Microfluidic assays for assessing oligonucleotide catalyst abundance and monitoring biomolecule concentration in real time
2023-10
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Microfluidic assays for assessing oligonucleotide catalyst abundance and monitoring biomolecule concentration in real time
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2023-10
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Microfluidic platforms control and manipulate very small volumes of liquid, typically at the microliter or nanoliter scale. By replacing pipettes and flasks with microfluidic channels and chambers, routine laboratory processes can be scaled down and sped up. Microfluidic platforms can mix, react, incubate, separate, extract, and detect solutions with high throughput and reproducibility, measuring the natural world at physical scales and timescales that would be inaccessible using traditional laboratory techniques. This thesis describes the development of microfluidic assays to address two bioanalytical challenges. First, a droplet microfluidic platform was developed to quantify the abundance of catalytic molecules in pools of random-sequence DNA. Although catalytic oligonucleotides are attractive as sensors and therapeutic agents, the full scope of their catalytic activity is largely unknown. The microfluidic platform described here encapsulates a library of DNA sequences in droplets with a fluorogenic substrate. Droplets that contain a catalytic sequence will become fluorescent after a period of incubation, while droplets without a catalyst will remain dark. The frequency of catalysts in the original library can be calculated from the ratio of fluorescent and non-fluorescent droplets. This thesis describes the technical design of a droplet microfluidic platform, its performance in library screening experiments, and its application for the detection of a known DNA catalyst. A versatile microfluidic platform for oligonucleotide library screening could assess catalyst abundance across a wide variety of reactions and conditions, creating a new framework for understanding the catalytic potential of oligonucleotides.
Second, an aptamer affinity assay was developed for continuous cytokine quantification using micro free-flow electrophoresis (µFFE). Affinity assays are a prominent tool for biomolecule quantification because of their excellent sensitivity and specificity. However, traditional affinity assays use discrete samples and are poorly suited for measuring dynamic changes in an analyte’s concentration. The ultimate aim of the aptamer assay is to continuously quantify cellular cytokine secretion in real time using µFFE, a continuous separation technique that can detect free aptamer and bound aptamer complexes in a flowing sample stream. This thesis describes the characterization of µFFE devices fabricated in cyclic olefin copolymer as well as initial development of a µFFE aptamer assay for continuous quantification of tumor necrosis factor α (TNFα).
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University of Minnesota Ph.D. dissertation. October 2023. Major: Chemistry. Advisor: Michael Bowser. 1 computer file (PDF); xv, 189 pages.
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Douma, Cecilia. (2023). Microfluidic assays for assessing oligonucleotide catalyst abundance and monitoring biomolecule concentration in real time. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/260118.
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