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 poly¿uoroalkyl 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 micro¿uidic technology to continuously monitor water for PFASs. In order to demon-
strate technical feasibility, QBI will perform these Speci¿c Aims:
Speci¿c 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 speci¿cally 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 speci¿city 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 micro¿uidic platform.
Speci¿c 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 micro¿uidic-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 ¿uores-
cence response. Finally, QBI will optimize a micro¿uidic device to facilitate this assay and maximize the
cellular ¿uorescence 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 end-users' needs.