PFI (MCA): Developing Safe and Affordable Alternatives to Horseshoe Crab Blood for Detecting Harmful Bacterial Toxins

This Mid-Career Advancement project develops affordable and accurate tests for detecting bacterial endotoxins—harmful substances released from the outer membrane of certain bacteria that can cause serious infections and inflammatory responses in humans. This advance will help the biomanufacturing industry reduce quality control costs and eliminate the need for horseshoe crab blood, which is currently used in testing but raises sustainability concerns. The project will benefit pharmaceutical companies, medical device manufacturers, healthcare facilities, environmental testing labs, and regulatory agencies by improving safety and efficiency in detecting endotoxins. The project will integrate commercialization, education, and outreach by mentoring undergraduates in transitioning research into commercial products, training a skilled biomanufacturing workforce, and developing a senior-level course on biomedical entrepreneurship. By enhancing scientific understanding and promoting cost-effective testing, this project supports industry growth while benefiting public health and sustainability. This project leverages biological sensing mechanisms by using natural transcription factors to develop artificial transcription factors (ATFs) for in vitro endotoxin detection. The research focuses on designing, modeling, and constructing ATFs through a modular approach to identify endotoxin-specific effector domains and linker peptides, optimizing them and conducting fluorescence polarization screening, assembling ATF-regulated gene circuits using a cell-free synthesis system, and ultimately implementing and commercializing the ATF-based sensor. Key innovations include on-chip ATF development using droplet microfluidics and a fluorescence polarization detector to identify high-affinity variants, cell-free expression of ATF-regulated gene circuits to enhance detection sensitivity, and integration with paper substrates and portable instruments for cost-effective, field-deployable testing. By providing a low-cost, alternative to traditional horseshoe crab blood-derived tests, this research has potential to transform endotoxin detection across multiple industries. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.