Infectious diseases cause morbidity and mortality worldwide, with the COVID-19 pandemic, caused by a coronavirus, underscoring this fact. Rapidly mutating and proliferating infectious agents perpetuate the threat of future pandemics, making pathogen monitoring imperative; however, a lack of scalable and convenient multiplexed diagnostic technologies makes this challenging. To address this deficit, new methods and technologies for nucleic acid diagnostics were investigated in this work. Centrifugal microfluidic chips were developed which aliquoted liquid to multiple reaction chambers with 97.5% accuracy. These chips were fabricated using variotherm desktop injection molding, a method innovated in this project capable of faithfully molding 100-micron wide channels with a coefficient of va...[ Read more ]

Infectious diseases cause morbidity and mortality worldwide, with the COVID-19 pandemic, caused by a coronavirus, underscoring this fact. Rapidly mutating and proliferating infectious agents perpetuate the threat of future pandemics, making pathogen monitoring imperative; however, a lack of scalable and convenient multiplexed diagnostic technologies makes this challenging. To address this deficit, new methods and technologies for nucleic acid diagnostics were investigated in this work. Centrifugal microfluidic chips were developed which aliquoted liquid to multiple reaction chambers with 97.5% accuracy. These chips were fabricated using variotherm desktop injection molding, a method innovated in this project capable of faithfully molding 100-micron wide channels with a coefficient of variation averaging only 3.6%. Microfluidic designs prototyped using this method are guaranteed scalable, since they can be mass-manufactured with minimal alteration via industrial injection molding. Loop-mediated isothermal amplification (LAMP) and reverse transcription LAMP (RT-LAMP) were used for detecting DNA and RNA fragments, respectively. A colorimetric method for SARS-CoV-2 detection was initially developed utilizing triarylmethane dyes for assay readouts and had a limit of detection of 170 RNA copies per reaction. Next, halochromic indicators were used to generate readouts in minimally buffered RT-LAMP reactions, followed by the novel usage of fluorescent pH sensors comprising poly(2-hydroxyethyl methacrylate) conjugated with fluorescein isothiocyanate. The acidification caused by positive diagnostic LAMP reactions was converted into a fluorescent signal by the optical pH sensors. Sensor precursor solution was drop-cast into the centrifugal microfluidics’ reaction chambers and using a custom-built imaging rig, allele-specific LAMP (AS-LAMP) and RT-LAMP assays could be performed for multiplexed discrimination of human CYP2C19 alleles and respiratory virus RNA, respectively. Assays were completed within an hour and sensitivities of only 50 RNA copies per reaction were obtained for SARS-CoV-2. This technology should therefore help public health officials, medical practitioners, and even home users to diagnose genetic predispositions and disorders and monitor infectious agents.