BioE PhD Proposal Presentation- Jiho Seok

Advisor: Mark Styczynski, Ph.D. (School of Chemical and Biomolecular Engineering, Georgia Tech)

Committee Members:

John Blazeck, Ph.D. (School of Chemical and Biomolecular Engineering, Georgia Tech)

Lily Cheung, Ph.D. (School of Chemical and Biomolecular Engineering, Georgia Tech)

Gabriel Kwong, Ph.D. (Wallace H. Coulter Department of Biomedical Engineering, Georgia Tech )

Brian Hammer, Ph.D. (School of Biological Sciences, Georgia Tech) 

Multifaceted Rolling Circle Amplification Strategies to Reduce False Positives in Point-of-Care Biosensors

In modern warfare, developing sensors that can be used in the field to address the risks associated with biological weapons and infectious diseases is essential. Densely populated military bases enable the rapid spread of infectious diseases, requiring rapid and accurate detection despite limited resources. Particularly, false positives in a sensor result in a significant waste of workforce, time, and resources. Therefore, developing sensors with minimal false positives and ease of use is essential. Rolling Circle Amplification (RCA) is an isothermal DNA amplification technology making it ideal for point-of-care (POC) biosensors. However, RCA performed in a minimal-equipment POC setting operates at a temperature low enough to increase the rate of false positives, presenting an inherent limitation to its utility at the POC. Therefore, I propose a multifaceted approach to reduce false positives in RCA through the multiplexed detection of various targets while maintaining suitability for field deployment. First, I propose a strategy to enhance input selectivity to detect target pathogens accurately without false positives. I propose a novel approach using an AND gate to detect multiple genes simultaneously. Next, I will improve selectivity for a single gene by combining a gap-filling technique with mismatch endonucleases to require precise sequence complementarity for amplification. Then, I will reduce false positives by diversifying output signals for multiple targets within the same pathogen by combining Rolling Circle Transcription (RCT) with RNA fluorescent aptamers. For all of these approaches, I will aim to achieve field deployability by integrating the multiple reaction steps of RCA into one step and demonstrating functionality after lyophilization. Once successfully completed, the work proposed here will enable the development of biosensors that minimize false positives, are user-friendly, and present practical sensor technology applicable in the field.