is true is because these molecules are very diffuse, highly reactive, and exist at low concentrations, ranging from nM to [mu]M, respectively. Discussed in this dissertation is an electrochemical approach to detect these biogenic species, in vitro. Here, transparent carbon ultramicroelectrode arrays (T-CUAs) were fabricated using a facile and cheap method. iii The electrodes are biocompatible and it is demonstrated that they can be easily modified to enhance their response and selectivity to NO•. The T-CUAs are transparent, therefore cellular behavior could be examined or optical spectroscopic experiments could be performed as the concentration of the analyte is electrochemically determined. The optical transparency of the electrodes also allows for the in situ mechanistic and kinetic characterization of heterogeneous electrochemical and intermediate homogeneous chemical reactions while simultaneously using spectroscopic techniques with high electroanalytical sensitivity. The figures of merit determined for each of the biogenic analytes will be used to determine the concentrations of cellularly-derived biogenic species of interest, in vitro to observe concentration dependent sociomicrobial phenomena. Discussed in more detail is the simultaneous fluorescent and electrochemical detection of NO• at the T-CUAs, in vitro. Thus, the dual capabilities of the electrochemical platform for biological applications are demonstrated.