Abstract

Diagnostic tests can dramatically improve and save lives. Survival rates and patient outcomes related to cancer and other diseases can be improved when routine testing guides personalized treatment. Highly accurate laboratory tests exist for thousands of biomarkers, including those of interest in this dissertation, but cost and complexity limit access to life-saving lab tests in many cases throughout the world. To fill this need and increase accessibility of diagnostics, in this report we report our engineering advancements towards portable biosensors for colorimetric detection of glutamine, endocrine disrupting compounds (EDC), and pathogenic RNA which are key biomarkers in cancer treatment monitoring, cancer prevention, and pathogen containment, respectively. The scope of this dissertation spans from sample collection to delivery of results, featuring optimization related to sample pre-processing, test reagent design, test output engineering, and sensitivity improvements. This dissertation is organized as follows. Chapter 1 provides an introduction to the field of cell-free biosensors and introduces the reported research in the following chapters. Chapter 2 reports our work to engineer blood sample processing with portable dilution and filtration technologies to integrate blood samples with cell-free biosensors. Chapter 3 reports a paper-based luminescent biosensor in a proof-of-concept study to detect SARS-CoV-2 RNA in spiked saliva. Chapter 4 reports the mechanistic discoveries of a fusion protein biosensor to detect EDC hormone binding compounds. Chapter 5 reports the engineering of the fusion protein to reduce costs and improve colorimetric capabilities. In the culminating work of this dissertation, Chapter 6 reports our work to leverage genetic engineering, metabolic engineering, process engineering, and the versatility of cell-free biosensors to create a paper-based glutamine biosensor with dramatically improved signal resolution. The open cell-free protein synthesis platform is well-suited for biosensing owing to the open and accessible reagent pool which can be designed specifically for a desired diagnostic application, and mixed directly with a sample for production of a colorimetric reporter protein. Importantly, the cell-free synthetic biology platform used in this dissertation has been successfully optimized by others in the scientific community to detect a wide variety of clinically valuable biomarkers. Taken together, the projects described in this dissertation represent important progress toward overcoming current limitations of portable biosensing technology. Improved access to portable diagnostics could help patients to receive personalized treatment to promote the best outcomes.

Degree

PhD

College and Department

Ira A. Fulton College of Engineering; Chemical Engineering

Rights

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