Esther Stopps's PhD Comprehensive Exam Presentation, ChBE
- Wednesday, January 12, 2022 from 11:00am to 12:00pm
- Roberts Hall, 210 or Via Webex - view map
Meeting Link: https://montana.webex.com/montana/j.php?MTID=me3526f6b2704b079947ed036801ca8f7
Meeting number (access code): 2624 674 1689
Meeting password: ccRCPcgU388
Leveraging cooperative binding kinetics to tune the sensitivity and specificity of isothermal amplification reactions
There is a need within the medical community for more sensitive and robust biosensors to diagnose diseases quickly and accurately. Nucleic acid (NA) biosensors are advantageous because their complementary base-pairing structure makes them highly tunable and they have the potential for sensitive discrimination between molecular targets. NA biomarkers are also cheaper than imaging methods and more definitive than self-reported symptoms. While established techniques such as Polymerase Chain Reaction (PCR) generally provide high sensitivity and specificity, they require more time and specialized equipment for thermocycling. Alternatively, isothermal amplification reactions occur at a single temperature and therefore are often faster and more applicable to limited resource settings; however, nonspecific amplification can raise the limit of detection and reduce specificity.
To address these challenges, we aim to design isothermal NA hybridization reactions that exhibit cooperative kinetics to achieve more rapid, definitive responses to specific concentrations of miRNA biomarkers, compared to current biosensors. Cooperativity has been demonstrated to result in a steeper, switch-like response to a target as the receptor goes from an unbound state to a bound state; however, the kinetics of this reaction have not been well characterized. We hypothesize that cooperative DNA receptors can be fine-tuned through multiple target binding sites, creating a unique thermodynamic and kinetic profile for hybridization reactions and a distinct response outcome. To test this hypothesis, we have designed cooperative DNA binding pairs and will measure their binding parameters using Surface Plasmon Resonance and Isothermal Titration Calorimetry techniques. We will also study the effects of common fluorescent dyes on the binding kinetics. We will then integrate cooperativity into isothermal amplification detection assays, specifically Ultrasensitive DNA Amplification Reaction and Rolling Circle Amplification, to detect potential miRNA biomarkers for Alzheimer's disease. This exploration into DNA hybridization reactions will provide valuable insight into biosensor mechanisms and reaction modeling, while progressing the field towards more rapid, point-of-care testing.
- Department of Chemical and Biological Engineering