Advisor: Dr. Mark Styczynski (School of Chemical and Biomolecular Engineering)

Committee Members:

Dr. John Blazeck (School of Chemical and Biomolecular Engineering)

Dr. Julie Champion (School of Chemical and Biomolecular Engineering)

Dr. Brian Hammer (School of Biological Sciences)

Dr. Valeria Milam (School of Materials Science and Engineering)

Engineering Modular Target Recognition for Cell-Free Biosensing in One-Pot PURE

Point-of-care (POC) diagnostics benefit from low-cost biosensing platforms that offer simple readouts and can be readily adapted to detect diverse analytes. Cell-free protein synthesis (CFPS) offers a programmable environment for combining molecular recognition elements and genetic circuits for biosensing. One such approach, the T7 RNA polymerase-Linked ImmunoSensing Assay (TLISA), uses nanobodies to reassemble split polymerase fragments upon simultaneous binding to a target protein, driving reporter expression. A more sensitive version of TLISA has been implemented in PURE, a CFPS system reconstituted from purified components. However, commercial PURE costs $1,110 per mL of reaction, making biosensor optimization expensive and limits affordability for POC deployment. One-pot PURE simplifies preparation by jointly purifying the 36 PURE proteins from a co-culture, reducing costs to $90 per mL. I hypothesize that one-pot PURE will improve TLISA’s affordability, while interchangeable nucleic acid recognition elements will enable modular detection of protein and nucleic acid targets. These elements would address a separate limitation: adapting TLISA to new targets requires cloning new affinity domain–polymerase fusions, slowing sensor development. First, I will establish and optimize one-pot PURE in-house and validate its support for TLISA sensing. Second, I will engineer an RNA hairpin-aptamer TLISA architecture that tethers target-binding aptamers to split polymerase fragments through specific protein-RNA interactions. This design would allow target specificity to be changed by switching out aptamers while retaining the same polymerase fusion proteins, reducing the need for cloning. Third, I will adapt TLISA to detect microRNA, extending the platform to sense nucleic acid biomarkers. Together, this work aims to establish an affordable, modular cell-free biosensing platform that accelerates sensor development and supports future POC diagnostic applications.