Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, May, 2020Includes bibliographical references.In 2016, nearly 5.5 million deaths were attributed to infectious and parasitic diseases. Although many of these diseases are preventable and treatable, resource-constrained regions often lack access to rapid and accurate diagnostic tests to appropriately diagnose and treat these diseases. In order to improve the accessibility of diagnostics, the development of low-cost, simple, and rapid diagnostic tests is vital. Antibodies have been widely used as the binding reagents in these tests to detect a target biomarker from the patient sample. These tests are often designed as a sandwich assay, which requires a pair of antibodies as complementary capture and reporter reagents. However, antibodies have some limitations for use in in vitro applications, including variable stability from clone to clone, long developmental timelines, and structural complexity.In this thesis, we investigated the use of the reduced-charge Sso7d (rcSso7d) binding scaffold as an antibody replacement in diagnostic tests due to its intrinsic stability, inexpensive production in bacteria, and ease of genetic modification. In order to identify unique rcSso7d clones specific to different target biomarkers, we used directed evolution techniques by screening through a yeast surface display library of 1.4 x 10⁹ different clones. Through this process, we identified multiple high affinity variants against target biomarkers for Zika virus, malaria, inflammation and infection, and a foodborne pathogen. We also demonstrated flexibility of the in vitro surface display selection process by incorporating additional selective pressures based on the desired properties, e.g. complementary binding pairs, minimal off-target binding, or binding to a conserved epitope.In order to integrate rcSso7d into diagnostic assays, we incorporated the scaffold into a reporter reagent format to associate a signal in the presence of the target biomarker. We then demonstrated applicability and translatability of the rcSso7d scaffold for use in different diagnostic assay formats, including paper-based, bead-based, well plate ELISA-based, and agglutination assays. Finally, we found that the rcSso7d scaffold retained full functionality in 100% human serum. This work demonstrates that the rcSso7d binding scaffold is a promising alternative binding reagent for the development of robust, low-cost, rapid diagnostic tests to reduce the large global burden of infectious diseases.by Ki-Joo Sung.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Chemical Engineering