MEMS-enhanced solid-phase isothermal amplification for rapid, multiplexed molecular diagnostics

ABSTRACT RNA viruses are responsible for substantial morbidity and mortality worldwide, from HIV to Influenza, to Coronavirus. As the clinical presentation for disease may be similar or asymptomatic, accurate and rapid diagnosis is essential for monitoring outbreaks and administering of effective therapy. Despite this, there remains an unmet need for nucleic acid amplification tests that are rapid (lt30 minutes), highly multiplexed, compact, and cost-effective. In this project, we will use a MEMS technology for microfluidic agitation to accelerate solid phase isothermal amplification by at least 10x. The focus of this Phase I proposal is the development of the amplification reaction chamber. Solid-phase (SP) amplification is a well-known potential solution to achieve multiplexing in single-pot INAA. In SP-INAA, one or both primers for each target is immobilized on a surface, while the other reagents remain in solution. Unfortunately, solid-phase amplification is dramatically less efficient than liquid-phase reactions, because template needs to diffuse to the primer location in order to be amplified. SP-INAA is therefore slower and has a lower LOD than standard liquid-phase NAATs. We aim to demonstrate SP-INAA that is at least 10x—and as much as 100x—faster than prior implementations of SP-amplification. Our module will be a self-contained, all-in-one amplification module, with an INAA master mix lyophilized inside the chamber in addition to immobilized primers, so the only addition required will be a sample ready for amplification. In success, this project will deliver a breakthrough in nucleic acid amplification testing (NAATs) by eliminating the tradeoff between speed, multiplexing, and device complexity.