Development of a Highly Sensitive Protein Diagnostic Device using Isothermal Strand Displacement Amplification

Thesis (Master's)--University of Washington, 2018The lateral flow test (LFT) is the preferred tool for detection of protein biomarkers in low-resource settings. While low-cost and rapid, these immunoassay devices often suffer from limited sensitivity in the single chemical step format. Our group has addressed this deficiency by coupling a lateral flow test to isothermal strand displacement amplification (iSDA), a nucleic-acid amplification test (NAAT). This novel method is called LFT-iSDA and uses an oligonucleotide-labelled detection antibody in the immunoassay as the target template for iSDA. In its current embodiment, LFT-iSDA is ~10,000x more sensitive than gold nanoparticle-based LFTs, but is highly manual and relies on laboratory infrastructure. Here we present preliminary development of a diagnostic device integrating the highly sensitive LFT-iSDA into a cartridge supporting rapid time-to-results and minimal user-steps— a format amenable to decentralized test settings. We characterized performance limitations of this assay, developed a real-time fluorescence detection system, and used a two-dimensional paper network (2DPN) to semi-automate this multi-step chemical process. These results show important progress toward enabling highly sensitive protein detection in a user-friendly, low-cost device, at the point of care