Engineering human liver fatty acid binding protein for detection of poly‐ and perfluoroalkyl substances

Per- and polyfluoroalkyl substances (PFAS) are a large group of synthetic fluorinated chemicals with surface active and water-repellent properties. The combination of wide-spread use in numerous consumer and industrial products and extended biological half-lives arising from strong carbon-fluorine bonds has led to significant accumulation of PFAS in humans. As most human interaction with PFAS comes from ingestion, it is important to be able to detect PFAS in drinking water as well as in agricultural water. Here we present an approach to designing a fluorescence-based biosensor for the rapid detection of PFAS based on human liver fatty acid binding protein (hLFABP). Introduction of solvatochromic fluorophores within the ligand binding pocket (L50) allowed for intrinsic detection of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) via blue-shifts in fluorescence emission spectra. Initially, a single tryptophan mutation (L50W) was found to be able to detect PFOA with a limit of detection (LOD) of 2.8 ppm. We improved the sensitivity of the biosensor by exchanging tryptophan for the thiol reactive fluorophore, acrylodan. The acrylodan conjugated C69S/F50C hLFABP variant is capable of detecting PFOA, PFOS, and PFHxS in PBS with LODs of 112 ppb, 345 ppb, and 1.09 ppm, respectively. The protein-based sensor is also capable of detecting these contaminants at similar ranges in spiked environmental water samples, including samples containing an interfering anionic surfactant sodium dodecyl sulfate. Overall, this study demonstrates engineered hLFABP is a useful platform for detection of PFAS in environmental water samples and highlights its ease of use and versatility in field applications.