Paper microfluidic tools to improve the sensitivity and dynamic range of point of care immunoassays

Abstract

Over the past decade, developing affordable home-based tests to diagnose infectious diseases has become a pressing need. The lateral flow immunoassay (LFIA) is the most successfully commercialized point-of-care immunoassay. However, it suffers from poor sensitivity compared to conventional laboratory techniques such as enzyme-linked immunosorbent assay (ELISA). Consequently, traditional LFIAs fail to deliver on the promise of bedside diagnostic testing for many applications. Paper-based microfluidic devices provide an alternative platform for performing molecular diagnosis at a low cost and have become popular for their simplicity. My research aimed to develop a portable paper-based signal-enhanced immunoassay device that satisfies WHO s ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users) criteria. Using the malarial antigen, PfHRP2, as a model analyte, we developed a gold nanoparticle-based LFIA to determine a baseline limit of detection (LOD). To improve upon the baseline LOD, we ported the ELISA assay into a paper microfluidic device using HRP and poly-HRP enzymes. In addition, we also explored the gold-based enhancement of the signal generated in gold nanoparticle-based LFIAs. Finally, we compared all the colorimetric signal enhancement techniques. While we observed a 4-fold improvement in LOD using the gold enhancement technique, the HRP and the poly-HRP based enhancement did not improve the LOD as expected. This was contrary to the popular belief that enzyme-based signal amplification would produce an improved LOD compared to gold nanoparticle-based LFIAs (despite the fact that a direct comparison was never performed). Using time-lapse imaging, we elucidated that the poor sensitivity in the paper-based ELISA platform is because of the kinetic limitations of the enzymatic amplification system. Finally, we built a 3D printed device housing Arduino-controlled electromagnets to automate the multiple steps of signal-enhanced immunoassays,...