UC Riverside

Rapid, sensitive, and accessible pathogen diagnostics are urgently needed to address the global health burden of infectious diseases and the growing threat of antimicrobial resistance. Conventional methods, such as culture-based assays and PCR, remain limited by long turnaround times, sophisticated instrumentation, and restricted multiplexing capabilities, which make them impractical for deployment in resource-limited or point-of-care (POC) settings. This dissertation presents the development of integrated microfluidic platforms coupled with biochemical assays based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection for rapid, portable, and instrument-free diagnostics. A series of microdevices were engineered to overcome limitations in fluid transport, sample processing, and assay integration. First, a Rotation-Chip was designed as a low-cost, reusable platform that enables high-throughput multiplexing through manual fluid transfer. Leveraging superhydrophobic coatings and simple rotational mechanics, the device achieves uniform reagent distribution across 60 wells and supports accurate pathogen detection without pumps or valves. Second, a nano-sieve microdevice incorporating deformable bead stacks was developed to selectively concentrate bacteria from complex samples, increasing sensitivity when paired with RPA/CRISPR assays. Third, an instrument-free duplex microfluidic system was created for simultaneous detection of Leishmania species, demonstrating robustness, leak prevention, and compatibility with lyophilized reagents for field deployment. Finally, a silicon micropillar platform was engineered for one-pot RPA/CRISPR assays, where micropillar surface chemistry and structure enhanced probe immobilization, reduced premature cleavage, and improved assay signal. Together, these platforms highlight a systematic effort to address the challenges of multiplexing, portability, reagent stability, and assay sensitivity. The work demonstrates that combining biochemical assays with engineered microstructures enables diagnostic solutions that meet the World Health Organization’s ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable). This dissertation not only provides new insights into integrating CRISPR-based detection with microfluidics but also establishes versatile device designs adaptable for diverse infectious pathogens, offering significant promise for the future of point-of-care diagnostics.