DEVELOPMENT OF MICROFLUIDIC AND ELECTROCHEMICAL TECHNQIUES FOR THE ASSESSMENT OF MITOCHONDRIAL RESPIRATION IN SCARCE SAMPLES
In 2021, cancer, Alzheimer’s disease, and diabetes accounted for more than a combined 800,000 deaths according to the Centers for Disease Control and Prevention. These diseases have been the focus of large amounts of research and a focus of multiple funding agencies for many years. It is also publicized that these diseases all have connections to the function of mitochondria. While a relationship is established, the physiological mechanisms of mitochondrial dysfunction and its relationship to these chronic diseases remains a largely unresolved topic. This is in large part due to the techniques that are currently used to study mitochondria in a physiological setting. Current technologies rely on measuring mitochondrial respiration by tracking changes in oxygen concentration in a sample. This is due to the final protein complex in the mitochondrial electron transport chain consuming oxygen. This allows for measurement of mitochondrial respiration; however, this presents two major problems for physiological studies. First, measuring changes in oxygen concentration is a metric that only allows for direct measurement of the activity of one of the four complexes that compose the mitochondrial electron transport chain. Any assessment of the first three complexes requires either indirect assessment using a combination of chemical inhibitors and substrates or requires the complex to be isolated from the mitochondrial electron transport chain, causing a loss of context that is the other complexes. Second, traditional techniques require large samples (~mL volumes) that make working with scarce samples, such as mitochondria isolated from cell lines or from human tissues, impractical and expensive at best, and impossible at worst. In this thesis, I propose two techniques that can open the door for further physiologically relevant studies on scarce mitochondrial samples. The first of these techniques relies on taking advantage of the inherently electrochemical nature of the mitochondrial electron transport chain. By crafting a 3D printed, microfluidic device it was possible to incorporate electrodes into a reaction well that would reduce or oxidize complex IV in the electron transport chain using mediated microfluidic electrochemistry. I am able to demonstrate I can manipulate the rate of oxygen consumption of the mitochondria based on the applied potential, but am able to quantify the amount of oxygen being consumed by the mitochondria from measurements of current flowing across the electrode. This resulted in a reduction of volume demands from greater than a mL to less than 10 μL. This work also opens the door to other manipulations of the mitochondrial electron transport chain through different electrochemical pathways, yielding direct access to previously inaccessible complexes in the intact mitochondria. The second proposed pathway leans into the ability to detect changes in oxygen concentration using thin film fluorescent oxygen sensors. This work shows that by sandwiching μL sized droplets of solution between an oxygen sensing thin film and a glass cover slip it is possible to measure changes in oxygen concentration using 100s of nanograms of protein. This represents a greater than 500-fold decrease in sample demand compared to traditional techniques.
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- In Collections
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Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
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Theses
- Authors
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Frantz, Nathan
- Thesis Advisors
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Blanchard, Gary J.
- Committee Members
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Proshlyakov, Denis A.
Hegg, Eric L.
Swain, Greg M.
Hong, Heedeok
- Date Published
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2024
- Subjects
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Analytical chemistry
- Program of Study
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Chemistry - Doctor of Philosophy
- Degree Level
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Doctoral
- Language
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English
- Pages
- 82 pages
- Embargo End Date
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July 19th, 2026
- Permalink
- https://doi.org/doi:10.25335/spwm-1453
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