Diagnostics are critical tools to assist in the identification of pathogens, the assessment of medical conditions, and helping to inform therapeutic decisions. Nevertheless, commonly used molecular diagnostics often require sophisticated instruments and skilled technicians, and therefore can only be done in centralized, well-equipped laboratories, which leads to long turnaround times, increased costs, and limited accessibility. These limitations have motivated the development of rapid, low-cost, decentralized diagnostics that are more widely accessible, affordable, and suitable for point-of-care applications. Synthetic biology, by creating rationally designed biological components that can sense disease markers, provides innovative and promising diagnostic solutions to achieve highly sensitive and specific detection for targets of interest, while at the same time being time- and cost-efficient, field-deployable, and shelf-stable. This dissertation focuses on the development of novel biosensing elements and their diagnostic applications. First, I introduce the methods for the computational design of riboregulators using automated algorithms. Followed by that, I describe the development, optimization, and applications of toehold-switch-based platforms for the detection of coccidioides, noroviruses, and severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Next, I introduce the development of an ultra-specific riboregulator system termed single-nucleotide specific programmable riboregulators (SNIPRs) and their use for detecting different variants of concern of SARS-CoV-2. It is shown that riboregulators can be ideal solutions for various pathogen diagnostics with comparable accuracy and reduced cost. Lastly, I describe the use of peptide reporters derived from split protein systems to detect gene mutations. By incorporating peptide reporters into amplification primers, detection can be achieved by a quick isothermal amplification step and cell-free gene expression. Together, this research brings advancements in diagnostics based on riboregulators and cell-free systems that will increase the accessibility of these essential healthcare tools.

No description available.

No description available.