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Theoretical and Experimental Development of Bipolar Based Fluorescence Detection for Microchip Electrophoresis
Wijesinghe, Manjula Bandara
Wijesinghe, Manjula Bandara
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Abstract
Abstract Methods for the separation and detection of reactive oxygen and nitrogen species (RNOS) at the cellular level can be useful tools for the study of the biochemical mechanisms of neurodegenerative diseases. Microchip electrophoresis (ME) is a promising analytical separation technique that can be used to separate these short-lived RNOS since it offers sub-minute analysis times, low sample volumes, and the ability for single cell analysis. Amperometric detection is one of the most popular detection methods for ME and has been used for the detection of RNOS and related antioxidants. In this thesis, a dual-channel/dual-parallel electrode system is developed to identify electroactive species based on their redox properties without the need for complicated data correction procedures. This new strategy was applied to distinguish nitrite from azide in a cell sample. Azide is a contaminant that is introduced by the filters used to remove cell debris. Microchip electrophoresis can also be coupled to fluorescence detection (FL) for the investigation of RNOS production in macrophage cells using different fluorescent dyes for specific RNOS that exhibit similar excitation and emission wavelengths. Using ME-FL, the effect of engineered carbon nanoparticles on ROS production by microglia and lung epithelial cells was investigated. In this dissertation, a novel detection method for ME was developed that takes advantage of both electrochemical and fluorescence detection. This method involves transforming an electrochemical signal to a fluorescence signal using a bipolar electrode. The new method was evaluated with two model reducible analytes using 2,7-dichlorodihydrofluorescein (DCFH2) as the fluorescence reporter. In addition, modeling of the ME-bipolar electrochemistry/ fluorescence experimental setup was performed using COMSOL Multiphysics. Programs were developed to generate bipolar cell voltammograms and to model the effect of the flow rate on the size of the fluorescence plug formed at the detector electrode. As a result of these studies, a bipolar fluorescence detection method was developed that was able to obtain low micromolar detection limits for reductive analytes. The method was further developed to obtain the bipolar fluorescence response without a potentiostat and with a simplified experimental setup. This development will be extended in the future to detect oxidizable analytes such as RNOS in cells. Additionally, chemiluminescence reporting can be used instead of fluorescence reporting to obtain better detection limits. Lastly, this system could be coupled to a miniaturized optical detection system to develop a portable microchip device capable of detecting electroactive species on-site.
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Date
2019-05-31
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University of Kansas
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Keywords
Analytical chemistry, Bipolar electrode, Electrochemistry, Electrophoresis, Fluorescence, Microfluidics, Separation