The Exploration and Development of Focused Ultrasound Extraction (FUSE) for the Rapid Release of DNA from Complex Tissue Matrices

Over the past two decades, molecular detection platforms have seen rapid advancement, reshaping the way we monitor the safety and security of our environment and human health. One of the key drivers of this transformation has been the development of simpler, faster, and more accessible nucleic acid amplification tests (NAATs) that have enabled point-of-contact (POC) DNA testing, delivering real-time results in resource-limited settings. Despite these advancements, the DNA sample preparation process is laborious, resource-intensive, and often requires hazardous chemicals, preventing the performance of DNA extraction at the POC and severely limiting the potential of POC NAATs. Thus, DNA sample preparation methods have been the primary bottleneck restricting the widespread use and applicability of POC NAATs. To overcome this bottleneck, focused ultrasound extraction (FUSE) was recently introduced as a novel DNA extraction method capable of rapidly releasing DNA from complex tissue matrices without labor-intensive techniques or strong chemicals. This technology utilizes high-pressure focused ultrasound pulses that disintegrate tissue and release DNA through the control of acoustic cavitation. An initial feasibility study demonstrated the potential of FUSE for simple biological tissues, but the use of FUSE for preparing complex tissues has not been explored previously. Understanding the potential of the FUSE technology with diverse sample types is essential for developing versatile DNA preparation methods that can effectively protect both the environment and human health. This dissertation investigates the performance of FUSE in complex tissue matrices and evaluates the utility of a miniaturized FUSE system for streamlined DNA sample preparation. Specifically, this work addresses (1) the feasibility of FUSE in robust sample types with physical and chemical complexities that hinder DNA release, (2) the optimization of FUSE pulsing parameters to enhance the time efficiency of FUSE processing and improve the quality of released DNA, and (3) the development of a compact, accessible device for the performance of FUSE DNA sample preparation in resource-limited settings. The completion of this work will introduce a novel DNA sample preparation method to enable the use of POC NAATs.