Designing and optimizing a biosensor to detect per- and polyfluoroalkyl substances for real-time detection.

Abstract

Per- and polyfluoroalkyl substances (PFAS) are a highly stable and environmentally persistent class of compound that humans are frequently exposed to by contaminated wastewater sludge as fertilizer in agriculture. Current methods of detecting and processing PFAS in wastewater treatment plants are costly and require niche expertise, motivating the need to turn to protein-based biosensors as a quick, accessible, and on-site form of reliable testing. To detect and remediate PFAS types rapidly using an optical biosensor, this thesis seeks to screen varying proteins known to interact with PFAS as potential sensing modules for a biosensor in silico and study Acidimicrobium TMED77’s reductive dehalogenase (T7RdhA) in vivo as a potential fluorescent biosensor that can both detect and remediate varying PFAS types. Through screening, it was determined that peroxisome proliferated activated receptor  (PPAR-), T7RdhA, and Acidimicrobium Sp. 6 RdhA (A6RdhA) were proteins with the highest potential to serve as a sensing module for a biosensor. When circularly permuted green fluorescent protein (cp.GFP) was inserted into T7RdhA at a site of high variability and surface accessibility, it was determined that while there is a change in fluorescent signals, there is no clear gradient that directly correlates increasing PFAS concentrations to a gradient change in fluorescence. Overall, these results provide foundational preliminary work in studying how T7RdhA can serve as both a detector and remediator of PFAS through real-time biosensing.