Access to clean, reliable water supplies is critical to our quality of life and our economy, and ensuring this access for generations to come will involve developing novel approaches to determining the safety and composition of potable water that are practical and affordable. Per- and polyfluoroalkyl substances (PFASs) are among the most ubiquitous and persistent contaminants plaguing groundwater in the United States, and human epidemiological studies have found associations between PFASs in drinking water and a number of adverse health conditions, from liver and thyroid disorders to various forms of cancer. The main objective of this SBIR proposal is to develop a customizable biosensor platform that uses engineered microbial sensor strains paired with microfluidic technology to continuously monitor water for PFASs. In order to demon- strate technical feasibility, QBI will perform these Specific Aims: Specific Aim 1: To identify and characterize the binding kinetics of nanobodies for PFOA and PFOS. For an engineered bacterial strain to be maximally effective as a sensor, it must be able to specifically and strongly bind its target in the environment. QBI will identify nanobodies that bind PFAS molecules and characterize their capture potential when surface displayed in an adsorbing E. coli strain. QBI will work with a local nanobody company, Abcore, to isolate a set of nanobodies that are enriched for specificity to the two targets and will then bring these nanobodies to their facility, clone them into their E. coli nanobody display vector, and screen and characterize them within their multiplexed microfluidic platform. Specific Aim 2: To develop a prototype for continuous and batch PFAS sensing. In order to use the newly developed sensor strains in a continuous monitoring platform, QBI will need to develop a novel as- say for measuring agglutination on a microfluidic-scale from many individual strain banks. QBI will begin by developing and optimizing an agglutination assay using the reduced set of surface-displayed nanobody strains, and then they will build upon previous results to transduce the agglutination signal to a fluores- cence response. Finally, QBI will optimize a microfluidic device to facilitate this assay and maximize the cellular fluorescence signal so that they can quantify the amount of contaminant present in the water. Successful completion of these Aims will serve to validate the use of nanobody-based sensing strains to achieve sensitive, selective, and continuous contaminant detection, making it of great utility to monitoring efforts aimed at tracking and assessing potential hazardous exposures. Beyond the ability to detect many different targets with a single on-line sensor, which is highly unique, the customizability and expandability of the platform using synthetic biology to engineer strains is transformative. This will enable QBI to continually expand their customer base as they continue to add sensing capabilities tailored to meet ...