Engineering isothermal amplification solutions for improved point of care diagnostics

Since their inception nearly 60 years ago, molecular diagnostics have amassed impressive achievements with respect to sensitivity, specificity, cost, complexity, and time to-result. Consequently, researchers have become increasingly interested in point of care diagnostics, which seek to deliver results to the end user in an hour or less without need for laboratory instruments or trained technicians. While the potential benefits are obvious, the implementation is not always straightforward. Consider as an example the household pregnancy test, a transformative technology that enabled women to monitor their pregnancy status without a doctor, permeating markets so deeply as to now be considered commonplace. While lateral flow immunoassays like these are fantastic for some applications, their lack of signal amplification can limit sensitivity, making detection of rarer analytes challenging. Furthermore, these tests often require producing several antibodies that recognize discrete epitopes of a given target antigen, impeding the development of assays with novel targets. For this reason, nucleic acid amplification methods like the ubiquitous polymerase chain reaction (PCR) have entered the point of care market, improving limits of detection through the implementation of exponential signal amplification. While PCR requires equipment for thermal cycling and several hours to detect analytes, recent technologies have enabled isothermal amplification solutions that seek to circumnavigate these limitations by continuously replicating targets at a single temperature, resulting in shorter reaction times with improved sensitivity. Nevertheless, these nascent technologies have their own impediments, often requiring multiple enzymatic activities to enable continuous replication that can lead to spurious amplification.
Within this document, I summarize the current achievements and limitations of these isothermal amplification technologies in the context of molecular point of care diagnostics from an academic and economic perspective. Next, I present my efforts in developing real-time detection circuits that provide both specificity and signal amplification to a variety of isothermal reactions, resulting in orders of magnitude improvement in limits of detection. I then explore the use of recombinases as a signal accelerator for said detection circuits, leading to nearly 10-fold increases in reaction rates with programmable background reduction. Finally, I develop a novel directed evolution platform to engineer polymerases with improved isothermal amplification performance, which I validate by evolving a thermostable, strand-displacing polymerase with unique capabilities. These achievements provide a variety of useful tools to assist researchers in developing next-generation diagnostics with unprecedented accuracy, sensitivity, and alacrity.