Technological advancements towards paper-based biomolecular diagnostics
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Clinically tractable diagnostics must be low-cost, rapid, sensitive, easy to use, and adaptable to new targets. With its rational design, synthetic biology holds promise for developing diagnostic technologies that can address these needs. In particular, progress in synthetic biology has led to improved circuit-building abilities and a large collection of biomolecular sensors. However, these technologies fundamentally require transcription and translation, limiting their applicability to cellular contexts In vitro cell-free expression systems that contain transcription and translation machinery provide the environment necessary for biologically-based technologies to function independently of living cells. Our lab recently developed a paper-based system for cell-free gene expression, which utilizes cell-free extracts that are freeze-dried on to paper and other porous substrates to allow for long-term preservation of synthetic circuits at room temperature. Our platform represents a scalable, cost-effective technology that is easy to use and is compatible with synthetic biology tools. In this dissertation, I present several advancements to this diagnostic platform that are geared towards improving the system’s clinical tractability. In the context of developing a diagnostic for Zika virus that could be deployed in low-resource settings, I demonstrate improvements to diagnostic sensitivity and rapid sample processing that allow for detection of low femtomolar quantities of active virus directly from blood plasma samples. I also describe preliminary results towards a streamlined one-pot amplification-sensing reaction, and propose the development of a paper-based diagnostic for antibiotic susceptibility testing.